Textbook of Pediatric Hematology and Hemato-Oncology (PDFDrive)

Textbook of
Pediatric Hematology and

Hemato-Oncology
Textbook of
Pediatric Hematology and
Hemato-Oncology
Editor-in-Chief
MR Lokeshwar
Consultant Pediatrician and Pediatric Hematologist Oncologist
Shushrusha Citizens Co-operative Hospital and
Lilavati Hospital and Research Centre, Mumbai, India
Editors
Nitin K Shah Bharat R Agarwal
President, Indian Academy of Pediatrics, 2006 Head
Consultant Pediatrician, PD Hinduja Hospital, Mumbai, India Department of Pediatric Hematology and Oncology
Hon. Pediatric Hematologist Oncologist BJ Wadia Hospital for Children Institute of Child Health
BJ Wadia Hospital and Lions Hospital, Mumbai, India and Research Centre, Mumbai, India
Co-editors
Mamta Vijay Manglani Anupam Sachdeva

Professor and Head Director, Pediatric Hematology-Oncology and
Department of Pediatrics Bone Marrow Transplantation, Institute for Child Health
Chief, Division of Hematology-Oncology Sir Ganga Ram Hospital, New Delhi, India
Program Director, Pediatric Center of Excellence for HIV Care Recipient, Dr BC Roy Award
Lokmanya Tilak Municipal Medical College and Recipient, Silver Jubilee Research Award
General Hospital, Mumbai, India
Publication Editor
Asha Pillai
Medical Officer
Kashyap Nursing Home
Mumbai, India
Forewords
SS Kamath
Vijay N Yewale
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Textbook of Pediatric Hematology and Hemato-Oncology
First Edition: 2016
ISBN: 978-93-5152-143-3
Printed at
Dedicated to
Zinet Currimbhoy
Teacher of Teachers
in Pediatric Hematology and Oncology
for whom
Children with blood disorders and cancer were the greatest teachers!
Contributors
Aditya Kumar Gupta Anand Deshpande

MD FNB (Pediatric Hematology Oncology, SGRH) Consultant
Assistant Professor Hematopathologist and In-Charge
Division of Pediatric Hematology- Transfusion Medicine
Oncology PD Hinduja National Hospital
Department of Pediatrics Mumbai, India
Institute of Medical Sciences [email protected]
Banaras Hindu University
Varanasi, India Anupa A Joshipura
[email protected] Fellow Pediatric Hemato-Oncology
BJ Wadia Hospital for Children
Ajay Kumar Institute of Child Health and Research Centre
Senior Specialist Mumbai, India
Department of Neonatology [email protected]
Lady Hardinge Medical College
and Kalawati Saran Children’s Hospital
New Delhi, India Anupama S Borker
[email protected] Consultant Pediatric Oncologist
Somaiya Ayurvihar
Aman Chauhan Asian Institute of Oncology
Internal Medicine and Pediatrics KJ Somaiya Hospital Campus
Combined Resident Mumbai, India
Louisiana State University Health Science [email protected]
Center
New Orleans, USA
Anupam Sachdeva
[email protected]
Director
Pediatric Hematology-Oncology and
Ambreen Pandrowala Bone Marrow Transplantation
Clinical Associate Institute for Child Health
Lilavati Hospital Sir Ganga Ram Hospital, New Delhi, India
Mumbai, India Recipient, Dr BC Roy Award
[email protected] Recipient, Silver Jubilee Research Award
[email protected]
Amol Dongre Aparna Vijayaraghavan

Assistant Professor Registrar
Department of Medical Oncology Department of Pediatrics
Jawaharlal Nehru Medical College Chennai, India
Wardha, India [email protected]
[email protected]
viii Textbook of Pediatric Hematology and Hemato-Oncology
AP Dubey Brijesh Arora

Director Professor
Professor and Head Division of Pediatric Oncology
Department of Pediatrics Tata Memorial Hospital
Maulana Azad Medical College and Mumbai, India
Associated Lok Nayak Hospital [email protected]
New Delhi, India
[email protected] Deepak K Changlani
Pediatric Interventional Cardiologist
Arvind Saili Lilavati Hospital and Research Centre
Director Mumbai, India
Professor and Head [email protected]
Department of Neonatology
Lady Hardinge Medical College and
Kalawati Saran Children’s Hospital Dilraj Kaur Kahlon
New Delhi, India Consultant Pediatric Hemato-Oncologist
[email protected] Amandeep Hospital
Amritsar, India
[email protected]
ATK Rau
Pediatric Hematologist-Oncologist
Professor, Department of Pediatrics
Dinesh Yadav MD
MS Ramaiah Medical College
Consultant Pediatrician
Bengaluru, India
Vivekanand Hospital, Bhadra, India
[email protected]
Formerly Assistant Professor
Department of Pediatrics
Sri Aurobindo Institute of Medical
Bharat R Agarwal
Sciences, Indore, India
Head
[email protected]
Department of Pediatric Hematology and
Oncology
BJ Wadia Hospital for Children Farah Jijina
Institute of Child Health and Research Consultant Hematologist
Centre, Mumbai, India PD Hinduja Hospital, Mahim, India
[email protected] Professor
Department of Hematology
Seth GS Medical College and
Bhavna Dhingra KEM Hospital, Mumbai, India
Assistant Professor [email protected]
All India Institute of Medical Sciences Gaurav Narula
Bhopal, India Associate Professor (Pediatric-Oncology)
[email protected] Department of Medical Oncology
Tata Memorial Hospital
Mumbai, India
Bipin P Kulkarni [email protected]
Scientist ‘B’
National Institute of
Immunohaematology (Indian Council of Girish Chinnaswamy
Medical Research, ICMR) Associate Professor (Pediatric-Oncology)
KEM Hospital Campus Department of Medical Oncology
Mumbai, India Tata Memorial Hospital
[email protected] Mumbai, India
www.niih.org.in [email protected]
Contributors ix
Jagdish Chandra Malobika Bhattacharya

Director-Professor of Pediatrics Consultant Pediatrics
In-Charge, Pediatric Hematology and Kailash Hospital
Thalassemia Day Care Centre Greater Noida, India
Programme Director, Pediatric Centre of [email protected]
Excellence (HIV)
Lady Hardinge Medical College and
Kalawati Saran Children’s Hospital
Mamta Vijay Manglani
New Delhi, India
Professor and Head
[email protected]
Chief, Division of Hematology-Oncology
Jayashree Mondkar Program Director, Pediatric Center of
Professor and Head Excellence for HIV Care, Lokmanya Tilak
Department of Neonatology Municipal Medical College and General
Director Hospital, Mumbai, India
Human Milk Bank [email protected]
Lokmanya Tilak Municipal Medical
College and General Hospital
Mumbai, India Manas Kalra
[email protected] Fellowship
Pediatric Oncology and BMT-Sydney
Kana Ram Jat Consultant, Pediatric Hematologist
Assistant Professor Oncologist and BMT Unit
Department of Pediatrics Sir Ganga Ram Hospital
All India Institute of Medical Sciences New Delhi, India
New Delhi, India [email protected]
[email protected]
Maya Prasad
K Ghosh Assistant Professor
Director Department of Medical Oncology
National Institute of (Pediatrics)
Immunohaematology (ICMR) Tata Memorial Hospital, Mumbai, India
KEM Hospital Campus [email protected]
Mumbai, India
[email protected]
MMA Faridi
Professor and Head
KN Aggarwal MD FIAP FAMS FNA
Former Professor and Director
In-Charge
Institute of Medical Sciences
Division of Neonatology
Banaras Hindu University
University College of Medical Sciences
Varanasi, India
Guru Teg Bahadur Hospital
Former Director
New Delhi, India
SGPGIMS, Lucknow, India
[email protected]
Professor
UCMS, Bhairwah, Nepal MR Lokeshwar
Consultant Pediatrician and Pediatric
Madhulika Kabra Hematologist Oncologist
Professor, Division of Genetics Shushrusha Citizens
All India Institute of Medical Sciences Co-operative Hospital and Lilavati
New Delhi, India Hospital and Research Centre
[email protected] [email protected]
x Textbook of Pediatric Hematology and Hemato-Oncology
Mukesh M Desai MD Nirmalya D Pradhan

Hon. Hematologist Oncologist and Senior Pediatric Oncologist
Immunologist Department of Pediatric Oncology
Professor Tata Memorial Hospital
Department of Pediatric Hematology- Mumbai, India
Oncology (DNB) [email protected]
Department of PHO
Chief Division of Immunology Nita Radhakrishnan
BJ Wadia Hospital for Children Consultant Pediatric Hematology
Honorary Consultant Hematologist Oncology
B Nanavati Hospital Institute of Child Health
Sir HN Hospital Sir Ganga Ram Hospital
Saifee Hospital New Delhi, India
Asian Heart Institute, India [email protected]
[email protected]
Nitin K Shah
Narendra Chaudhary President
Assistant Professor Indian Academy of Pediatrics, 2006
Pediatric Hematology-Oncology Consultant Pediatrician
Department of Child Health PD Hinduja Hospital, Mumbai, India
Christian Medical College Hon. Pediatric Hematologist Oncologist
Tamil Nadu, India BJ Wadia Hospital and Lions Hospital
[email protected]
Neerja Gupta
Assistant Professor Pankaj Dwivedi
Division of Genetics Fellow in Pediatric, Hematology and
Department of Pediatrics Oncology
All India Institute of Medical Sciences Hospital for Sick Children
New Delhi, India Toronto, Canada
Neha Vilas Dighe Pooja Balasubramanian

Fellow Clinical Associate
Department of Pediatrics Lilavati Hospital and Research Centre
BJ Wadia Hospital for Children Mumbai, India
Institute of Child Health and Research Centre [email protected]
Mumbai, India
[email protected]
Pooja Dewan
Nirav Buch MD FNB
Assistant Professor
Pediatric Hematologist-Oncologist
[email protected]
University College of Medical Sciences
[email protected]
Guru Teg Bahadur Hospital
Delhi, India
[email protected]
Nirav Thacker Priti Desai

Senior Registrar Pediatric Oncology Associate Professor
Department of Pediatric Oncology Department of Transfusion Medicine
Tata Memorial Hospital Tata Memorial Hospital
Mumbai, India Mumbai, India
Contributors xi
PS Patil MD FIAP Rashmi Dalvi

Neo Clinic Consultant Pediatric Oncologist
Samarth Nagar Bombay Hospital and Medical Research
Aurangabad, India Centre
Rajesh B Sawant MD Path Ratna Sharma

Consultant—Transfusion Medicine Professor (Pediatrics)
PD Hinduja National Hospital and MRC In-Charge
Mumbai, India Pediatric Hematology-Oncology
[email protected] Dr DY Patil Hospital
[email protected] Navi Mumbai, India
[email protected]
Rajiv Kumar Bansal Renu Saxena

Consultant Pediatrician Professor and Head
In-Charge Thalassemia Unit Department of Hematology
Santokba Durlabhji Hospital All India Institute of Medical Sciences
Jaipur, India New Delhi, India
Raj Warrier MD FAAP FIAP Revathi Raj

Section Head Consultant Pediatric Hematologist
Ochsner, Peds Hem/Onc Apollo Hospitals
Ochsner for Children Chennai, India
Professor Emeritus and Clinical Professor [email protected]
Louisiana State University Health
Sciences Center (LSUHSC)
New Orleans, USA Rhishikesh Thakre
Professor Consultant Neonatologist
Department of Pediatrics Neo Clinic
Tulane University School of Medicine Aurangabad, India
New Orleans, USA [email protected]
Professor
University of Queensland, Australia Sadhna Arora
Manipal University, India Genetics Unit
[email protected] Old OT Block
[email protected]
All India Institute of Medical Sciences
(Late) Ram Kumar Marwaha New Delhi, India
Professor [email protected]
In-Charge, Division of Pediatric
Saroj P Panda
Hematology-Oncology
DM Trainee (Pediatric Oncology)
Advanced Pediatric Centre
Department of Medical Oncology
Postgraduate Institute of Medical
Education and Research (PGIMER) Tata Memorial Hospital
Chandigarh, India Mumbai, India
xii Textbook of Pediatric Hematology and Hemato-Oncology
Satya P Yadav Shripad Banavali

Senior Consultant and Head Professor and Head
Pediatric Hemato-Oncology and BMT Department of Medical Oncology
Unit Tata Memorial Hospital
Fortis Memorial Research Institute Mumbai, India
Gurgaon, India [email protected]
[email protected]
[email protected]
Sonali Sadawarte
SB Rajadhyaksha Consultant Hematologist and BMT
Professor and Head Physician Royal Hobart Hospital
Department of Transfusion Medicine Hobart, Tasmania, Australia
Tata Memorial Hospital [email protected]
Mumbai, India
[email protected]
Sonika Agarwal
Seema Gulia
Pediatric Neurology Fellow
Assistant Professor
Baylor College of Medicine
Department of Medical Oncology
Houston
Tata Memorial Hospital
TX, USA
Mumbai, India
[email protected]
[email protected]
Shanaz Khodaiji Soundarya M

Consultant Hematology and Transfusion Associate Professor
Medicine Department of Pediatrics
PD Hinduja National Hospital and MRC Kasturba Medical College
Mumbai, India Mangalore, India
Consultant Hematologist [email protected]
PD Hinduja National Hospital and
Medical Research Centre
Mumbai, India Sriram Krishnamurthy
[email protected] Associate Professor
Shilpa Sanjay Borse MD Jawaharlal Institute of Postgraduate
Pediatrics Medical Education and Research
Dattatraya Nagar (JIPMER)
Nagpur, India Puducherry, India
Sunil Gomber
Shrimati Shetty Director Professor
Scientist E Department of Pediatrics
National Institute of In-Charge Hematology-Oncology
Immunohaematology (ICMR) University College of Medical Sciences
KEM Hospital Guru Teg Bahadur Hospital
Mumbai, India New Delhi, India
Contributors xiii
Sunil Udgire FNB Vineeta Gupta

Fellow Associate Professor and In-Charge
Department of Hemato-Oncology Pediatric Hematology-Oncology
BJ Wadia Hospital for Children Department of Pediatrics
Institute of Child Health and Research Centre Institute of Medical Sciences
Mumbai, India Banaras Hindu University
[email protected] Varanasi, India
[email protected]
Swati Kanakia MD DCH PhD
Pediatric Hematologist-Oncologist VP Choudhary
Kanakia Health Care, Lilavati Hospital MD FIAP FIMSA FIACM FISHTM
and Research Centre Former Professor and Head
Raheja Fortis Hospital Department of Hematology
Lion Tarachand Bapa All India Institute of Medical Sciences
Hospital, Mumbai, India New Delhi, India
[email protected] Director
Sunflag Hospital
Faridabad, India
[email protected]
Foreword
It is both a pleasure and a privilege to write a foreword for this first edition of Textbook of Pediatric Hematology and
Hemato-Oncology. Immense advancement has been made over the past decade in the field of hematology and hemato-
oncology providing not only enhanced accuracy in the diagnosis of inherited and acquired malignant and nonmalignant
blood disorders but also new therapeutic strategies that have resulted in improved patient outcomes. The book with
its illustrations, tables, figures and clinical photographs provides a concise yet thorough comprehension of pediatric
hematology and will definitely become a reference book for the students and the practitioners.
I congratulate the Editor-in-Chief, MR Lokeshwar; Editors, Nitin K Shah and Bharat R Agarwal; Co-editors, Mamta
Vijay Manglani and Anupam Sachdeva; Publication Editor, Asha Pillai and the 72 reputed and dedicated pediatric
hematologists and hemato-oncologists from across the world who after 3 years of exhausting brainstorming sessions,
brought out the remarkable book with 50 chapters spread over 7 sections.
I am sure that the information contained herein will be a benchmark in the understanding of the best approaches to
the patients that we evaluate and manage.
SS Kamath
President, 2015
Indian Academy of Pediatrics (IAP)
[email protected]
Foreword
I am delighted to write the foreword for the first comprehensive book Textbook of Pediatric Hematology and Hemato-
Oncology. Postgraduate students look up to their teachers for a book by the editorial team of Doynes in the field of
Pediatric Hematology and Hemato-oncology with their vast experience in the field and past experience in writing will
fill in this void and meet the expectations of the postgraduate students. The Editor-in-Chief, MR Lokeshwar has been
well supported by the other editors, Nitin K Shah, Bharat R Agarwal, Mamta Vijay Manglani and Anupam Sachdeva.
The descriptive text along with clinical pictures make it an interesting reading material. It covers a vast spectrum of
conditions making it very useful to the reader. I congratulate the entire team which includes the contributors, section
editors, editorial board and M/s Jaypee Brothers Medical Publishers (P) Ltd, New Delhi, India, who have worked
relentlessly to achieve this milestone for the book. Above all, my hearty congratulations to the ever-enterprising respected
Dr MR Lokeshwar who thought of this brilliant idea of Textbook on Pediatric Hematology and Hemato-Oncology. I wish
the best for the success and popularity of the publication among both postgraduate students and practitioners. It will be
a landmark publication on Pediatric Hematology Oncology in the history of medical literature.
Vijay N Yewale
President, 2014
Indian Academy of Pediatrics (IAP)
Preface
Pediatric Hematology and Hemato-Oncology as a pediatric specialty has developed rapidly in the Western countries since
last few decades and is now catching-up in developing countries. Gone are the days when the ‘adult’ hematologists and
hemato-oncologists used to treat pediatric patients in need of specialized services. To begin with several pediatricians
got self-trained in pediatric hematology and hemato-oncology and pioneered training in field of pediatric hematology
and hemato-oncology in the form of several informal and formal courses throughout the country. This created interest
in budding pediatricians to join this field by undergoing fellowships or even formal training in pediatric hematology
and hemato-oncology abroad and now in India. The last step towards the growth of this specialty has been starting of
2 years of Post-Doctorate Fellowship in Pediatric Hematology and Hemato-Oncology by the National Board in India
with several pioneering centers now offering this course since last 7 years, as well as 1 year Post-Doctorate Fellowship
by the Maharashtra University of Health Sciences (MUHS) and even by the Indian Academy of Pediatrics (IAP). Several
formally trained Pediatric Hematologists and Pediatric Hemato-oncologists are now providing the specialized services
in private and public hospitals.
With the interest created in the field of pediatric hematology and hemato-oncology, there was also a felt need by the
students as well as practitioners alike to have a dedicated textbook on pediatric hematology and hemato-oncology. While
there are several reputed textbooks on pediatric hematology and hemato-oncology, many of them are also elaborate
and at times bogged down with details of molecular science which may not necessarily fulfill the needs of students and
practitioners who are looking at complete yet concise book.
With the single aim in mind of having a complete and yet easy-to-read textbook on pediatric hematology and hemato-
oncology, we have attempted to bring out the first edition after 3 years of brain-storming and grueling process. The 50
chapters spread over 7 sections and authored by 72 reputed pediatric hematologists and hemato-oncologists from India
and abroad make the book elaborate enough to give enough to all the readers, yet curtailing it to more than 500 pages
making it concise enough! Further powered by illustrations, tables, figures and clinical photographs will make reading
a unique and memorable event for the readers. Each chapter is further enriched by references at the end and is peer-
reviewed making it scientifically as complete as possible. This being the first edition is bound to have some errors which
might have escaped our attention in spite of our best efforts. We would request all our readers to send their feedback to
us which will help us improve upon in the subsequent editions.
We are sure that the book will become a reference book for the students and a desk companion to the practitioners!
Editors
Acknowledgments
We would like to express our gratitude to many people who saw us sail through the publication of Textbook of Pediatric
Hematology and Hemato-Oncology by providing support, talking things over, read, write, offering comments and helping
us in bringing out the book.
We wish to especially thank the following people for their contributions:
• Shri Jitendar P Vij (Group Chairman), Mr Ankit Vij (Group President), Mr Tarun Duneja (Director-Publishing)
without whom the book would have not found its way, Ms Samina Khan (Executive Assistant to Director-Publishing)
for guiding us throughout, Mr KK Raman (Production Manager), Mr Sunil Dogra (Production Executive) and other
staff of M/s Jaypee Brothers Medical Publishers (P) Ltd, New Delhi, India, for assisting us in the editing, proofreading
and designing skills.
• All the authors/contributors mentioned in the list of contributors.
• Special thanks to Mr Harish Raut for assisting us in the editing, proofreading and designing skills.
• All the patients and families for allowing us to continue to learn the subject.
• We would also like to thank our family members for their support.
Last but not least, we beg forgiveness of all those who have been with us over the course of years and whose names,
we have failed to mention.
Editorial Board
Contents
Section 1 PHYSIOLOGY
1. Ontogeny of Erythropoiesis 3
Nirav Buch
2. Physiology of Blood Coagulation 10
Shrimati Shetty
3. Structure, Function and Physiology of Platelets 15
K Ghosh, Bipin P Kulkarni
Section 2 NEONATAL HEMATOLOGY
4. Variation in the RBC Parameters in the Newborn 23

MR Lokeshwar, Ambreen Pandrowala, Jayashree Mondkar
5. Physiological Anemia of Newborn, Anemia of Prematurity and Role
of Erythropoietin in the Management 29
Rhishikesh Thakre, PS Patil
6. Effect of Maternal Iron Status on Placenta, Fetus and Newborn 36
KN Aggarwal, Vineeta Gupta, Sonika Agarwal
7. Developmental Aspects of Hemostasis in the Fetus and Newborn 41
Bhavna Dhingra, Renu Saxena
8. Anemia in the Newborn 45
Jayashree Mondkar, Shilpa Sanjay Borse, MR Lokeshwar
9. Polycythemia and Hyperviscosity Syndrome 57
MMA Faridi, Sriram Krishnamurthy
10. Vitamin K Deficiency: Bleeding in Newborns 64
Arvind Saili, Ajay Kumar
11. Bleeding Neonate: Approach and Management 68
Mamta Vijay Manglani, Neha Vilas Dighe, Ratna Sharma, MR Lokeshwar
12. Approach to Neonatal Thrombocytopenia 77
Nitin K Shah
Section 3 RBC AND WBC DISORDERS
13. Introduction and Classification of Anemias in Children 87

Manas Kalra, Satya P Yadav, Anupam Sachdeva
14. Nutritional Anemia in Infancy, Childhood and Adolescents 100
MR Lokeshwar, Nitin K Shah
xxiv Textbook of Pediatric Hematology and Hemato-Oncology
15. Megaloblastic Anemia 126

Anupa A Joshipura, Nitin K Shah
16. Anemia of Chronic Disease 149
Dilraj Kaur Kahlon, Satya P Yadav, Anupam Sachdeva
17. Thalassemia Syndromes 163
Mamta Vijay Manglani, Ambreen Pandrowala, Ratna Sharma, MR Lokeshwar
18. Sickle Cell Anemia in Children 190
Swati Kanakia, Pooja Balasubramanian, MR Lokeshwar
19. Antenatal Diagnosis of Hemoglobinopathies 204
Neerja Gupta, Sadhna Arora, Madhulika Kabra
20. Red Cell Membrane Disorders (Spherocytosis, Elliptocytosis, Stomatocytosis) 213
Sunil Gomber, Pooja Dewan
21. Red Cell Enzymopathy 219
Bhavna Dhingra, Dinesh Yadav, Jagdish Chandra
22. Autoimmune Hemolytic Anemia 227
Rajiv Kumar Bansal
23. Paroxysmal Nocturnal Hemoglobinuria 238
Farah Jijina, Sonali Sadawarte
24. Diagnosis and Management of Acquired Aplastic Anemia in Children 247
Nitin K Shah
25. Inherited Bone Marrow Failure Syndromes 255
Revathi Raj
26. Benign Disorders of Neutrophils 258
Bharat R Agarwal
Section 4 BLEEDING DISORDERS
27. Approach to a Bleeding Child 275

Raj Warrier, MR Lokeshwar, Aman Chauhan
28. Diagnosis and Management of Hemophilia Patients 285
Farah Jijina
29. von Willebrand Disease and Other Rare Coagulation Disorders 296
Kana Ram Jat, Ram Kumar Marwaha
30. Acquired Inhibitors of Coagulation 311
ATK Rau, Soundarya M
31. Immune Thrombocytopenic Purpura—Diagnosis and Management 318
MR Lokeshwar, Deepak K Changlani, Aparna Vijayaraghavan
32. Platelet Function Disorders 332
Shanaz Khodaiji
33. Pediatric Thrombosis 348
Rashmi Dalvi
34. Disseminated Intravascular Coagulation in Neonates 356
VP Choudhary
Contents xxv
Section 5 TRANSFUSION MEDICINE
35. Blood Components in Pediatric Practice 363

Nitin K Shah, Sunil Udgire
36. Nucleic Acid Amplification Testing 372
Anand Deshpande, Rajesh B Sawant
37. Transfusion Transmitted Infections 376
AP Dubey, Malobika Bhattacharya
38. Noninfectious Hazards of Blood Transfusion 384
SB Rajadhyaksha, Priti Desai
Section 6 HEMATO-ONCOLOGY
39. Pediatric Acute Lymphoblastic Leukemia 395

Pankaj Dwivedi, Shripad Banavali
40. Pediatric Acute Myeloid Leukemia 408
Maya Prasad, Shripad Banavali
41. Chronic Myeloid Leukemia 419
Nirav Thacker, Brijesh Arora
42. Juvenile Myelomonocytic Leukemia 430
Gaurav Narula, Nirmalya D Pradhan
43. Pediatric Hodgkin Lymphoma 439
Amol Dongre, Brijesh Arora
44. Non-Hodgkin Lymphoma in Children and Adolescents 451
Seema Gulia, Brijesh Arora
45. Langerhans Cell Histiocytosis 462
46. Hemophagocytic Lymphohistiocytosis: Revisited 470
Mukesh M Desai, Sunil Udgire
47. Bone Marrow Transplantation 479
Nita Radhakrishnan, Satya P Yadav, Anupam Sachdeva
Section 7 GENERAL
48. Gene Therapy 491

Aditya Kumar Gupta, Nita Radhakrishnan, Anupam Sachdeva
49. Monoclonal Antibodies in Pediatric Hematology and Oncology 496
Saroj P Panda, Girish Chinnaswamy
50. Biological Response Modifiers 501
Anupama S Borker, Narendra Chaudhary
Index 511
S E C T I O N 1
Physiology
CHAPTERS OUTLINE
1. Ontogeny of Erythropoiesis
Nirav Buch
2. Physiology of Blood Coagulation
Shrimati Shetty
3. Structure, Function and Physiology of Platelets
C H A P T E R 1
Ontogeny of
Erythropoiesis
Nirav Buch
DEVELOPMENT OF HEMATOPOIESIS vasculature.1 Initially, clusters of mesodermal cells called

hemangioblasts develops in the extra-embryonic region.
Hematopoiesis occurs in three different waves in humans: They are initially solid but later inner cells disappear
yolk sac liver and the bone marrow named so based upon and peripheral cells acquire morphology of vascular
the main sites of hematopoiesis. Their characteristics endothelium, opening up vessel lumens. Cells adhering to
reflect the oxygen needs and the characteristics of the these endothelium form hematopoietic cells and are called
developing embryo (Fig. 1).1 blood islands (Figs 2A to C). These cells (endothelium
and hematopoietic precursors) show CD 34 expression
Yolk Sac Phase (Figs 3A and B).2
After 19 days of fertilization islands of hematopoietic They are macrocytic and contain embryonal hemo
tissue appear in the yolk sac and develop within the globins: Gower I (ζ2ε2), Gower II (α2ε2) and Portland
Fig. 1 Chronology of appearance of hematopoietic stem cells in the developing human embryo2
4 Section-1 Physiology
A B C
Figs 2A to C Sequence of emergence of hematopoietic stem cell cluster within the human embryo. (A) Beginning from 27 days of
development, scattered groups of a few hematopoietic stem cells appear, adhering to the aortic endothelium in the preumbilical region.
Groups of 2 to 3 cells are also often detected in a more rostral region, where the aorta is still bifurcated; (B) From day 30, the hematopoietic
cell clusters increase in size and groups of cells are also encountered at the bifurcation of the vitelline artery, always associated with the
ventral aspect of the vascular endothelium; (C) The size of hematopoietic progenitor clusters attains several hundreds of cells at 36 days
of development. At subsequent stages, stem cell clusters undergo gradual decrease till 40th day
Abbreviations: A: Aorta; V: Vitelline duct; U: Umbilical Arteries; H: Heart.
I (ζ2γ2)—and later in the development hemoglobin

F (α2γ2). Cells are large and are nucleated. These consist
mainly of erythroid precursors but during 6th and 7th
week few megakaryocytes also develop.1
This first wave of erythrocyte production in the yolk
sac is known as primitive, and erythrocyte production that
takes place in is known as definitive erythropoiesis.2
A
Onset of Blood Circulation
Primitive erythrocytes are detected in embryo at day 21
(3 somite stage) suggesting vascular connections between
the yolk sac and the embryo.2
Transition from Yolk Sac to Hepatic

B
Erythropoiesis
Primitive nucleated erythroblasts are seen in early hepatic
rudiment from 4.5 to 5 weeks onwards. They rapidly decrease
in number and are replaced by definitive macrocytes. At day
23, rare CD 34 negative cells of erythromyeloid lineage are
Figs 3A and B Possible origin hematopoietic stem cell emergence
detected followed by CD 34 +ve cells at day 30 suggesting
within the human embryo.2 (A) Redifferentiation hypothesis at 27
two distinct waves of hepatic colonization.2
days of development, pre-existing endothelial cells in the ventral
aspect of human intraembryonic arteries differentiate locally
Hepatic Phase
into blood cell progenitors; (B) Migration hypothesis scattered
mesodermal CD34-CD45–cell precursors colonize the ventral Hepatic colonization of hematopoietic progenitors start
vascular wall and give rise to (blood-forming?) endothelial cells by 6 weeks and the liver becomes a major hematopoietic
and hematopoietic cell clusters organ in the 2nd trimester with about half of nucleated
Chapter-1 Ontogeny of Erythropoiesis 5
cells of liver being erythroid precursors. These are

smaller than their yolk sac predecessors and result in
forming anucleate red cells that are megaloblastic. Chief
hemoglobin is HbF. The maturation of cells is extravascular
in association with macrophages of the erythroid islands.
The hematopoiesis now is multilineage and has erythroid
myeloid megakaryocytic and lymphoid precursors.1
Bone Marrow Phase (Table 1)

With hepatocyte proliferation hematopoiesis becomes
restricted in the liver and bone marrow hematopoiesis
begins in the fetal bone marrow by 16th to 18th weeks.
Fetal marrow becomes major site for hematopoiesis by
6th month of gestation. Hematopoiesis is multilineage with
normoblastic maturation. Chief hemoglobin contents are
HbF and HbA. Fetal marrow has dominant erythropoiesis Fig. 4 The phases of embryonal and fetal hematopoiesis. There is
with M : E ratio at about 1:4.1 a considerable overlap and gradual transition from stage to stage1
CYTOKINE REGULATION OF ONTOGENY

AND HEMATOPOIESIS
Transcriptional Regulation of Early
Several cytokines play an important role in hema
topoiesis. They are granulocyte colony-stimulating Hematopoietic Development (Fig. 5)
factor, interleukin (IL)-6, IL-1, IL-4, IL-9, insulin growth
factor-1 and EPO. EPO plays an important role in
erythropoiesis. Loss of EPO/EPO receptor leads to failure
of fetal erythropoiesis causing fetal death. In adults,
EPO provides antiapoptotic and proliferative signals to
erythroid precursors (Fig. 4).1
Hematopoietic Cytokines, Transcription

Factors and Lineage Commitment
Close Relation with Endothelium
The endothelium and hematopoietic progenitor cells Fig. 5 Important transcription factors for primitive and definitive
share several antigens supporting theory of hemogenic hematopoiesis are SCL (stem cell leukemia hematopoietic
endothelium or hemangioblasts. They are SCL, GATA-2, transcription factor), GATA-2 and Lmo-2. AML-1 is required for
C-kit, AA-4.1, CD34, Flit-3 ligand, Sca-1, VEGFR-1 and -2, definitive hematopoiesis4
only with the exception of CD45 (Fig. 5).4
Regulation of Self-renewal and
Differentiation of HSCs
Table 1 Comparison of embryonic, fetal and adult Transcription factors HoxB4 and Ikaros, activated nuclear
erythropoiesis3 form of Notch1, cell, cycle inhibitor P21, and TGF/BMP-
4 family members, TNF-a receptor P55 signaling may
Yolk sac Liver Bone marrow
be important in the maintenance or promotion of the
Lineages Erythroid All All
hematopoietic stem cell renewal. Adjacent cells stromal
Stem cell Cycling G0 G0
cells, endothelial cells and local cytokines are thought
Erythroid site Yolk sac Liver Bone marrow
to play an important role in regulation of HSC cell into
Nucleated RBC Yes No No renewal or differentiation. TGF-b1, p21, p27, IL-3, GM-CSF,
a-globulin za1, a2 a1, a2 a1, a2 BMP-4 and TNF-a are some of the important cytokines
b-globulin e gA, gD bd involved.4
Commitment to Lymphoid and

Myeloid Lineage (Fig. 6)
After 10 to 15 divisions, the descendent cells daughter
cells become fixed towards a single lineage. First cells
are believed to be either common lymphoid precursor
or common myeloid precursor. These have been
differentiated using specific expression patterns.4 It is
believed to be brought about by differential expression of Fig. 7 Chromosome map of human globin chains9
transcription factors. Several candidate transcriptional
factors differentially expressed in committed cells have been
identified using cDNA library and RT-PCR technologies.4
ONTOGENY OF HEMOGLOBIN
Hemoglobin production “switches” from embryonic to
fetal hemoglobin at 6 to 7 weeks of gestation and finally
to adult hemoglobin at birth (Fig. 8). This is brought
about of sequential activation of z and e-genes on
chromosomes 16 and 11 respectively. This is unrelated to
site of erythropoiesis (Fig. 7).5 The pattern of expression
of genes occurs in 5’ to 3’ region as development
Fig. 8 Production of globin chains during the fetal and neonatal

period9
proceeds from embryonic, fetal and then adult life. In

fetus, z and e genes are expressed forming Gower 1,
Gower 2, and Portland hemoglobins and are seen in
yolk sac, para-aortic region and then the liver. Later
2a genes and the 2g genes are expressed forming
hemoglobin F. Later they are downregulated and adult
hemoglobins predominate (Table 2 and Fig. 10).6
Reduction of MCV, erythroblast count and CD 71
count is believed to be due to switch of erythropoiesis from
liver to bone marrow and maturation of hematopoietic
tissues.5
Fig. 6 Transcriptional regulation of common myeloid precursor

Hemoglobin and Hematocrit Rise
(CMP) commitment. CMPs differentiate into either common
precursors for granulocytic and monocytic lineages (GMPs) or
in Fetal Period (Fig. 9)7
common precursors for both erythroid and megakaryocytic It has been determined that hemoglobin concentration
lineages (EMPs). A separate pathway leading to eosinophils is increases by 0.21 g/dL and hematocrit by 0.64
shown may be possible percent each week from 22 weeks to 40 weeks. Thus
reference values of hemoglobin and hematocrit can (GA × 0.2087) where GA is the gestation age in weeks.
be predicted using the formula : hematocrit = 28.59 + Hematologic values of normal fetuses are described in
(GA × 0.6359) and hemoglobin concentration = 9.92 + Table 3.
Table 2 Hemoglobins in embryo fetus and adult life8

Developmental Hemoglobin name Chain composition
phase
Embryo Portland z2Gg2
z2Ag2
Gower I z2e2
Gower II a2e2
Fetus F a2Gg2
a2Ag2
A a2b2
Adult A a2b2
A2 a2d2
F a2Gg2
a2Ag2
Fig. 9 Hemoglobin production during the fetal and neonatal
period9
Fig. 10 Relative electrophoretic mobilities on starch gel electrophoresis at pH 8–6 of human hemoglobin variants10 positions of HbA2
and HbA are marked as red and yellow respectively
Table 3 Hematologic values of normal fetuses

WBC count corrected (×
Weeks of RBC Count (× 1012/L) Hemoglobin (g/dL) Hematocrit (%) MCV (fL) Platelet count (× 109/L)
109/L)
gestation
Mean + 2 SD –2 SD Mean + 2 SD –2 S D Mean + 2 SD –2 SD Mean + 2 SD –2 SD Mean + 2 SD –2 SD Mean + 2 SD –2 SD
Reference Millar et al
15 2.43 2.17 2.69 10.9 11.6 10.2 34.6 38.2 31 143 151 135 1.6 2.3 0.9 190 221 159
16 2.68 2.89 2.47 12.5 13.2 11.7 38.1 40.2 36 143 155 131 2.4 4.1 0.7 208 265 151
17 2.74 2.97 2.51 12.4 13.3 11.5 37.4 40.2 34.6 137 145 129 2.0 2.8 1.2 202 227 177
18 2.77 3.1 2.44 12.4 13.6 11.2 37.3 41.5 33.1 135 146 126 2.4 3.3 1.5 192 237 147
19 2.92 3.19 2.65 12.3 13.5 11.1 37.5 40.6 34.4 129 135 123 2.5 3.3 1.7 211 259 163
20 3.12 3.48 2.76 13.0 14.1 11.9 39.3 43.4 35.2 126 132 120 2.6 3.8 1.4 170 230 110
21 3.07 3.49 2.65 12.30 13.1 11.5 37.3 40.8 33.8 123 131 115 2.7 3.4 2 223 284 162
Reference Forestier et al
18–21 2.8 3.22 2.38 11.69 12.96 10.42 37.3 41.62 32.98 131.1 142.07 120.13 2.57 2.99 2.15 234 291 177
22–25 3.09 3.43 2.75 12.20 13.8 10.6 38.59 42.53 34.65 125.1 132.94 117.26 3.73 5.9 1.56 247 306 188
26–29 3.46 3.87 3.05 12.91 14.29 11.54 40.88 45.28 36.48 118.5 126.46 110.54 4.08 4.92 3.24 242 311 173
> 30 3.82 4.46 3.18 13.64 15.85 11.43 43.55 50.75 36.35 114.38 123.72 105.04 6.40 9.39 3.41 232 319 145
REFERENCES 6. Schechter AN. Hemoglobin research and the origins of

molecular medicine. Blood. 2008;112:3927-38.
1. Proytcheva MA. Issues in neonatal cellular analysis. Am J 7. Jopling J, Henry E, Wiedmeier SE, Christensen RD. Reference
Clin Pathol. 2009;131:560-73. ranges for hematocrit and blood hemoglobin concentration
2. Tavian M, Péault B. Embryonic development of the human during the neonatal period: Data from a multihospital health
hematopoietic System. Int J Dev Biol. 2005;49:243-50. care system. Pediatrics. 2009;123(2):e333-7. Accessed on
3. Zon LI. Developmental biology of hematopoiesis. Blood. pediatrics.aappublications.org, on October 7, 2012.
1995;86(8):2876-91. 8. Wood WG, Weatherall DJ. Developmental genetics of the
4. Zhu J, Emerson SG. Hematopoietic cytokines, transcription human haemoglobins. Biochem J. 1983;215:1-10.
factors and lineage commitment. Oncogene. 2002;21:3295- 9. Ohls RK. Core Concepts: The Biology of Hemoglobin.
313. Neo Reviews 2011;12:e29-e38. Accessed from http://
5. Al-Mufti R, Hambley H, Farzaneh F, et al. Fetal and neoreviews.aappublications.org/cgi/content/full/
embryonic hemoglobins in erythroblasts of chromoso neoreviews;12/1/e29 on 06/04/2011.
mally normal and abnormal fetuses at 10–40 weeks of 10. Huehns ER, Shooter EM. Human haemoglobins. Med
gestation. Hematologica. 2000;85:690-3. Genet. 1965;2:48.
C H A P T E R 2
Physiology of Blood Coagulation
Shrimati Shetty
The three major components of blood coagulation are platelet, plasma and the endothelium. The platelets adhere to damaged
endothelium with the help of von Willebrand factor (VWF) and when activated, they aggregate and make a platform for coagulation
factors which then initiate a series of reactions on the damaged blood vessels. The reaction begins with the activation of contact factors
which then results in the sequential activation of these clotting factors, resulting in thrombin generation, which converts fibrinogen to
fibrin clot. A series of inhibitors to these coagulation factors keep them under check to maintain the thrombohaemorrhagic balance.
The most important of these inhibitors are protein C, protein S, antithrombin, tissue factor pathway inhibitor (TFPI) and heparin
cofactor II . The fibrinolytic system has an important role in the removal of clot formed, thus maintaining the hemostatic balance.
Thrombin has both pro- and anticoagulant role in the coagulation cascade and it is thrombomodulin which converts thrombin into an
anticoagulant enzyme by a negative-feedback regulation of its prothrombotic activity through its association with activated protein
C (APC). It also has an important role of linking coagulation with fibrinolysis through thrombin activable fibrinolytic inhibitor (TAFI).
The deficiency of factors in the Kallekrein-kinin system does not result in a bleeding phenotype; have a role in various noncoagulant
functions including apoptosis, proinflammatory and prothrombotic manifestations.
PLATELET ACTIVATION 4), resulting in the release of adenosine diphosphate

(ADP) and thromboxane A2 (TXA2).1,2 Thus both
Upon breach of vasculature, platelets get exposed to platelet activation and coagulation are interdependent.
collagen and VWF which facilitate their adhesion to the Platelets have a wide array of receptors on their surface
subendothelium through GP-1b-V-IX receptors. The which help both in adhesion and aggregation and it
adhesion of the platelets to the subendothelium results is these receptors which have become the target for
in platelet activation which results in an “inside out”
anti-platelet therapies. Platelet aggregation requires
signal, causing the exposure of phosphatidyl serine to
the platelet membrane glycoprotein receptor i.e. GP
the outer surface, which forms catalytic surface for its
IIb-IIIa, which helps in platelet aggregation by binding
procoagulant activities. The activation also results in
to fibrinogen and fibronectin.3 Once the platelets are
secretion of platelet contents along with the exposure
activated, they release different types of granules into
of fibrinogen receptors resulting in platelet aggregation
at the site of injury. Platelets when activated also the circulation which include ADP, platelet-activating
trigger the coagulation reaction by providing a catalytic factor (PAF), VWF, serotonin, TXA2 and platelet factor
surface which results in the formation of thrombin. 4.4 These stimulate and activate more and more platelets
More than 250 active substances are released into resulting in the primary hemostatic platelet plug at the
circulation from the granules present within platelets, site of wound. It is the same nature of the platelets of
when platelet gets activated. Thrombin further showing a quick response to the vascular breach, which
stimulates platelet activation through G-protein- is responsible for myocardial infarction or stroke under
coupled protease activated receptors (PAR-1 and PAR- pathological conditions.
Chapter-2 Physiology of Blood Coagulation 11
THEORIES OF BLOOD COAGULATION Flow chart 1 The coagulation cascade
Morawitz in 1905 put forward the first theory of blood

coagulation which is also referred as “four clotting factor”
theory.5 According to this theory, blood clotting is possible
due to the presence of four clotting factors, namely,
thromboplastin, prothrombin, thrombin and calcium.
The thromboplastin gets released on tissue injury which
converts prothrombin to thrombin in the presence of
calcium. Subsequent to the identification of the remaining
coagulation factors, two groups independently put
forward a revised blood coagulation model referred to as
the “waterfall cascade” model.6,7 According to this model,
all the blood coagulation factors remain in an inactive
state and they get activated by the upstream clotting factor.
The coagulation cascade shows two distinct pathways i.e.
extrinsic pathway initiated by the interaction of tissue
factor (TF) with the circulating serine protease factor
VIIa. This increases the catalytic activity of FVIIa several
fold which then activates factor X to Xa. Similarly in the
intrinsic pathway, there is a sequential activation of factors
XII, XI, IX the activated form of which then activates factor
X to Xa . Thus the two pathways converge into the common
pathway by the activation of FX to FXa which then binds
to FVa to form prothrombinase, which rapidly converts
prothrombin to thrombin. Calcium and phospholipids
are required for these sequential activation of coagulation
factors. Both extrinsic and intrinsic pathways cannot
work independent of each other as deficiencies of any
of these factors in either of the two pathways (excluding
Initiation Phase
contact factors and factor XII) can lead to lifelong bleeding This phase begins with TF bearing cells and is referred
tendency (Flow chart 1). to as the ‘extrinsic’ pathway. TF binds to activated FVII
(FVIIa) which in turn activates factor IX to Fix factor X
Cell Based Model of Blood Coagulation to Xa. Subsequently FXa activates FV to FVa forming
the “prothrombinase complex” on TF bearing cells.
Though the waterfall hypothesis of blood coagulation The dissociation of FXa from these TF bearing cells is
has expanded our understanding of blood coagulation, prevented by inhibitors like antithrombin and tissue
more recent observations demonstrate that the cascade/ factor inhibitor (TFPI). The FV can either come from
waterfall hypothesis does not fully simulate the in vivo activated platelets at the sites of injury or from plasma,
hemostasis. The cascade model of blood coagulation both of which can be activated by factor Xa.12 When
highlights the importance of coagulation factors in the present on the TF bearing cells FXa is resistant to its
generation of thrombin and overall hemostasis and the inhibitor i.e. antithrombin. It has also been reported that
role of cells is only to provide phospholipids surface for some amount of FVIIa remains bound to TF even in the
the coagulation factors to generate thrombin. The cell absence of injury thus facilitating mild activation of FX
based model of coagulation shows the significance of cells and FV all the time.13
in coagulation. While the extrinsic pathway is initiated on
the TF bearing cells the intrinsic pathway takes place on
the platelets. The contact factors and factor XII are not
Amplification Phase
included in the cell based model of coagulation as the The amplification phase involves the activation of platelets
deficiency of these factors does not result in bleeding.8-11 by the initial thrombin generated on the TF bearing cells.
According to this model coagulation takes place in 3 Besides thrombin also activates coagulation factors V, VIII
phases i.e. initiation phase, amplification phase and and XI on the platelet surface resulting in small amounts
propagation phase. of thrombin on the platelet surface.14
Propagation Phase protein Z). Two enzymes take part in these processes–
vitamin K epoxide reductase (VKORC1) and gamma
This phase takes place on the surface of activated platelets. carboxylase (GGCX). The gamma carboxylase enzyme
FIXa produced during the first step binds to FVIIIa to adds the gamma carboxyl group to the glutamate residues
activate FX to FXa (Tenase complex). FXa then activates during which Vitamin K epoxide gets reduced. The
FV to FVa which then activates prothrombin to result in VKORC1 enzyme converts it into its active form. The
thrombin burst (prothrombinase complex). As more gamma carboxylation is required for an effective binding
and more platelets are recruited to the site of injury, the of these serine proteases to the phospholipid surfaces.
thrombin generation gets amplified several fold resulting
in a thrombin burst which then acts upon fibrinogen to
form fibrin. The fibrin clot also consists of erythrocytes,
Blood Coagulation Inhibitors
leukocytes and platelets which are held together by fibrin Blood coagulation inhibitors as well as fibrinolytic
chains. The thrombin activated FXIII then cross links these inhibitors neutralize the coagulant or fibrinolytic proteins
fibrin chains to form a firm fibrin clot. to prevent excessive thrombosis or hemorrhage . The major
anticoagulant pathway is the protein C anticoagulant
The Kallekrein-Kinin System pathway in which both FVIIIa and FVa required for tenase
and prothrombinase complex formation get neutralized.
The Kallekrein-kinin system consists of 3 factors: Protein C shows a very high homology to all the remaining
prekallekrein (PK), high molecular weight kininogen vitamin K dependant clotting factors. Protein C shows
(HMWK) and factor XII (FXII). Their role in the initiation a very high homology to all the remaining vitamin K
of the intrinsic pathway of coagulation is still not clear
dependant clotting factors. Protein S is an important
as FXII can get autoactivated even in the absence of
cofactor for activated protein C (APC) in its anticoagulant
prekallekrein.15 The deficiency of any of these factors does
action on FVa and FVIIIa. A mutation at Arg 506 in factor
not result in bleeding phenotype. Though the absence
V gene factor V Leiden) makes it resistant to APC cleavage.
of these factors prolongs the in vitro investigations like
Homozygotes for this mutation show 20-30 fold increased
activated partial thromboplastin time (APTT), their
risk of thrombosis.20 As the concentration of thrombin
physiological significance in hemostasis is unclear.
Recently there are several reports to support their role increases it binds to another protein i.e. thrombomodulin
in thrombosis.16 A polymorphism in FXII i.e. 46C/T has which then activates the inactive protein C to its activated
shown strong association with arterial thrombosis.17 The form. The APC inactivation by thrombin-thrombomodulin
homozygous carriers of this polymorphism were found complex takes place on the surface of endothelial cells
to have reduced FXII levels which in turn will result in , where the APC is bound by a receptor i.e. endothelial
reduced fibrinolytic activity, thus being implicated in protein C receptor (EPCR).
thrombosis. Though protein S has initially been reported as a
FXIIa also activates FXI to FXIa which then activates cofactor for APC in neutralizing FVIIIa and FVa, studies
FIX to Fix. FXIa along with PK also releases bradykinin, the have shown that PS can independently neutralize these
proinflammatory factor from HMWK.18 factors.21 This is possible because PS generally remains
combined to C4BP protein. Besides, PS also has inhibitory
COAGULATION PROTEASES AND action against FXa by facilitating the interaction of FXa
with TFPI.22 It has also been reported that FV acts as a
COFACTORS cofactor in the inactivation of FVa by APC and PS.23
Biochemically, coagulation factors can be classified into Antithrombin is another strong inhibitor which
two groups i.e. serine proteases–FVII, FIX, FX, FXI and inactivates both FXa and thrombin, besides its inhibitory
cofactors-FVIII and FV. The two main cofactors required action against IXa, XIa and XIIa along with some of the
in most of the steps in blood coagulation including the factors in the fibrinolytic pathway. The activity of AT is
formation of tenase and prothrombinase complexes increased several fold in the presence of heparin. Though
are phospholipids and calcium. Calcium also has an rare, AT deficiency leads to one of the most severe form
important role of binding the coagulation factors to the of thrombophilia. Heparin cofactor II is another inhibitor
platelet membrane.19 of thrombin. Protein Z is another inhibitor which is an
Vitamin K is an important cofactor for coagulation inhibitor of FXa.
factors which require post translational modification i.e. The major inhibitor of tissue factor is tissue factor
gamma carboxylation of their glutamic residues which is pathway inhibitor (TFPI) which is expressed on the
important for their efficient functioning (factors II, VII, endothelial cells. The extrinsic pathway involving the
IX, X and their inhibitors i.e. protein C, protein S and activation of X to Xa gets inhibited and there is a shift in the
Chapter-2 Physiology of Blood Coagulation 13
balance towards the intrinsic pathway of coagulation. TFPI the formation of tenase complex. The binding of FXIa and
inhibits both TF-VIIa complex and FXa. Thus the initial FXIIa to the platelet surface however is not clear. Among
phase of extrinsic pathway is inhibited. However once the anticoagulant proteins both PC, PS and TFPI can bind
Xa takes part in the prothrombinase complex, it becomes to the platelet surface to neutralize the corresponding
resistant to the action of TFPI. Various strategies have proteins. Thus platelets have a important role in blood
been used to use inhibitors of TFPI as a novel therapeutic coagulation.
protocol for haemophilia.24
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2001;1:59-74.
by Arg506 to Gln mutation in factor V as a pathogenic
risk factor for venous thrombosis. Thromb Haemost. 26. Maroney SA, Cooley BC, Ferrel JP, et al. Murine
1995;74(1):139-48. hematopoietic cell tissue factor pathway inhibitor
21. Maurissen LF, Thomassen MC, Nicolaes GA, et al. Re- limits thrombus growth. Arterioscler Thromb Vasc Biol.
evaluation of the role of the protein S-C4b binding protein 2011;31:821–6.
complex in activated protein C-catalyzed factor Va- 27. Gould WR, Silveira JR, Tracy PB. Unique in vivo
inactivation. Blood. 2008;111:3034–41. modifications of coagulation factor V produce a physically
22. Heeb MJ, Mesters RM, Tans G,et al. Binding of protein S and functionally distinctplatelet-derived cofactor. J Biol
to factor Va associated with inhibition of prothrombinase Chem. 2004;279:2383–93.
C H A P T E R 3
Structure, Function and
Physiology of Platelets
Platelets are one of the formed elements of blood. These are anucleate, disc-shaped cells 2 to 4 m in diameter and are present in blood
at a conc. 1,50,000 to 4,50,000/mL. The function of platelets is to bring about primary hemostasis and localize the active coagulant
enzymes which develop by a cascade of reactions from inert procoagulant precursors at the site of tissue injury. Platelets admirably
performs this job and when somebody gets severe thrombocytopenia (<10,000/mL) or, inherited or acquired platelet defects, their
silent but admirable function becomes apparent in the form of microcapillary or more serious cerebral bleeding and on the other
extreme, when their function is over—done various types of thrombosis may be the end result.
Platelets are produced from megakaryocytes in bone marrow. Megakaryocytes are naturally occurring giant cells where ploidy
value can reach up to 128N, but modal ploidy value for megakaryocytes are 16N to 32N. Megakaryocytes are produced from their
precursor progenitor cells by extensive proliferation and endoreduplication of the nuclei. A specific growth factor, thrombopoietin,
acts on committed megakaryocyte precursors by acting on c-MPL (CD110) receptor and can produce large number of megakaryocytes
and platelets.1 Presently nonpeptide analogs of thrombopoietin are available (eltrombopag®) and are being used for various causes of
thrombocytopenia.
Human hemopoiesis being extravascular, the megakar ball with small surface projections. These projections
yocytes once matured, produce very long pseudopod become irregular and longer when platelets are activated
like structures through depolymerization of cellular (Fig. 2A).2
microtubules and microfilaments. These structures are
called proplatelets. Eventually, these proplatelets throw
off platelets through the endothelial gaps in the marrow
sinusoids.
STRUCTURE AND ULTRASTRUCTURE

OF PLATELETS
In a well-stained blood film made from EDTA anticoagu
lated blood and stained with Romanowsky’s stain,
platelets appear as single 2 to 4 m anucleate cells which
are purple in color. These cells have granules in the
center (Chromomere) and denser peripheral nongranular
hyalomere (Fig. 1). If the blood smear is made directly from
fingerprick then the platelets are found in small and large
aggregates. Occasionally, the platelets can form satellites
around neutrophils (Satellitism) due to EDTA dependent
cold antibody present in some of the blood samples. Under
scanning electron microscope, platelets appear as a small Fig. 1 Peripheral blood showing platelet clumping
A B
Figs 2A and B SEM images of platelets adhered onto topographically structured polymethylmethacrylate (PMMA) surfaces
Transmission electron microscope shows detailed Function of Platelets

cyto- architecture of platelets (Fig. 2B). It has a trilaminar
cell membrane, of which outermost layer is carbohydrate Platelets main function is its involvement in primary
rich and it invaginates the whole cell as surface canalicular hemostasis, and in addition through release of growth
system, thus enormously increasing the surface area of the factors such as platelet derived growth factor, platelets also
cell membrane on which various glycoprotein receptors help in wound healing. Hemostatic function of platelet
are located. Platelets can also secrete a large number takes place in the following stages. Each of the stage is possible
of peptides and active chemicals and ions into this through enactment of a series of biochemical reactions
canalicular system. Under the cell membrane of platelets involving ligand-receptor interaction, signal transduction,
the myosin heavy chain, actin and other tubular proteins release of cations like Ca2+ and active chemicals and
are arranged in parallel array. peptides, reverse signal transduction leading to changes
In the cytoplasm of platelet, (1) Dense Granule (called in receptor conformation and further reinforcement of
delta granule), (2) Alpha granule, and (3) Lysosomes and interaction and finally activation of coagulation cascade on
mitochondria are seen. Alpha and Delta granule contains the surface of the platelet membrane, largely localizing the
large number of peptides and other chemicals (Table 1). clot at the site of tissue injury.5
These chemicals are responsible for various platelet func Platelet function can be envisioned to take place
tions. Some of the chemicals such as 5-hydroxytryptamine in vivo as:
(5HT) and peptides are absorbed by the platelets from • Adhesion to injured capillary endothelium and sub-
plasma. While some of the proteins and chemicals are endothelium
synthesized internally.3 • Shape change, aggregation and secretion
• Further aggregation and activation of coagulation.
Table 1 Platelet granules and their contents4 Adhesion to Injured Capillary Endothelium
a granules Dense granules Lysosomes and Subendothelium
PDGF ATP Acid hydrolases
With this reaction, platelets are initially activated to adhere
TGF-b ADP
to the collagen of capillary/vascular endothelium at the site
CTAPIII GTP of the injury. Adhesion reaction is initiated by interaction
PF4 GDP of Glycoprotein Ib/IX on the platelet membrane with
TSP Serotonin activated von Willebrand factor (Activated by shear stress
Fibronectin Calcium at the site of injury) and collagen in the injured vessel.
Fibrinogen Magnesium The interaction is strengthened by interactions of platelet
Vitronectin receptor Glycoprotein IIb/IIIa with fibrinogen.6,7
vWF
Albumin Shape Change, Aggregation and Secretion
FV, FVIII
At the site of tissue injury several platelet agonists are
Protein S
released in small amounts, i.e. Thrombin, ADP, ATPase,
PAI-1
etc. Injury to the endothelium also exposes subendothelial
HMWK collagen which is an aggregation promoting ligand for
C1 inhibitor platelets. Small amounts of ADP from surrounding tissue
Chapter-3 Structure, Function and Physiology of Platelets 17
(red cells) cause platelets to change its shape through preventing the entry of arachidonic acid into prostaglandin
contraction of microtubules and microfilaments. This synthesis pathway. Normally, healthy endothelium
leads to appearance of pseudopodia like structures, produces many antiaggregatory and anticoagulant sub
centralization of granules. As the process continues and stances. One such antiaggregatory substance is called
platelets are activated through activated von Willebrand prostacyclin. When endothelial cells are damaged,
factor and fibrinogen via its GP Ib/IX and GP IIb/IIIa postacyclin production is reduced or stopped and this
receptors, a graded amount of secretion takes place leads proaggregatory thromboxane A2 to take the upper
leading to release of ATP, ADP, 5HT from dense granules, hand. Healthy endo thelium also produces anti-aggre
and various peptides involved in coagulation, fibrinolysis, gatory nitric oxide and anti-coagulant heparin.3-7
wound healing from alpha granules. This causes initial With mild stimulus, dense granule products are
weak aggregation reaction between platelets into a strong released and with stronger stimulus alpha granules and
irreversible aggregation of adhered platelets at the site finally acid hydrolases from lysosome are released.
of tissue injury. Platelets also activates production of
Thromboxane A2 during its initial activation and this is Further Aggregation and Activation of Coagulation
another pathway through which platelets are activated
and this pathway can be inhibited by drugs like aspirin, As the reinforcement of adhesion and aggregation conti
which inhibits the enzyme cyclo-oxygenase irreversibly nues at the site to tissue injury, the liquid phase of blood
A B
C D E
Figs 3A to E Platelet aggregometry (A) Born principle, (B) ADP-induced platelet aggregation, (C) Collagen-induced platelet
aggregation, (D) Ristocetin-induced platelet aggregation, and (E) Arachidonic aggregation acid-induced platelet aggregation
coagulation is also activated. Initial liberation of tissue phospholipids on its surface membrane which in resting
thromboplastin produces a small amount of thrombin. state is electroneutral or slightly electropositive. Activation
This thrombin and some ADP from tissue (mainly red causes the inner membrane phospholipids of platelet
cells) initiate platelet activation. As part of activation to express outside. This phospholipid which is largely
of platelets, the platelets change the nature of exposed phosphotidyl serine is electronegative and supports on it
A
Fig. 4A Platelet flow cytometry: Normal platelet rich plasma (PRP) sample
Chapter-3 Structure, Function and Physiology of Platelets 19
Fig. 4B Platelet rich plasma (PRP) of Glanzmann’s thrombasthenia sample
the assembly of Tenase complex (Factor IXa, factor VIIIa, photocell. When an agonist is added the platelets aggregate
calcium and phospholipids) and prothrombinase complex and absorb less light and so the transmission increases and
(Factor Va, Xa and prothrombin). These complexes this is detected by the photocell.
ultimately produce explosive amount of thrombin locally All the facets of platelet function can be investigated
and strengthens locally adhered and aggregated platelets in the laboratory by using platelet aggregometry (Figs
by a blood clot.8 3A to E). Glycoprotein antigen expression and activation
With passage of time, this aggregated platelet along on platelet surface can be quantitatied and visualized
with clot, contracts through platelets actin-myosin by platelet flow cytometry (Figs 4A and B). Retraction of
machinery and the clot is consolidated. platelets can be tested simply in the coagulation laboratory
In the Born aggregometer, PRP is stirred in a cuvette by testing the changes in the volume of blood clot after it
at 37°C and the cuvette sits between a light course and a has been incubated for 2 hours at 37ºC. Ultra-structural
Fig. 5 Schematic diagram of the platelet activation showing the major receptors and effectors.6-8 Biochemistry of platelet activation
(expert review of cardiovascular therapy)
study of platelets can show presence or absence of various degradation of the thrombopoietin receptor c-Mpl. Blood.
granules or granular contents. Secretory functions of 2010;(115)6:1254-63.
platelets can be tested by either seeing 3H labeled 5 HT 2. Minelli C, Kikuta A, Tsud N, Ball MD, Yamamoto A. A
release or by quantitation of ADP and ATP release by microfluidic study of whole blood behaviour on PMMA
topographical nanostructures. J Nanobiotechnology.
Chemiluminescence assays.
2008;6:3.
Platelets are biochemical dynamos. Activation of
3. Orr MW, Boullin DJ. The relationship between changes
platelets leading to its final aggregation and adhesive in 5-HT induced platelet aggregation and clinical state in
state relates to complex chemical interaction of ligands patients treated with Fluphenazine. Br J Clin Pharmacol.
and agonists in vivo. A large part of such reactions are still 1976;3(5):925-8.
unknown and is being continuously elucidated. 4. Rendu F, Brohard-Bohn B. The platelet release reaction:
But what we already know is substantial (Fig. 5) and granules’ constituents, secretion and functions. Platelets.
several reactions of this pathway are already exploited in 2001;12(5):261-73.
therapy and are useful in understanding hereditary and 5. Holmsen H. Physiological functions of platelets. Ann Med.
acquired platelet dysfunction. 1989;21(1):23-30.
To summarize, platelets are of utmost importance 6. Jurk K, Kehrel BE. Platelets: physiology and biochemistry.
Semin Thromb Hemost. 2005;31(4):381-92.
in the hemostatic system for the formation of primary
7. Aslan JE, Itakura A, Gertz JM, McCarty OJ. Platelet shape
hemostatic plug. The granule secretions help recruit more
change and spreading. Methods Mol Biol. 2012;788:91-
platelets to the primary plug, which eventually provides a 100.
surface on which the secondary hemostatic system forms 8. Heemskerk Johan WM, Bevers Edouard M, Lindhout
a sturdy clot. The ruptured endothelium gradually repairs Theo. Platelet activation and Blood coagulation. Thromb
itself and the vessel wall structure is restored. Dysfunction Haemost. 2002;88:186-93.
of these processes and systems may lead to inherited or
acquired platelet function distorders. BIBLIOGRAPHY
1. Sharathkumar Anjali A, Shapiro Amy. Platelet function
REFERENCES disorders, 2nd edn. Indianapolis Hemophilia and
1. Saur Sebastian J, Sangkhae Veena, Geddis Amy E, Thrombosis Center, Indianapolis, USA World Federation
Kaushansky Kenneth, Hitchcock Ian S. Ubiquitination and of Hemophilia (WFH), 2008.
S E C T I O N 2
Neonatal Hematology
CHAPTERS OUTLINE
4. Variation in the RBC Parameters in the Newborn
5. Physiological Anemia of Newborn, Anemia of Prematurity and Role
of Erythropoietin in the Management
6. Effect of Maternal Iron Status on Placenta, Fetus and Newborn
7. Developmental Aspects of Hemostasis in the Fetus and Newborn
8. Anemia in the Newborn
Jayashree Mondkar, Shilpa Borse, MR Lokeshwar
9. Polycythemia and Hyperviscosity Syndrome
10. Vitamin K Deficiency: Bleeding in Newborns
11. Bleeding Neonate: Approach and Management
12. Approach to Neonatal Thrombocytopenia
Nitin K Shah
C H A P T E R 4
Variation in the RBC
Parameters in the Newborn
The fetal and neonatal period is a most dynamic phase, Table 1 Hemoglobin concentration of cord blood3
as during this period there occur profound alterations
and adjustments, especially during transit of fetus Mean g/dL Hb g/dL range
from dependent hypoxic, intrauterine life—to totally Mollison (1951) 5
16.6 –
independent extrauterine existence. Erythrocytic system
Dochain et al. (1952) 6
17.9 14.4 – 21.6
undergoes serial adaptation to meet progressively
changing demands of oxygen in embryo, fetus and Walker et al. (1953)7 16.5
neonate. Marks et al. (1955)8 16.9 12.3 – 22
Hematology of newborn, remains a concern even
today, not only because of unique blood picture during this Guest et al. (1957) 9
17.1 13.6 – 25
period and normal variation in hematological parameters Sturgeon (1956) 10
15.7 –
but also in no other period of life is anemia known to occur
Dalal and Lokeshwar 3
16.2 13.2 – 22
due to such varied causes.
Although the fetus is nourished and protected by the Rama Rao11 15.13 14.16 – ± 3.26
mother during this period, fetus may suffer adverse effects
related to maternal malnutrition, illnesses, infections,
drug ingestions, etc. Shortly after birth the Hb concentration increases by
Moreover, the proximity of two circulatory systems as much as 2.5 to 6 gm%/dL depending on the amount of
(mother and the baby) also permit the free passage of placental transfusion. The redistribution of body fluids
formed blood elements between mother and fetus as with decrease in plasma volume after birth also accounts
seen in fetomaternal hemorrhage leading to anemia and for this rise. Failure of hemoglobin to rise during this
sensitization to RBC antigens. Also maternofetal hemorrhage period is a marker of blood loss.
may occur, leading to hyperviscosity syndrome. • Hemoglobin levels return to cord blood values by the
end of the first week.
NORMAL HEMATOLOGICAL VALUES • A significant Hb decrease during this time even if
IN THE NEWBORN absolute values of Hb are within the normal range is
also suggestive of hemorrhage or hemolysis.
During first several hours after birth, there is increase
Hemoglobin in Hb concentration. Hb increases by 17 to 20 percent of
Various authors have reported values for the normal mean the initial level in the first 24 hours of life but then falls
hemoglobin concentration of the cord blood ranging from slightly during the next 24 hours.3,19,25
15.7 to 17.9 gm% (Table 1). Approximately, 95 percent of At the end of the first week of life, the Hb concentration
all values fall between 13.7 and 20.1 g/dL1,3,5-11 is as high as it was in the cord blood.
24 Section-2 Neonatal Hematology
Anemia Table 3 MCV, MCH, MCHC and normoblast count in the full term
Anemia during the first week of life is defined as a normal infant studied at LTMG Hospital, Mumbai3
hemoglobin value less than 14 g/dL. MCV (fL) MCH (Pg) MCHC (%) Normoblasts
Beyond the first week of life many factors influence (cells/mm3)
what is considered as normal hematological parameters Cord blood 113.04 ± 5.3 34.33 ± 1.4 33.9 ± 0.8 600 ± 186
in newborn period. Naiman and Oskin,1 Mollison et al,5,31 12–18 hours 108.96 ± 5 35.1 ± 1.9 34.4 ± 0.6 283 ± 122
Lokeshwar et al.3 and others10,12,13,15 have suggested that
72 hours 98.54 ± 2.9 35.82 ± 0.8 34.9 ± 0.5 36 ± 48
13.5 g/dL be considered as lowest normal value for
cord blood Hb. Most authorities suggest that an Hb 7 days 96.0 ± 3.4 34.0 ± 1.0 34.6 ± 0.8 –
concentration of 13.5 g/dL in cord blood be considered 15 days 95.5 ± 4.0 33.2 ± 9.0 34.54 ± 0.5 –
as the lower limit of normal. Hb value for umbilical artery 28 days 96.1 ± 3.2 31.6 ± 0.93 34.2 ± 0.7 –
blood tend to be about 0.5 g/dL higher than sample
obtained from umbilical vein.8
In a study (Lokeshwar et al.) of 100 newborn babies,
Table 4 Normal hematological values during the first-two
only 2 percent of neonates had Hb level less than 13 gm%
weeks of life in the term infants1
in cord blood.2,3
Hb concentration decreases in both term and preterm Value Cord blood Day 1 Day 3 Day 7 Day 14
infants to reach minimal levels of 9.4 to 14.5 g/dL in term Hb g/dL 16.8 18.4 17.8 17.0 16.8
infants by 7 to 9 weeks of age. This “physiological” anemia
occurs because of a decline in erythrocyte mass due to the Hematocrit (%) 53.0 58.0 55.0 54.0 52.0
following reasons: Red cells (mm3) 5.25 5.8 5.6 5.2 5.1
• In utero the fetal oxygen saturation is low at around
MCV (fL) 107 108 99.0 98.0 96.0
45 percent, erythropoietin levels are high and RBC
production is rapid. Reticulocyte counts are 3 to 7 MCH (Pg) 34 35 33 32.5 31.5
percent reflecting erythropoiesis.
MCHC (g/dL) 31.7 32.5 33 33 33
• With improved oxygen saturation to 95 percent after
birth, the erythropoietin levels become undetectable Reticulocytes 3–7 3–7 1–3 0–1 0–1
hence RBC production stops, reticulocyte counts are Nucleated RBCs 500 200 0–5 0 0
low and the hemoglobin level falls.
• This factor coupled with a reduced life span of fetal
RBCs results in anemia that is not a functional one as
oxygen delivery to the tissue is adequate as the levels of Blood Volume
Hb A and 2,3DPG increased. Immediately after birth, the blood volume of term
• At 8 to 12 weeks, hemoglobin levels reach their nadir infants may range from 50 to 100 mL/kg, with mean of
(Tables 2 and 3), oxygen delivery to the tissues is 85 mL/kg.4,5,7 If cord clamping is done early for instance
impaired, erythropoietin production is stimulated and at 30 minutes of age, blood volume is 78 mL/kg as
hemoglobin starts increasing. The hemoglobin and compared to 98.6 mL/kg in case of delayed cord clamping.
RBC count fall earlier and to a greater extent in preterm By 72 hours, this difference in blood volume decreases.
infants leading to “anemia of prematurity”. The blood volume of premature infants ranges from
89 to 105 mL/kg during first few days of life (Table 4).6,7
This is mainly because of increase in plasma volume, with
Table 2 Hematological parameters in the full term normal infant the total RBC volume per kg of body weight being the same
studied at LTMG Hospital, Mumbai3 to that of term infants.
Hb (g%) Hematocrit (%) RBC (million/mm3) By 1 month of age, this value remains at 73 to 77 mL/kg.
Newborns with tight cord around neck and with hyaline
Cord blood 16.2 ± 3.6 46.66 ± 5.1 4.9 ± 1.2 membrane disease have low blood volume and those born
12–18 hours 18.79 ± 2.8 49 ± 4.8 5.3 ± 0.8 after late intrauterine asphyxia have higher blood volume.
72 hours 17.38 ± 3.0 46.9 ± 5.3 5.2 ± 0.6
Hematocrit
15 days 16.36 ± 2.2 43.4 ± 4.1 5.01 ± 0.9
Normal values of hematocrit ranges from mean of 51.3 to
28 days 14.17 ± 2.4 42.1 ± 3.8 4.7 ± 1.0
56 percent.1,3,13,20 Just as Hb value, hematocrit value also
Chapter-4 Variation in the RBC Parameters in the Newborn 25
shows increase during first few hours of life and reaches

original value of cord blood by one week and mean
capillary hemotocrit value is two percentage point higher
than the mean venous hematocrit value at one week age.
Gatti et al.16 reported capillary hematocrit on first
day of life to be 62.9 ± 3.2 percent reaching 56.6 ± 2.6 by
day 7 and 53.7 ± 2.5 percent by day 10, whereas Guest
and Brown17 recorded mean cord blood hematocrit of
52.3 percent and 58.2 percent on first day, 54.31 percent
on 3rd day and 54.9 percent on 7th day. Mean capillary
hematocrit value is two percentage points higher than the
mean venous hematocrit values1 at 1 week of age.
RED CELL COUNT AND RED CELL INDICES7,8
Red Blood Cell Count Fig. 1 Normocytic normochromic RBC in newborn

Red blood cell count also shows a great variability at
the time of birth and ranges from 4.6 to 5.2 million/
counts, with values ranging between 6 and 16 percent in
cumm.1,3,8,9,18
infants born between 30th and 34th week (Fig. 2).
Our study of 100 newborn babies2,3 showed red blood
Term infants have an average 7.3 nucleated RBC/100
cell count in cord blood 4.9 ± 1.2 million/cmm reaching
WBC with normal range of 0 to 24 at birth.
5.3 ± 0.8 million/cmm after 12 to 18 hours and 5 ± 1.12
Infants born prematurely have higher retic count with
million/cmm at 7th day and stabilizing at same level
values ranging between 6 and 16 percent in infants born
thereafter throughout the neonatal period. Other studies
between 30 and 36 weeks of gestation.
also have reported similar changes in red blood cell
This increased retic count in first 2 to 3 days of life
count.1,8,9,18
reflects very active erythropoiesis during newborn period
and value drops to about 1 percent by 7th day of life.1,17,26,27,29
Mean Corpuscular Volume7,21-24
Persistent reticulocytosis in cord blood suggests
Newborn red cells are generally much larger than adults • Hemolytic process1
and average diameter of RBC is 8.5 to 9.3 cumm at birth • Hypoxia
reaching the adult value of 7.5 Cu around 6 months of • Blood loss.
age.19-21 Relative macrocytosis is observed in the newborn The term infant has approximately 500 nucleated
period. Mean corpuscular volume (MCV) at birth ranges RBCs/cμm at birth (0.1% of red cell population) which
from 104 to 118 fL. Compared to normal adult value of drops to 50 percent by 12 hours and 20 to 30 nucleated
82 to 92 fL. Mean corpuscular volume rapidly decreases RBC/cμm by 48 hours (Fig. 1). It is unusual to see nucleated
in first week of life and at the age of 2 months, cell size is RBCs in the peripheral smear of term infant after the age of
comparable to those in adult cells.7,21-24 4 days.1,18 In other words the term infants have 7.3
MCV values less than 92 fL should strongly suggest nucleated red cells per 100 leukocytes at birth (ranging
alpha thalassemia trait or iron deficiency. MCV is higher
from 0 to 24). In premature infants the average figures are
in preterm infants 115 ± 5 fL1,30 and decline to mean value
21 NRBC/100 WBC.28
of 95 ± 5 by 7th week of life.31
In premature babies nucleated red cells may vary 1000
MCH is also increased in newborn period, values
to 1500/mm3 at birth1 and decreases rapidly during first
ranging from 33.5 to 41.4 Pg as compared to adult values
week of life. However occasional nucleated RBCs may be
of 27 to 31 Pg.1,3 However, MCHC in the newborn period is
seen in the peripheral blood smear of 7-days-old infant.
quite similar to that in adult ranging from 30 to 35 percent
Increased number of nucleated RBCs are seen in
in newborn and 30 to 36 percent in adults.1,3
following:1,28
• After hemorrhage
Reticulocyte Count and Nucleated RBC1,3,14,19,30 • Hypoxia
Average reticulocyte count at birth ranges from 1.6 to • Hemolytic disease of newborn
6.2 percent, infants born prematurely have higher retic • Down’s syndrome and congenital anomalies.
age. Diagnosis of anemia can be missed by evaluating

capillary blood Hb.
Moe et al.33 (1967) in their study of 54 infants with
erythroblastosis fetalis 25 out of 41 infants found to anemic
whereas only 14 could be considered as anemic according
to the value obtained from capillary sample.34
Capillary values should not be compared to previously
obtained cord venous blood values when one is looking
for changes in Hb concentration during first week of life
and venous blood should be obtained for this purpose.
The selection of the vein is unimportant as blood from
different sites of vein gives similar results.15,31
Time of sampling
During the first few hours after birth, an increase in
hemoglobin concentration takes place (as great as 2.5–6
Fig. 2 Reticulocytes count g/dL)1,31,33 which is due to placental transfusion that occurs
during the time of delivery. Readjustment of the blood
volume after birth resulting in increased red cell count,
Factors affecting normal hematological values in
hematocrit and Hb concentration. Magnitude of increase
newborn1,3
depends on the amount of placental transfusion.31
• Site of sampling
• Time of sampling Treatment of umbilical vessels
• Treatment of umbilical vessels at the time of delivery The amount of blood received by the neonate depends
• Position of neonate after delivery upon time of clamping umbilical cord at birth.36
• Gestational age of the infant At birth by allowing complete emptying of placental
• Fetomaternal transfusion vessel before cord is clamped, the blood volume of infant
• Maternofetal transfusion. may be increased by as much as 61 percent.43 Placental
Various variables influence the interpretation of vessels contain 75 to 125 cc of blood, i.e. 1/3rd to 1/4th
normal values of Hb, HCT, RBC indices, reticulocyte count fetal blood volume.37
at the time of birth and during early weeks of life. Within 15 seconds of birth about a quarter and at
the end of 1 minute, about half of placental transfusion
Site of sampling
takes place. Placental transfusion occurs more rapidly in
Capillary sampling collected by skin prick from heal or the
women who receive ergometrin derivatives at the onset of
toe has a 5 to 10 percent higher hemoglobin concentration
3rd stage of labor.1,3,35,37-40
than simultaneously collected venous sample.1
During first hour of birth, plasma leaves the circulation.
Oettinger and Mills29 reported this difference around
Greater the placental transfusion the greater plasma loss.
during 1st hour of life 3.6 to 8 gm%. However, in some
On the third day of life, there are only small differences in
instances the capillary hemoglobin–venous Hb difference
total blood volumes regardless of method of cord clamping.
may exceed 5 to 10 gm%.29-33
In the group with late cord clamping, at 24 hours of age the
Stasis of the blood in the peripheral vessels–because of
red cell mass was approximately 32 percent greater and
sluggish circulation and resultant transudation of plasma
hematocrit was 15 percent higher.41,42
is believed to be the cause of higher capillary hemoglobin
Hb percent difference can range from 2 to 4 gm%
as compared to venous hemoglobin.1
between early clamping and delayed clamping with
Capillary/venous hematocrit ratio is greater than 1 in
hematocrit difference of 2 to 16 percent by various
virtually all infants. A ratio higher than 1.2 is observed in
authors37,40,43,44 during first week of life. By 6 weeks the
premature infants (before 30 weeks of gestation), infants
difference are no longer apparent.37,43,44
with acidosis (pH less than 7.2) and hypotension.32
Thus capillary Hb and hematocrit are falsely elevated Position of the neonate after delivery
in sick infants with altered microcirculation. However, As umbilical arteries generally constrict shortly after birth,
an accurate determination of Hb concentration is most no blood flows from the infant to mother. However, as the
important in the clinical management. Capillary venous umbilical vein remains dilated it permits the blood flow in
HCT ratio gradually decreases with increasing gestational the direction of gravity.
Chapter-4 Variation in the RBC Parameters in the Newborn 27
Infants held below the level of placenta, as it happens 6. Dochain J, Lemage L, Lambrechts A. Cited in Oski and
during normal delivery, continue to gain blood from Naiman. Normal blood values in newborn period.
placenta, whereas infants held above the placenta which Hematologic problems in the newborn. WB Saunders Co,
is often seen during cesarean delivery, infant may bleed Philadelphia. 1982.pp.1-31.
into mother, thus leading to anemia in the neonate.35,37-41 7. Walker JL, Turnbull EPN. Hemoglobin and red cells in
human foetus and their relation to the oxygen content of
In infants delivered at term with cesarean section,
the blood in the vessels of umbilical cord. Lancet.1953;2:
maximal placental transfusion is achieved in seconds after
312.
birth.37 Delay of 3 minutes in cord clamping after cesarean 8. Marks J, Gairdner D, Rescoe JD. Blood formation in infancy
section has been associated with signs of respiratory and III cord blood. Arch Dis Child. 1955;30:117.
metabolic acidosis indicating that earlier clamping may 9. Guest GM, Brown EW. Erythropoiesis and hemoglobin of
be preferable.37 Infants with delayed cord clamping had an blood in infancy and childhood. Am J Dis. 1957;93:486.
average red cells mass of 49 mL/kg at 72 hours as compared 10. Sturgeon P. Iron metabolism—a review with special
to 31 mL/kg in infants with immediate cord clamping.38-42 consideration of iron requirement during normal infancy.
However, in infants in whom cord ligation was delayed, Pediatrics. 1956;18:267.
decreased incidence of RDS has been reported and 11. Rama Rao BR, Krishnamurthy PN, et al. Study of neonatal
hence delayed cord clamping is indicated for premature hematological values in relation to maternal and foetal
infants. Premature infants with increased red cell mass, as factors and incidence of transplacental passage of foetal
consequence of delayed cord clamping, have been found erythrocytes. Ind Pediatr. 1979;16:591-5.
12. Wegelius R. On changes in the peripheral blood picture
to have higher serum bilirubin level.43,45 There have been
of newborn infant immediately after birth. Acta Pediatr.
reports of circulatory overload and congnitive cardiac
1948;35:1.
failure in the setting of delayed clamping (symptomatic 13. Chaplin H Jr. Cited by Mollison PL. Blood transfusion
neonatal plethora). in clinical medicine, 3rd edn. Springfield III, Charles
However, when there is obvious evidence of feto- (Thomas). 1961.p.581.
placental or maternal bleeding before or during the birth 14. Wough JF, Merchant FT, Maugham GB. Blood studies in
and infant appears pale and in shock, cord clamping should newborn–determination of Hb, PCV, reticulocyte and
be delayed, if resuscitation can be given simultaneously. fragility of erythrocytes over 98 days period. Am J Med Sc.
Thus numerous physiological changes occur in 1939;198:646.
succession and rapidity in fetus and neonates to adapt 15. Gairdner D. Cited by Oski FA and Naiman JL. Hematologic
to the changing pattern of life. This leads to rapid change problems in newborn. WB Saunders Co, Philadelphia,
in normal hematological parameters from fetal period to 1982.
immediately after birth and throughout neonatal period 16. Gatti RA. Hematocrit value of capillary blood in the
newborn infants. J Pediatr. 1967;70:117.
even hours, days and weeks after birth.
17. Guest GM, Brown EW, Wing M. Erythrocytes and Hb of
Interpretation of laboratory findings and institution
blood in infancy and in childhood, variability in number,
of appropriate therapy requires understanding of the size and Hb content of erythrocytes during first five years
maturational process and normal physiological variations of life. Am J Dis Child. 1938;56:529.
that takes place during this period.1-3 18. Lippman HS. Morphologic and quantitative study of blood
corpuscles in newborn period. Am J Dis Child. 1924;27:
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1. Oski FA, Nathan JL. Normal blood values in newborn 19. Heissen A, Schallo GR. Cited Oski FA, Naiman JL. Normal
period-hematological problems of the newborn. Vol IV blood value in newborn period–hematological problems
in the series. Major Problems in Clinical Pediatrics WB in newborn Vol.1 in the series Major problems in clinical
Saunders Company. 1982.pp.1-31. pediatrics. WB Saunders Co. 1982.pp.1-31.
2. Dalal R. Hematological parameters in the newborn 20. Saragea T. Cited Oski FA, Naiman JL. Normal blood value
period. Thesis submitted for MD (Ped) Exam., University in newborn period – hematological problems in newborn
of Bombay, under the guidance of Dr MR Lokeshwar. Vol.1 in the series Major problems in clinical pediatrics.
1986.p.12. WB Saunders Co. 1982.pp.1-31.
3. Lokeshwar MR, Dalal R, Manglani M, Shah N. Anemia in 21. Breathnach GS. Red cell diameters in human cord and
newborn. Ind J Pediatr. 1998;65:651-61. neonatal blood. Quaterly J Exp Physiol. 1962;47:148.
4. Usher R, Shepard M, Lind J. The blood volume of newborn 22. Kato (1933). Physiological variations in reticulocyte in
infant and placental transfusion. Acta Paediatric Scand. newborn study of 219 cases. Cited Oski FA, Naiman JL.
1963;52:497-512. Normal blood value in newborn period – hematological
5. Mollison PL, Veall N, Cutbush M. Red cell and plasma problems in newborn Vol. 1 in the series Major problems
volume in newborn infants. Arch Dis Child. 1950;25:242-53. in clinical pediatrics. WB Saunders Co. 1982.pp.1-31.
23. Faxen 1937. Red blood picture in healthy infants. Acta 34. Rivara LM, Rudulph N. Postnatal persistence of capillary
Pediatr. 1937;19:1. venous difference in hematocrit and Hb values in low birth
24. Schmairen AH, Haner HM. All thalassemic screening weight and term infants. Pediatrics. 1982.
in neonate by mean corpuscular volume and mean 35. Usher R, Shephard M, et al. The blood volume of the
corpuscular mean hemoglobin concentration. J Pediatr. newborn infant and placental transfusion. Acta Pediatr
1973;83:794. Scand. 1963;52:497.
25. Marks J, Gardner D, Rosecoe JD. Blood formation in 36. Yao AC, Moinian M, Lind J. Distribution of the blood
infancy III cord blood. Arch Dis Child. 1955;30:117. between infants and placenta after birth. Lancet.
26. Seyfarth C, Jurgen SR. Cited Oski FA, Naiman JL. Normal 1969;2:871.
blood value in newborn period – hematological problems 37. Yao AC, Wist A, Lind J. The blood volume of the newborn
in newborn Vol. 1 in the series Major problems in clinical infant delivered by cesarian section. Acta Pediatr Scand.
pediatrics. WB Saunders Co. 1982.pp.1-31. 1967;58:585.
27. Seip M. The reticulocyte level and erythrocyte production 38. Yao AC, Hirvansalo M, Lind J. Placental transfusion rate
judged from reticulocyte studies in the newborn infants and uterine contraction. Lancet. 1968;1:380.
during first week of life. Acta Pediatr. 1955;44:355. 39. Hasselhost G, Allmeling A. Cited by – Oski FA, Naiman JL.
28. Anderson GW. Studies on nucleated red cell count in Normal blood value in newborn period – hematological
chorionic capillaries and cord blood of various types of
problems in newborn Vol. 1 in the series Major problems
frequency. Am J Obstet Gynaecol. 1941;42:1.
in clinical pediatrics. WB Saunders Co. 1982.pp.1-31.
29. Oettinger L Jr, Mills WB. Simultaneous capillary and
40. Colozzi AE. Clamping the umbilical cord – its effect on
venous hemoglobin determinations in newborn infants. J
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Pediatr. 1949;35:362.
41. Mc Cue C, Garner FB, Hurt WB, et al. Placental transfusion.
30. Vehlqust B. Cited by Oski FA, Naiman JL. Normal blood
value in newborn period–hematological problems in J Pediatr. 1968;72:15.
newborn Vol. 1 in the series Major problems in clinical 42. Yao AC, Lind J, Tiisala R, et al. Placental transfusion in the
pediatrics. WB Saunders Co. 1982.pp.1-31. premature infant with observation on clinical courses and
31. Oh W, Lind J. Venous and capillary hematocrit in newborn outcome. Acta Pediatr Scand. 1969;58:561.
infants and placental transfusion. Acta Pediatr Scand. 43. Demarch QB. Alt HL and Windle WF. Factors influencing
1966;38:55-60. the blood picture of the newborn. Am J Dis Child. 1948;
32. Linderkamp O, Versmold HT, Strohacker I, et al. Capillary 75:860.
hematocrit difference in newborn infant. Eur J Pediatr. 44. Lanzkowosky P. Effect of early and late clamping of
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evaluation of anemia in erythroblastosis fetalis. Acta 45. Wu PC, Ku TS. Early clamping of umbilical cord: a study of
Pediatr Scand. 1967;31:391. its effects on the infants. Clin Med J. 1960;80:351.
C H A P T E R 5
Physiological Anemia of Newborn,
Anemia of Prematurity and Role of
Erythropoietin in the Management
Anemia is the most common hematological abnormality in the newborn. Anemia is defined as a hemoglobin or hematocrit value that
is more than two standard deviations below the average for a particular gestational as well as chronological age.1 As a “rule of thumb”
a hemoglobin value less than 13 g/dL in first 2 weeks of life is labelled as anemia and warrants evaluation.
Anemia is a sign and not a diagnosis. There are two Table 1 Postnatal changes in Hb
categories of anemia: Maturity Hb at nadir (g%) Time of nadir (weeks)
1. Pathologic Term 9.5–11 6–12
2. Physiologic
1200–1500 gm 8–10 5–10
Pathologic anemia in newborns results from accele
< 1200 gm 6.5–9 4–8
rated blood loss, destruction of red blood cells, or a defect
at some stage of red blood cell production. the fact that erythroid precursors present in both the bone
Physiologic anemia is common and a normal marrow and the blood are highly sensitive to EPO. It is not
physiologic process in term infants. It is typically known why the bone marrow does not respond adequately
asymptomatic requiring no intervention. to this hypoxic stimulus. The maximum decline in Hb
Anemia of prematurity (AOP) is an exaggerated and is reached by 4 to 12 weeks and is earlier and of greater
pathologic response of the preterm infant (<32 weeks severity in preterms compared to terms2 (Table 1).
gestation) to the transition from fetal to postnatal life. The Hb concentration continues to decrease until
The etiology, symptomatology, diagnosis, treatment and tissue oxygen needs are greater than oxygen delivery.
prevention of anemia of prematurity are addressed. Term infants remain asymptomatic and tolerate the
physiologic process well, but very preterm infants, <32
PHYSIOLOGIC ANEMIA OF INFANCY weeks gestation, may become symptomatic or the Hb
Immediately after birth with successful transition from fetal drops to a critical level warranting a blood transfusion.
to neonatal circulation, there is increase in blood oxygen Further in preterms several other factors contribute in
content and tissue oxygen delivery. This downregulates exacerbating the physiologic fall in Hb leading to AOP
erythropoietin production so that erythropoiesis is which are discussed later in the article.
suppressed. As a result of this, the hemoglobin concen
tration in healthy full-term and premature infants ANEMIA OF PREMATURITY
undergoes typical changes during the first weeks of life.
All infants experience a decrease in hemoglobin and
Background
hematocrit concentrations after birth. This physiological First described by Shulman in 1959, anemia of prematurity
decrease in Hb level is principally due to a reduced response (AOP) is a common hematological problem of premature
capacity for erythropoietin (EPO) production in the face of infants. AOP is an exaggerated and pathologic response of
anemia, even in the presence of symptoms and in spite of the preterm infant to the transition from fetal to postnatal
life. It is often, unlike the physiological anemia in term is due to the lower levels of intracellular ATP, enzyme
babies, a true anemia since oxygen delivery is diminished. activity, and carnitine levels as well as the increased
AOP is a normocytic, normochromic, hyporegenerative susceptibility to lipid peroxidation and susceptibility
anemia that is characterized by the existence of a low of the cell membrane to fragmentation. This shorter
serum EPO level in an infant who has what may be a life span means that the bone marrow must provide
remarkably reduced hemoglobin concentration. new red blood cells at a very high rate just to maintain
adequate hemoglobin and hematocrit levels.8
Incidence3 • Increased blood loss: Preterm infants loose blood
in different situations, some more avoidable than
AOP typically is not a significant issue for infants born
others. The most common form of blood depletion in
beyond 32 weeks’ gestation. Frequency of AOP is related
the preterm population is iatrogenic losses secondary
inversely to the gestational age and/or birth weight.
to blood sampling. Phlebotomy losses for blood
Approximately 80 percent of very LBW (VLBW) infants
tests are directly related to the length of an infant’s
(<1500 g) and 95 percent of extremely LBW (ELBW)
stay in the NICU and the severity of illness. Hidden
infants (<1000 g) receive at least one red blood cell (RBC)
blood loss (e.g. blood adherent to sampling syringes,
transfusion during their stay in the neonatal intensive care
gauze pads, bedding, tubing) is inevitable and under
unit (NICU). AOP spontaneously resolves by the time most
recognized. Accurate assessment of the volume of
patients are aged 3 to 6 months.
blood removed for laboratory testing is imprecise
leading to underestimation of laboratory losses. One
Pathophysiology milliliter of blood represents 1 percent of total blood
The three basic mechanisms which occur singly or in volume, especially in preterm babies. As a result very
combination for the development of AOP include: preterms can lose close to their entire blood volume
• Inadequate red blood cell (RBC) production for within a few days of life, necessitating frequent blood
a growing premature infant. Fetal and maternal transfusions.9
erythropoiesis occurs independently throughout Nutritional deficiencies of iron, vitamin E, vitamin
gestation. Erythropoietin does not cross the placenta. B12 and folate may exaggerate the degree of anemia.
Erythropoietin is the main growth factor responsible Several other factors predispose to anemia of
for erythropoiesis. The liver is the principal site of EPO prematurity.6,8,10 Some of these factors are common to
production during fetal life. By 32 weeks’ gestation, both the term and the preterm infant, but may be more
RBCs are produced approximately evenly from both the profound in the premature infant.
liver and bone marrow. Production of erythropoietin Lists factors that can contribute to the exaggerated
shifts to the peritubular cells of the kidney after term physiologic anemia of prematurity (Table 2).
gestation.4 Interrupting a pregnancy prematurely does
not alter these ontological processes. Erythropoietin Clinical Manifestations11
(EPO) production is thought to be controlled by an
oxygen-sensing mechanism in the liver and kidney Infants with anemia of prematurity may be completely
and both anemia and hypoxia stimulate mRNA asymptomatic. The signs and symptoms listed in Table 3 are
transcription and EPO protein production. The liver commonly seen but nonspecific and are not diagnostic
is less sensitive than the kidney in response to these
stimuli.5 Because it remains the major source of EPO in
Table 2 Factors contributing to exaggerated physiologic
the preterm infant, RBC production may be blunted.
anemia of prematurity
Premature infants also have relatively poor iron
stores. Erythroid progenitors of premature infants • Decreased RBC mass at birth
• Rapid body growth resulting in hemodilution
are quite responsive to EPO when that growth factor
• Poor iron stores along with limited ability to build iron
finally is produced or administered, but the response stores
may be blunted if iron stores are insufficient.6 • Increased iatrogenic losses from laboratory sampling
Iron stores are accrued during the last trimester of • Shorter RBC life span
pregnancy; premature infants therefore miss out on • Inadequate erythropoietin production
this opportunity. • Initial reliance on the liver as the primary site of
• Shortened RBC life span or hemolysis: The average erythropoietin production
life span of neonatal RBCs is approximately a half to • Abrupt decline in reticulocyte counts after the first few days
two thirds that of the adult RBC.7 This shorter life span of life
Chapter-5 Physiological Anemia of Newborn, Anemia of Prematurity and Role of Erythropoietin… 31
Table 3 Signs and symptoms of anemia of prematurity Prevention Strategies

• Tachycardia • Apnea and bradycardia • Delay in clamping the premature infant’s umbilical
• Tachypnea • Poor feeding cord during birth contributes to reducing the number
• Lethargy • Poor growth of transfusions to which they will later be subjected.13
• Pallor • Metabolic acidosis It may be beneficial to maintain the fetus 20 cm below
the level of the placenta during 30 seconds after birth.
by themselves of AOP.5,7,11,12,17 Historically these clinical Although a tailored approach is required in the case of
symptoms have been attributed to anemia by clinicians cord clamping, the balance of available data suggest that
despite the fact that there is no relationship established delayed cord clamping should be the method of choice.
between low levels of hemoglobin and onset of symptoms. • A direct relationship exists between the volume of
Asymptomatic premature infants may tolerate hemoglobin blood extracted and the number of transfusions
levels as low as 6.5 g/dL without showing any compromise performed. It is therefore necessary to conscientiously
of their physiologic functions. Although some studies limit the number of laboratory tests performed.
conclude that the use of red blood cell transfusions in • To avoid excess iatrogenic blood loss, the amount of
anemic premature newborns revert these symptoms, blood collected should be recorded.
others affirm that this association is not so clear. It is now • Administration of iron may begin at 1 month of age
recognized that resolution of these symptoms may be a (2 mg/kg/d) and continued until 6 to 12 months of age.
maturational process, not a hematologic issue. However, iron supplementation should not be started
in growing premature infants until adequate vitamin
E is supplied in the diet or supplemented; otherwise,
Diagnosis12 iron may increase blood cell hemolysis.14,15
In preterm infants, the hemoglobin and hematocrit levels • Vitamin E is an antioxidant that is vital to the integrity of
may be followed periodically, although no specific criteria erythrocytes. In its absence, these cells are susceptible
exist as to how often to monitor these levels. A rule of to lipid peroxidation and membrane injury. The
thumb is to monitor the hemoglobin and the hematocrit logical conclusion is that vitamin E deficiency might
if symptoms of anemia of prematurity are present. The contribute to the anemia of prematurity.16
CBC count demonstrates normal white blood cell and • The use of noninvasive monitoring devices, such
platelet series. The hemoglobin is less than 10 g/dL but as transcutaneous hemoglobin oxygen saturation,
may descend to a nadir of 6 to 7 g/dL; the lowest levels partial pressure of oxygen and partial pressure of
generally are observed in the very premature. RBC indices carbon dioxide, may allow clinicians to decrease blood
are normal (e.g. normochromic, normocytic) for age. The drawing; however, no data currently support such an
reticulocyte count is low for the severity of anemia. The impact of these devices.3
finding of an elevated reticulocyte count is not consistent
with the diagnosis of AOP. Peripheral blood smear shows Role of Blood Transfusion
no abnormal cells. Essentially, AOP is a diagnosis of
Despite disagreement regarding timing and efficacy,
exclusion.
packed red blood cell transfusions continue to be the
mainstay of therapy for AOP. The frequency of blood
MANAGEMENT OF ANEMIA OF transfusions varies with gestational age, degree of illness,
PREMATURITY and hospital practices. The goal of transfusion in infants
Options available to the clinician treating an infant with with anemia of prematurity is to ‘restore or maintain
anemia of prematurity (AOP) are prevention, blood oxygen delivery’ without increasing oxygen consumption.
transfusion, and recombinant EPO treatment.
Rationale for not Transfusing
Goal of Anemia of Prematurity Therapy Based on Hb Value Alone
• Maintaining the premature newborn’s erythrocyte There is no trustworthy marker for tissue hypoxia, hence
mass as “intact” as possible. it should be considered that the appearance of symptoms
• Provide the growing neonate with adequate oxygen attributable to reduced tissue oxygen delivery may not
carrying capacity to prevent symptomatic manifesta be solely due to low Hb levels, but also to other non-
tions of anemia while minimizing morbidity from hematological factors such as cardiac output and oxygen
treatment. partial pressure, the percentage of fetal Hb and the activity
of 2,3 diphosphoglycerate. Therefore considering the Hb Issues in Blood Transfusion11,12

level alone when making a transfusion decision, would
appear to be inadequate. There is no consensus as to • Donor exposure: A concern for infants who might
whether transfusions alleviate the signs and symptoms of need multiple transfusions is exposure to multiple
anemia of prematurity.17 donors. The use of multiple donors increases the risk
of infection and transfusion reactions. Donor exposure
can be reduced for infants who need small volume
Indications for Blood Transfusion transfusions (<15–20 mL/kg) by using stored packed
red blood cells (PRBCs) from a single unit. This unit
Transfusion practices vary markedly across units and is divided into multiple aliquots that are reserved for
there is a lack of evidence- based studies to guide a specific infant. This procedure has reduced donor
practice.18 None of the clinical signs has been consistently exposure to one or two donors for most infants.
useful either alone or as a group in determining when to • Preservatives: Use PRBCs stored in preservatives
transfuse an infant with low hemoglobin of (physiologic) (e.g. citrate-phosphate-dextrose adenine [CPDA-1])
anemia of prematurity and iatrogenic losses. The most and additive systems (e.g. Adsol). Preservatives and
common problem associated with anemia of prematurity additive systems allow blood to be stored safely for up
is identifying the threshold for transfusion. The guidelines to 35 to 42 days. The additives having mannitol should
use specific hematocrit levels, clinical status, and not be used for neonates.
sometimes infant’s age to determine when to transfuse19,20 • Screening: The risk of cytomegalovirus (CMV)
(Table 4). In many cases, the hematocrit levels are lower transmission can be reduced dramatically (but not
than those used previously. In the Premature Infants entirely) through the use of CMV-safe blood. This
in Need of Transfusion (PINT) study, Kirplani et al.19 can be accomplished by using either CMV serology-
demonstrated that transfusion threshold in ELBW infants negative cells or blood processed through leukocyte-
can be moved downwards by at least 1g/dL without reduction filters. This latter method also reduces other
incurring a clinically important increase in the risk of WBC-associated infectious agents (e.g. Epstein-Barr
death or major neonatal morbidity. virus, retroviruses, Yersinia enterocolitica).
Table 4 Transfusion guidelines

Hemoglobin Mechanical ventilation or symptoms of anemia PRBC volume
(g/dL)
11 or less Moderate or significant mechanical ventilation requirement (MAP >8 cm H2O and FiO2 15 mL/kg PRBCs
> 0.4) over 2–4 hours
10 or less Minimal mechanical ventilation requirement 15 mL/kg PRBCs
(any mechanical ventilation or CPAP >6 cm H2O and FiO2 >0.4) over 2–4 hours
8 or less No mechanical ventilation requirement and one or 20 mL/kg PRBCs
more of the following present: over 2–4 hours (divide
• 24 or more hours of tachycardia (HR >180) or tachypnea (RR >80) into two 10 mL/kg
• An increased oxygen requirement from the previous 48 hours volumes if fluid sensitive)
• An elevated lactate concentration (2.5 mEq/L or more)
• Weight gain <10 g/kg over previous 4 days while receiving 100 kcal/kg per day or
more
• An increase in episodes of apnea and bradycardia (10 or more episodes in a 24 hours
period or 2 or more episodes in 24 hours requiring bag-mask ventilation) while
receiving therapeutic doses of methylxanthines
• Undergoing some surgery
Less than 7 No symptoms and an absolute reticulocyte count 15 mL/kg PRBCs over 2–4
< 100,000 cells/mL (RBC x % reticulocyte count) hours
• Do not transfuse to replace blood removed for laboratory tests or low hematocrit alone unless above criteria are met
Abbreviations: CPAP: Constant positive airway pressure; HR: Heart rate; MAP: Mean airway pressure; PRBC: Packed red blood cell; RBC:
Red blood cell; RR: Respiratory rate.
Why Minimize Transfusions in Neonates? Current status of erythropoietin:3,11,12,20 Clearly, rEPO has
(Table 5) efficacy in stimulating erythropoiesis in preterm infants,
but success in the elimination or marked reduction in
Although transfusion may be lifesaving, like all medical the need for RBC transfusions has not been definitively
interventions, it is not without risk. Following are hazards demonstrated.
which need to be taken into account prior to transfusing • rEPO therapy must be carefully considered and used
blood: only when there is strong evidence of its need and
• The number of infectious agents that may potentially effectiveness.
be transmitted by this route continues to grow and is • There is no agreement regarding timing, dosing,
the most feared complication. route, or duration of therapy exists. Meta-analysis of
• Risk of fatal transfusions contaminated with bacterial controlled clinical trials show some benefit to EPO,
agents, hyperkalemia and graft versus host disease. but they cannot give firm guidelines on its use or
• Alterations of the immune system. recommend its routine use to prevent AOP. In short,
• Inferior quality of practices in developing countries – the cost-benefit ratio for EPO has yet to be clearly
collection, storage, screening policy and irradiation. established, and this medication is not accepted
The safest blood transfusion is the one not universally as a standard therapy for the individual
administered. with AOP.
There is lack of evidence that early EPO versus late EPO • When the family has religious objections to
confers any substantial benefits (Role of erythropoietin) transfusions, the use of EPO is advisable.
As a relative deficiency of EPO is present in the anemia of
prematurity it appears logical to supplement EPO in these CLINICAL GUIDELINES FOR TARGETED USE
infants. Recombinant erythropoietin (rEPO) has been
studied extensively since it became available for human
OF ERYTHROPOIETIN22-24
use in 1980. Despite several large EPO trials, there remains Indications for Erythropoietin
no clear consensus for the efficacy of EPO in neonates,
and currently its use remains inconsistent between The greatest hope of success in reducing need for RBC
centers.3,11,12,20 transfusions seems likely in preterm infants who are:
• < 28 weeks.
Early versus late erythropoietin: A Cochrane analysis • Infants 28 to 32 weeks who are <3rd centile for weight.
showed the use of early EPO (<7days of life) did not With phlebotomy loses expected to be >30 mL/kg.
significantly reduce the primary outcome of “use of
one or more red blood cell transfusions”, or “number Dose and Route of Administration
of transfusions per infant” compared to late EPO (>7
postnatal days) administration.20,21 In 750 Units/kg/week given as 3 doses on alternate days
by subcutaneous or intravenous injection beginning
Side effects20: Neutropenia and sepsis, rash, hypertension, at the end of the first week of life and for 6 weeks. The
convulsions, poor weight gain and SIDS have been reported subcutaneous route is preferred.
in some but not all studies. Apparently the problem of
aplastic anemia reported in adults, was related to a specific Iron supplementation: 0.4 to 1 mg/kg/day of iron dextran
type of EPO, and even to a single shipment of this drug sent is given via the parenteral nutrition solution. Once enteral
out by a single laboratory. Animal data and observational nutrition is initiated, 3 mg/kg/day of elemental iron should
studies in humans support a possible association between be provided by supplement. When full enteral nutrition is
treatment with EPO and the development of ROP. reached, elemental iron supplementation is increased to
6 mg/kg/day.
Vitamin E (Table 6) supplementation of 25 units/day
Table 5 Approaches to reducing transfusion needs should be given orally when EPO is administered.
• Development of, and adherence to, strict guidelines for Practice pointers in management of anemia of
transfusion prematurity (Table 7):
• Reducing iatrogenic blood loss • It seems clear that the major clinical goal for neonates with
• Autologous cord transfusion anemia is to reduce RBC transfusions, so as to minimize
• Targeted use of erythropoietin multiple donor exposures, infection, and immune risks.
• Iron supplementation EPO alone cannot achieve this goal reliably.
transfusion alternative? The answer may be yes for

Table 6 Medications used in the treatment of anemia of
those nurseries willing to apply restrictive transfusion
prematurity10,15,23
criteria and a single-donor system of blood banking.
Generic name Dosage/Route Common Comments For nurseries employing liberal transfusion practices,
adverse rEPO therapy using multidose vials may be a safe,
reactions
effective alternative to transfusion.25
Epoetin alfa 400–1400 Neutropenia, Supplement
U/kg/wk rash, with iron and
Key Points: Physiologic anemia of infancy
subcutane hypertension vitamin E
ously given convulsions, • N ewborns do not mount adequate erythropoietin response
every day or SIDS to hypoxia and anemia.
every other • The postnatal drop in Hb is universal in newborns. Term
day newborns reach a nadir of Hb between 6 and 12 weeks and
tolerate the physiologic process well. Few, if any, clinical
Ferrous sulfate 6 mg/kg/day GI upset Interferes
signs and symptoms are seen.
PO based on with vitamin E
• The exaggerated and pathologic response of the preterm
elemental iron absorption
infant to the transition from fetal to postnatal life is called
Folic acid 50 μg/day PO Caution May contain anemia of prematurity (AOP). It is seen by 4 to 10 weeks and
if used benzyl alcohol is more profound and occurs earlier than anemia of infancy.
concurrently as preservative • The clinical importance of knowing the postnatal drop in
with Hb is in curbing the tendency of subjecting these infants to
phenytoin un-necessary blood transfusions.
Vitamin E 25 IU/day PO May induce
vitamin K
deficiency REFERENCES
Vitamin B12 0.4 μg PO IM
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Table 7 Anemia of prematurity
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• Should rEPO therapy, as it is currently prescribed, treatment for anemia of prematurity. J of Perinat and
be discarded as a relatively useless and expensive Neonat Nur. 1997;11(3):57-68.
9. Walllgren G, Hanson JS, Lind J. Quantitative studies of 18. Bednarek FJ, Weisberger S, Richardson DK, et al. Variation
the human neonatal circulation. III. Observations on the in blood transfusions among newborn intensive care units.
newborn infant’s central circulatory response to moderate SNAP II Study Group. J Pediatr. 1998;133:601-7.
hypovolemia. Acta Paediatr Scand. 1967;179(Suppl): 19. Kirplani H, Whyte RK, Anderson C, et al. The premature
43-54. infants in need of transfusion (PINT) study: a randomized,
10. Gomella T, et al. Neonatology: Management, Procedures, controlled trial of a restrictive (low) versus liberal (high)
On-call Problems, Diseases, and Drugs, 4th edn. Stamford, transfusion threshold for extremely low birth weight
Connecticut: Appleton and Lange. 1999.pp.316-22. infants. J Pediatr. 2006;149:301-7.
11. Salsbury DC. Anemia of Prematurity. Neonatal Network. 20. Ohls RK. The use of erythropoietin in neonates. Clin
2001;20(5):13-20. Perinatol. 2000;27:681-96.
12. SM Aher, K Malwatkar, S Kadam. Neonatal Anemia. Seminars 21. Aher SM, Ohlsson A. Early versus late erythropoietin for
in Fetal and Neonatal Medicine. 2008;13:239-47. preventing red blood cell transfusion in preterm and/or
13. Levy T, Blickstein I. Timing of cord clamping revisited. J
low birth weight infants. Cochrane Database Syst Rev.
Perinat Med. 2006;34:293-7.
2006;3:CD004865.
14. American Academy of Pediatrics, Committee on Nutrition.
22. Irene Roberts. Management of neonatal anaemia: The role
Iron deficiency. In: Kleinman RE, ed. Pediatric nutrition
of erythropoietin. CME Bulletin Haematology. 1997;1(1):5-7.
handbook. Elk Grove Village, IL: American Academy of
23. Young T. Mangum O Neofax. A Manual of drugs used in
Pediatrics. 1998.pp.299-312.
15. Zenk K, Sills J, Koeppel R. Neonatal Medications and neonatal care, 20th edn. Montralle, New Jersey, Thompson
Nutrition: A Comprehensive Guide, 2nd edn. Santa Rosa, Healthcare, USA. 2007.p.86.
California: NICU INK. 2000.pp.184-5. 24. Ronald G. Strauss. Controversies in the management of the
16. Oski FA, Barness LA. Vitamin E deficiency: a previously anemia of prematurity using single-donor red blood cell
unrecognized cause of hemolytic anemia in the premature transfusions and/or recombinant human erythropoietin.
infant. J Pediatr. 1967;70:211-20. Transfusion 34 Medicine Reviews, 2006;20(1):34-44.
17. Atias D. Pathophysiology and treatment of anemia 25. Ellen M Bifano. Traditional and nontraditional approaches
of prematurity. Journal of Pediatric Hematology and to the prevention and treatment of neonatal anemia. Neo
Oncology. 1995;17(1):13-8. Reviews. 2000;1:69-73.
C H A P T E R 6
Effect of Maternal Iron
Status on Placenta,
Fetus and Newborn
Maternal anemia (hypoferremia) results in increased preterm and low birth weight deliveries and higher rate of stillbirths. There
are irreversible structural alterations in placenta. The transfer of iron to fetus is reduced in spite of gradient in relation to severity
of maternal hypoferremia. The fetal hepatic and brain iron contents were reduced. The brain iron reduction was irreversible on
rehabilitation and was associated with irreversible neurotransmitter and their receptor alterations.
The outcome of severe pregnancy anemia has been • Latent deficiency: As the bone marrow iron stores
associated with increased incidence of premature births, become absent, plasma iron decreases and bone
fetal distress, increased perinatal mortality and a higher marrow receives little iron for hemoglobin regeneration
frequency of maternal deaths.1 In the case of moderate to (bone marrow iron absent, serum ferritin <12 ug/L,
severe anemia—breathlessness, edema, congestive heart transferrin saturation <16 percent and free erythrocyte
failure and even cerebral anoxia have been observed. protoporphyrin is increased), however, hemoglobin
200 anemic pregnant women observed in the University concentration remains normal.
Hospital, Institute of Medical Sciences, Varanasi, showed: • Iron deficiency anemia: This is a very late stage of iron
reduced gestation; higher incidence of premature labor, deficiency with progressive fall in hemoglobin and
preterm, low birth weight and stillbirth deliveries. These mean corpuscular volume.
newborns had low Apgar score and there were increased
number of neonatal deaths. Maternal mortality was 13 PREVALENCE OF NUTRITIONAL ANEMIA IN
out of 200 anemic as compared to 1 in 50 controls. Similar PREGNANT WOMEN (INDIA)
findings were reported in other Indian studies. Anemic
mothers do not tolerate blood loss during childbirth; as National studies by the Indian Council of Medical
little as 150 mL can be fatal. Normally a healthy mother Research (ICMR)3—covering 11 states; reported in 1989,
during childbirth may tolerate a blood loss of up to prevalence of anemia by estimating hemoglobin using
1000 mL.2 cyanmethemoglobin method in pregnant rural women as
87.6 percent, hemoglobin being <10.9 g/dL. These anemic
CURRENT KNOWLEDGE IN THE women were given different doses of iron 60, 120 and 180 mg
with 500 ug folic acid daily for 90 days in 6 states; 62 percent
DEVELOPMENT OF IRON DEFICIENCY in spite of iron-folate therapy for 3 months, continued to
Iron deficiency is an end result of a long period of negative remain anemic.4 Thus indicating that short-term treatment
iron balance mainly due to poor dietary availability, rapid as recommended in the National Anemia Control Program
growth and blood loss. The pathological stages are; may not be sufficient to control anemia in pregnancy.
• Prelatent deficiency: Hepatic (Hepatocytes and macro However, it was observed that birth weight improved and
phages), spleen and bone marrow show reduced iron low birth weight deliveries were significantly reduced.5 The
stores (reduced bone marrow iron and serum ferritin). administration of higher dose 335 mg of ferrous sulfate and
Chapter-6 Effect of Maternal Iron Status on Placenta, Fetus and Newborn 37
500 ug of folic acid for 14 weeks as weekly or daily dose, both The placenta traps maternal transferrin, removes iron and
doses were found to be effective in control of pregnancy actively transports it across to the fetus where it becomes
anemia. This suggested that, even once a week iron—folate bound to fetal transferring and is distributed to the liver,
administration will be of help.6 spleen and other fetal hemopoietic tissues, maintaining
National Family Health Survey 1998–99 (NFHS-2) using higher levels of fetal iron as compared to the mother.
hemocue method reported prevalence of anemia as 49.7 Placenta plays an important role in maintaining iron
percent in pregnant women; 56.4 percent in breastfeeding transport to fetus. This process of iron transport is purely
nonpregnant women and 50.4 percent among nonpregnant a placental function over which mother and fetus has no
nonbreastfeeding women. Hemocue method estimates control, as placenta continues to trap iron even when
higher levels of hemoglobin thus difficult to compare with fetus is removed in animals.11 The placental trophoblastic
the other national studies. In 2005, NFHS-3 demonstrated membrane appears to act as an effective barrier against
increase in prevalence of anemia, suggesting marginal rise the further transport of iron to the fetus. In spite of this
in anemia nation wide.7 efficient protective mechanism the placental iron content
Nutrition Foundation of India in 2002–2003—studied reduces significantly in maternal hypoferremia.2,12-14 This
prevalence of anemia in pregnancy and lactation in was a very important finding as earlier studies on Swedish
7 states (Assam, Himachal Pradesh, Haryana, Kerala, and American women had shown that cord iron does not
Madhya Pradesh, Orissa, Tamil Nadu). The prevalence of change in iron deficient pregnant women.15,16 However,
pregnancy anemia was—86.1 percent (Hb <7.0 g/dL—in recent studies have confirmed that the maternal anemia
9.5%) and in lactation up to 3 months was 81.7 percent (Hb affects the placentofetal unit.17-20
<7.0 g/dL in 7.3%). The interstate differences responsible
Morphometry and biochemical alterations: Beischer et
for differences in prevalence of anemia were particularly
al.21 analyzed data (from Australia, India, New Guinea,
related to fertility, women education, nutrition status and
Singapore and Thailand) and demonstrated that in
occupation, availability of antenatal services and iron
all the studies placental weight in maternal anemia
folate tablets as possible factors.8,9
was higher than the control. This increase in placental
The ICMR, 1999–2000—conducted District Nutrition
weight was higher with increasing parity. The placental
Survey in 11 states covering 19 districts pregnancy anemia
hypertrophy did not correspond to fetal size and had
prevalence was 84.6 percent (Hb <7.0 g/dL—in 9.9%). The
no correlation with maternal serum protein. Ratten and
study also found 90 percent adolescent girls with anemia
Beischer22 confirmed that the placental weight exceeds
in these districts.10
the 90th centile in 20 percent of patients with hemoglobin
The prevalence as well as severity of anemia during
<8.2 g/dL and in 13.2 percent of those with hemoglobin
pregnancy and lactation is grave. This is the period when
8.2 to 9.1 g/dL. The placental hypertrophy is postulated to
brain cells grow and neurotransmitters develop, iron is
be due to hypoxia, which is supported by evidence of similar
essential for it.
phenomenon at higher altitudes. In our studies maternal
anemia was associated with low maternal serum albumin.
IRON STATUS IN PREGNANCY Both deficiencies were associated with reduced weight and
• Fetal growth depends, to a large extent, on the volume of placenta. Placentae in maternal anemia showed
availability of iron from the mother. reduced number of cotyledons and increase incidence of
• Normal nonpregnant woman needs iron 1.3 mg/day. ill-defined cotyledons and eccentric attachment of cord.
• Total pregnancy need of iron is 1000 mg or more. There was increased shrinkage in formalin in pregnancy
Absorption—6 mg/day in the last 2 trimesters. anemia.23-26 This reduction in placental weight was due to
• 350 mg of iron is transferred to the fetus and placenta. reduced DNA (cell number), however cell size was increased
• 250 mg is lost in blood at delivery. (weight/DNA). In maternal hemoglobin RNA content per
• 450 mg is needed to increase the RBC mass. cell remained constant.27 Placental succinic dehydrogenase
• Lastly around 240 mg is lost as basal losses. activity was decreased, total NADP-dependent ICDH was
• In cesarean delivery blood loss is almost twice (500 more than NAD+dependant ICDH in severe maternal
mL). In moderate and severe anemia mother will die, if hypoferremia; suggesting impaired citric acid cycle.2
blood loss is >150 mL.
• During lactation iron loss is 0.3 mg/day. Histology
There was decreased villous vascularity leading to fibrosis
PLACENTA IN IRON DEFICIENCY with increased endarteritis obliterans reflecting response
Iron transport: Normally ‘placental iron transfer’ to fetus to hypoxia. There was progressive decrease of surface
becomes 3 to 4 times during 20 to 37 weeks of gestation. area and volume of villi per unit volume of blood vessel
in relation to degree of anemia; suggesting maturational Brain ‘Glutamate metabolism’—(GAD, GDH, GABA-T) 35,37
arrest.2,26,28,29 On treatment with iron there was increase • Marked reduction in levels of brain GABA, L-glutamic
in hemoglobin, cord iron and placental (nonheme iron) acid and enzymes for biosynthesis of GABA and
and placental shrinkage in formalin reduced. However, L-glutamate like glutamate decarboxylase and gluta
the reduced villus vascularity, increased villus fibrosis mate transaminase.
and endarteritis obliterans in placenta of anemic mother • Binding of H3 Muscimol at pH 7.5 and 1 mg protein/
did not reverse. It was postulated that moderate-severe assay (GABA receptor) increased by 143 percent,
anemia present from the early days of pregnancy induces but glutamate receptor binding decreased in the
irreversible structural alteration, as iron is needed in 2nd vesicular membranes of latent iron deficient rats by 63
week of pregnancy for placenta formation.2 percent33,38
– Brain ‘TCA-cycle’ enzymes-mitochondrial NAD+
FETUS—NEWBORN linked dehydrogenase significantly reduced.
– Brain ‘5-HT metabolism’—tryptophan, 5-HT,
Cord serum iron and hemoglobin were reduced in preterm
5-HIAA significantly reduced.
as well as full term infants of hypoferremic mothers. There
– The whole brain and corpus striatum showed
is an increased gradient in presence of maternal iron
reduction in catecholamine, dopamine nor-epine
deficiency for transport of iron from mother to fetus but the
phrine, tyrosine and monoamine oxidase, while
transport remains proportionate to the degree of maternal
tyrosine aminotransferase increased in corpus
hypoferremia. The weight of full term singleton babies
striatum, in spite of reduction in whole brain
born of anemic mothers was reduced in direct relation
suggesting that latent iron deficiency induced
to hemoglobin level. Similarly these babies showed a
irreversible neurotransmitter alterations.39
progressive decrease in Apgar scores also.2 Fetal liver iron
– Brain ‘catecholamine metabolism’—whole brain-
stores are reduced significantly in maternal hypoferremia.
dopamine, neonephrine, tyrosine and TAT signi
Normally bigger the infant and more advanced the
ficantly reduced; in ‘corpus striatum’—same as in
gestational age higher was the amount of iron in fetal liver,
whole brain, except TAT was increased.40
spleen and kidney. The tissue iron content increases steeply
These changes were specific to iron deficiency
in last 8 weeks of gestation. Infant born before 36 weeks of
as neurotransmitter alterations in fetal brain due to
gestation, had half the iron content in hepatic reserve.30
malnutrition get normalized partially or completely on
Breast milk—iron content is increased in hypoferremic
rehabilitation.41,42
mothers, a phenomenon of “Physiological Trapping”.31,32
The significant effects on neurotransmitter receptors
To understand more a rat model was created with
(glutamate mediators) during early stages of iron
latent iron deficiency (low hepatic iron with out change in
deficiency clearly indicate the deficits in both excitatory
hematocrit) in pregnancy.33-38 Fetal brain iron content and
neurotransmitters in maternal (Rat) latent iron deficiency. and inhibitory pathways of the central nervous system,
showing an important role of iron in brain.33
Iron as a micronutrient is required for regulation of brain
To test the above findings in humans, babies born of
neurotransmitters by altering the pathway enzymatic
moderate to severely anemic mothers were examined
system. To study iron deficiency a rat model was developed
for “impact of iron deficiency on mental functions”. The
to create iron deficiency (low hepatic iron) without change
intrauterine growth retarded offspring’s of anemic as
in hematocrit.33
well as undernourished mothers showed hypotonia in
In postweaning rats iron decreased irreversibly in
72 percent and hypoexcitability in 56 percent.43-45
all brain parts except medulla oblongata and pons.
There was modification of responses in several
Susceptibility to iron deficiency showed variable reduction
neonatal reflexes, e.g. limp posture, poor recoil of limbs,
in different parts of the brain: corpus striatum-32 percent,
incomplete Moro’s and crossed extensor responses.
midbrain 21 percent, hypothalamus 19 percent, cerebellum
Their EEG had shortening of sleep cycle (REM AND
18 percent, cerebral cortex 17 percent and Hippocampus
NERM), the reduction was more marked for REM sleep.
15 percent.
There was some inter and intrahemispheric asymmetry
Alterations in brain iron content also induced—signi
and abnormal paroxysmal discharges; suggesting
ficant alterations in Cu, Zn, Ca, Mn, Pb and Cd.34
dysmaturity of brain.43,44
Fetal Latent Iron Deficiency (Rat) and The above findings were not specific to affects of
anemia on mental functions. Therefore effects of anemia
Brain Neurotransmitters (nutrition controlled) on mental functions were then
In latent iron deficiency there was irreversible reduction in studied in rural children during a period of three years.
neurotransmitters: Mental functions in nutrition controlled 388 rural
Chapter-6 Effect of Maternal Iron Status on Placenta, Fetus and Newborn 39
primary school (6–8 years of age), matched for social and 2. Agarwal KN. The effects of maternal iron deficiency
educational statuses were studied by WISC and arithmetic on placenta and foetus. In: Jelliffe DB, Jelliffe FEP,
test to assess “intelligence, attention and concentration”. eds. Advances in International Maternal Child Health.
Anemia does not affect intelligence, except subtest-digit Clarendon Press Oxford. 1984;4:26-35.
span. In arithmetic test, attention and concentration was 3. Indian Council Medical Research (1989). Evaluation of
the National Nutritional Anemia Prophylaxis Programme.
poor in anemic children.46
ICMR report, New Delhi.
Effects of iron deficiency and/or anemia—on brain: iron
4. Indian Council Medical Research (1992). Field supplemen
deficiency anemia in infancy has been consistently shown tation trial in pregnant women with 60, 120 and 180 mg of
to negatively influence performance in psychomotor iron and 500 ug of folic acid. ICMR report, New Delhi.
development. Short-term iron therapy did not improve the 5. Agarwal KN, Agarwal DK, Mishra KP. Impact of anemia
lower scores, despite complete hematological replenish prophylaxis in pregnancy on maternal hemoglobin, serum
ment. Neurological maturation was studied in infants ferritin and birth weight. Indian J Med Res. 1991;94:277-80.
6 months old, including auditory brainstem responses and 6. Gomber S, Agarwal KN, Mahajan C, et al. Impact of daily
naptime 18 lead sleep studies. The central conduction time versus weekly hematinic supplementation on anemia in
of the auditory brainstem responses was slower at 6, 12 and pregnant women. Indian Pediatr. 2002;39:339-46.
18 months and at 4 years, despite iron therapy beginning 7. NFHS-2 and- 3 India 1998-99-and 2005 National family
at 6 months. During sleep-wakefulness cycle, heart rate Health Survey-2 and 3 Anemia among women and children.
variability—a developmental expression of the autonomic Mumbai: International Institute for Population Sciences;
nervous system—was less mature in anemic infants. This is 2000.
8. Agarwal KN, Agarwal DK, Sharma A, et al. Prevalence of
possibly due to altered myelination of auditory nerves.47 It
anemia in pregnant and lactating women in India. Indian J
has been observed that these changes are resistant to iron
Med Res. 2006;124:173-84.
therapy in children <2 years of age with iron deficiency with 9. Sharma A, Agarwal KN. Author’s reply. Indian J Med Res.
anemia, but not in older children.48 2007;125:101.
These studies supported our earlier findings that 10. Teoteja G, Singh P. Micronutrient deficiency disorders in
brain functions are significantly affected in latent iron 16 districts of India. Report of an ICMR Task Force Study –
deficiency in the brain growth period and such changes District Nutrition Project, Part 1. 2001.
are irreversible. These have serious consequences, e.g. 11. Fletcher J, Suter PEN. The transport of iron by the human
poor cognition and learning disabilities. placenta. Clin Sci. 1969;36:209-20.
12. Singla PN, Chand S, Khanna S, et al. Effect of maternal
SUMMARY anemia on the placenta and the newborn. Acta Paediatr
Scand. 1978;67:645-8.
The above researches by our group are mainly on effects 13. Singla PN, Chand S, Agarwal KN. Cord serum and
of maternal hypoferremia on iron status of placenta, placental tissue iron status in maternal hypoferremia. Am
cord blood (hemoglobin and ferritin), and fetus (brain J Clin Nutr. 1979;32:1462-5.
and hepatic iron content). The rat model of “latent iron 14. Agarwal RMD, Tripathi AM, Agarwal KN. Cord blood
deficiency” showed irreversible brain iron reduction and hemoglobin iron and ferritin status in maternal anemia.
irreversible neurotransmitter alterations in ‘brain growth Acta Paediatr Scand. 1983;72:545-8.
period’. Once anemia sets in, the additional effects are 15. Vahlquist BC. Das serumerrisen, eine paediatrisch
due to anoxia. Our nation is faced with the problem of kinische und experimental studies. Acta Paeditr. 1941;28
iron deficiency that leads to anemia—a clinical condition (Suppl 6):1-4.
due to deficiency of many nutrients—mainly iron, folic 16. Rios E, Lipschitz DA, Cook JD, et al. Relationship of maternal
acid and vitamin B12. Folic acid is essential from prenatal and infant iron stores as assessed by determination of
period its deficiency causes neural tube defects. plasma ferritin. Pediatrics. 1975;55:694-9.
17. Emamghorashi F, Heidari T. Iron status of babies born to
iron-deficient anemic mothers in an Iranian hospital. East
ACKNOWLEDGMENTS
Mediterr Health J. 2004;10:808-14.
Thanks are due to Professor Dev K Agarwal for valuable 18. Paiva Ade A, Rondo PH, Paqliusi RA, et al. Ralationship
suggestions. The Indian National Science Academy sup between the iron status of pregnant women and their
ported part finances. newborns. Rev Saude Publica. 2007;41:321-7.
19. Kumar A, Rai AK, Basu S, Dash D, Singh JS. Cord blood
and breast milk iron status in maternal anemia. Pediatrics.
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22. Ratten GJ, Beischer NA. Significance of anemia in an 37. Shukla A, Agarwal KN, Shukla GS. Latent iron deficiency
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23. Sen S, Agarwal KN. Placental protein, free alpha amino 38. Mittal RD, Pandey A, Mittal B, et al. Effect of latent iron
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24. Khanna S, Chand S, Singla PN, et al. Morphological study 39. Shukla A, Agarwal KN, Chansuria JPN, et al. Effect of latent
of placenta in pregnancy anemia. Indian J Path Microbiol. iron deficiency on 5-hydroxytryptamine metabolism in rat
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25. Agarwal K N, Krishna M, Khanna S. Placental morphological 40. Shukla A, Agarwal KN, Shukla GS. Studies on brain
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after treatment. J Trop Paediatr. 1981;27:162-5. ency and subsequent rehabilitation. in rat. Nutr Res.
26. Marwah P, Singla PN, Krishna M, et al. Effect of pregnancy 1989;9:1177-86.
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C H A P T E R 7
Developmental Aspects of
Hemostasis in the Fetus and Newborn
Development of the hemostatic system in the human fetus has long been an area of interest and research. Hemostatic system starts
developing in the embryonic period and undergoes evolution in the postnatal period as well till adult levels are reached. As the
process spans over a long period of time, multiple age appropriate (gestational and postnatal age) reference ranges are necessary. An
understanding of development of hemostasis helps in diagnosis and management of hemostatic problems during childhood.
INTRODUCTION from 4th week onwards, with successive detection in the

fetal bone marrow and tissues over the next four weeks.
The hemostatic system is functionally intact in healthy Synthesis of coagulation proteins is among the most active
full-term newborns and clinical presentation of bleeding in the fetus after production of plasma proteins such as
or clotting is seen only in sick newborns especially the albumin.
preterm infants. All the screening tests of coagulation are
prolonged in the plasma of healthy infants compared with
adult values, in the absence of bleeding. Stable preterm
COAGULATION SYSTEM
infants have more prolonged values than the healthy full Coagulation Proteins
term infants.
Plasma concentrations of various coagulation proteins It has been demonstrated that the blood of fetuses does
mature at different rates in the fetus and newborn. The not clot before 10 to 11 weeks of gestation. Thereafter, the
normal adult range may be achieved as early as mid- clotting time rapidly becomes equal to the adult values
gestation for some proteins and as late as several months or even less. Fibrinolytic activity can be detected by 10
after birth for others. Thus, the fetus demonstrates a to 11 weeks gestation and thereafter is similar to adult
unique balance in levels of specific coagulation proteins values or even greater (indicating shorter lysis times). The
in the maintenance of hemostasis. coagulation proteins do not cross the placenta or do so in
The vitamin K status of a newborn is precarious at negligible quantities and need to be independently
birth and may cause significant bleeding in the absence synthesized by the fetus. Results obtained on healthy
of other problems. Complications of birth process may full-term infants are significantly higher than are those
result in birth asphyxia, which is an important cause of a documented on healthy full-term fetuses in utero.
consumptive coagulopathy with significant bleeding. Similarly, prematurely born infants have higher levels
of coagulation proteins than their age matched counter-
parts in utero indicating fast maturation of the coagulation
EARLIEST EVIDENCE OF THE FETAL
system soon after birth.
HEMOSTATIC SYSTEM Levels of factor V approximate the adult range by 12 to
Fibrinogen has been found in the fetal liver as early as 15 weeks. Plasma concentration of fibrinogen reaches 100
5th week of gestation. von Willebrand factor (vWF) can mg/dL by 12 to 15 weeks of gestation. Plasma concentration
be demonstrated in the endothelial cells of the placenta of most coagulation proteins are maintained at a constant
level throughout gestation until some time after 33 weeks, Plasma levels of thrombomodulin are increased in early
when a maturational burst happens. From 19 weeks childhood, and decrease to adult values by the late teenage
onwards, all the coagulation proteins except factor V, VII, years. Total tissue factor pathway inhibitor (TPFI) levels in
VIII, XII, and antithrombins circulate at 50 to 100 percent newborn infants are same as in older children or adults,
of the level achieved by full term. Factor V, VII, VIII, XII, but free TPFI is significantly lower. Thrombi in newborn
and antithrombins increase steadily throughout the infants do not propagate with the same propensity as in
second and third trimesters. adults, due to significantly low levels of fibrinopeptide A in
The levels of vitamin K dependent factors and the cord plasma.
contact factors (XI, XII, prekallikrein and high molecular
weight kininogen) gradually increase to those approaching PLATELETS
adult levels by six months of life. The low levels of contact
factors are partly responsible for the prolonged APTT Mega-karyocytes appear in the liver at 8 weeks gestation
during the first months of life. Plasma levels of fibrinogen, and platelets can be found in fetuses from 11 weeks
factor V, VIII, XIII and von Willebrand factor are similar gestation. Initially the platelets are large and beyond 12
to adult values at birth. Fibrinogen levels continue to weeks the mean platelet volume is essentially normal.
increase after birth. Plasma levels of factor VIII at birth Cord blood of preterm babies has increased number of all
are towards the higher range of normal and levels of both megakaryocyte precursors as compared with term babies
vWF and high molecular weight multimers are increased and term infants have higher circulating megakaryocyte
at birth and remain so till three months of life. progenitor numbers at birth compared with adults.
Reduced production of coagulation factors and After 20 weeks of gestation, the platelet counts and the
increased clearance of plasma proteins leads to the mean platelet volume are similar to those in adults with
differences in the level of various plasma proteins. values between 1.5 and 5.5 lakh per cu.mm and 7 to 9 fL
Premature infants have accelerated clearance of fibrinogen respectively. The platelet function in term and preterm
which can be attributed in some part to their increased babies has been found to be impaired in vitro.
basal metabolic rate. Fetal fibrinogen has an increased The most consistent abnormalities are reduced
content of sialic acid which accounts for the differences in aggregation in response to adrenaline, ADP and thrombin.
its structure and function as compared to adult fibrinogen. Electron microscopic studies on cord platelets have shown
normal number of granules, however, the concentration
of serotonin and adenosine diphosphate (ADP), which are
Regulation of Thrombin stored in dense granules, is less than 50% of adult values.
Newborns have delayed and decreased regulation of GP Ib is present on fetal platelet membrane in adult
thrombin compared with adults. The amount of thrombin quantities, however GP IIb/IIIa is significantly reduced.
generated is directly proportional to the concentration of Despite these differences, the bleeding time is normal in
prothrombin and the rate of thrombin generation depends term and preterm infants.
on the concentration of other procoagulants. Newborns have higher levels of thrombopoietin in the
Thrombin is directly inhibited by antithrombin (AT), cord blood as compared to adult values. The relatively
heparin cofactor II (HC II), and alpha 2 macroglobulin. higher hematocrit of neonatal blood contributes to
Alpha 2 macroglobulin is a more important inhibitor of increased blood clotting by increasing the number of
thrombin in plasmas from newborns as compared to platelets directed to the vessel wall by virtue of laminar flow
plasma from adults. The rate of inhibition of thrombin and by offering a larger surface for the formation of fibrin
is slower in newborn infants than it is in adults plasma clots. vWF facilitates adhesion of platelets to collagen, and
concentrations of protein C are very low at birth, and it reaches normal adult values by 20 weeks gestation. At
remain decreased during the first six months of life. full term, it has a higher total concentration and increased
Neonates have a two fold increase in the single chain form number of larger (stickler) UlvWF.
of protein C, compared with the double chain form that is Platelet receptors in the fetus mature around 12
predominant in adults and no difference has been found to 16 weeks’ gestation except for decreased coupling
in the functioning of the two forms. Newborns have lower of epinephrine receptors. In response to activators,
levels of total protein S at birth but functional activity is the granular release is decreased in the fetus due to
similar to that in the adult because in newborns, protein diminished calcium channel transport and impaired
S is completely present in the free, active form due to the signal transduction. Platelets get activated during the
absence of C4 binding protein. The interaction of protein S birth process by interplay of various factors which
with activated protein C in the newborn plasma is regulated include thermal changes, hypoxia, acidosis, adrenergic
with by the increased levels of alpha 2 macroglobulin. stimulation and the thrombogenic effects of amniotic fluid.
Chapter-7 Developmental Aspects of Hemostasis in the Fetus and Newborn 43
Cord plasma levels of thromboxane B2, thromboglobulin enzymatic activity of fetal plasmin and its binding to
and platelet factor-4 are increased, the granular content cellular receptors for fetal plasminogen is decreased but
of cord platelets is decreased and epinephrine receptor still clot lysis is more rapid in fetal plasma due to decreased
availability is reduced. inactivation of fetal plasmin by alpha-2 antiplasmin. The
proof of activation of the fibrinolytic system at birth lies
Bleeding Time in the short whole blood clotting times, short euglobulin
lysis times (ELTs) and increased plasma concentrations of
Infants during the first week of life have significantly fibrin related peptides.
shorter bleeding times as compared to those in adults.
Enhanced platelet and vessel wall interaction, higher
CONCLUSION
plasma concentrations of vWF, enhanced function of vWF
due to a disproportional increase in the high molecular Decreased concentration and activities of coagulation
weight multimeric forms, active multimers, large red cells proteins is responsible for the mild prolongations of all
and high hematocrits all contribute to the shorter bleeding the screening tests detected in healthy full-term infants.
time in infants. Except for vWF, factors V, VIII and fibrinogen. All other
plasma coagulation proteins are generally low in the
VESSEL WALL newborn. These four coagulation proteins which are
all within or above the normal adult range at full-term
The endothelial cells and extracellular matrix components gestation, together with hematocrit, platelet number
of the vessel wall have procoagulant and anticoagulant and platelet adhesiveness make a major contribution to
properties which are significantly influenced by age and hemostatic potential in the neonate and are to a certain
have a significant bearing on the hemostasis. One of these extent responsible for the hypercoagulability observed in
anticoagulant properties is mediated by lipoxygenase and sick infants.
cyclo-oxygenase metabolites of unsaturated fatty acids. Full-term newborn infants have a balanced and
Prostaglandin I2 (PGI2) production from cord vessels intact hemostatic system, despite apparent deficiencies
is higher than that of adult vessels. Levels of soluble of procoagulant, regulatory and fibrinolytic activities.
endothelial cell adhesion molecules and selectins are The neonatal hemostatic system lacks adequate reserve
also age dependent, due to differences in development capacity to cope with massive stresses of low blood flow,
of endothelial cell expression and secretion of these acidosis and sepsis, which becomes a clinical challenge in
molecules. Nitric oxide (NO) modulates vascular tone in the sick preterm infant.
fetal and postnatal lungs and is responsible for the normal Fetal hemostasis is a dynamic system that gradually
decline in pulmonary vascular resistance at birth. NO is a evolves by stages towards the adult state, but always
potent inhibitor of platelet adhesion, aggregation maintains equilibrium between activators and inhibitors
and stimulates disaggregation of platelet aggregates. throughout intrauterine life until birth. The vascular
NO interacts with PGI2 and other metabolites of the and hemostatic systems of the fetus and neonate are
lipoxygenase pathway to modulate platelet function. continually evolving and this must be taken into account
when evaluating these systems for dysfunction.
ANGIOGENESIS
Angiogenesis plays an important role in development SUGGESTED READING
of the alveoli in the fetal and neonatal lung. Angiogenic 1. Andrew M, Paes B, Milner R, Johnston M, Mitchell L,
factors-angiogenin, basic fibroblast growth factor (bFGF) Tollefsen DM, Castle V, Powers P. Development of the
and vascular endothelial growth factors (VEGF) in the human coagulation system in the healthy premature
serum increase soon after birth. infant. Blood. 1988;72:1651-7.
2. Andrew M, Paes B, Milner R, Johnston M, Mitchell L,
Tollefsen DM, Powers P. Development of the human
FIBRINOLYTIC SYSTEM coagulation system in the full-term infant. Blood.
Plasminogen levels in the neonates are approximately 50 1987;70:165-72.
percent and antiplasmin (AP) levels are approximately 80 3. Elizabeth A Chalmers, Michael D Williams, Thomas A.
Acquired disorders of hemostasis. In: Robert J Arceci, Ian
percent of the normal adult values. Plasma concentrations
M Hann, Owen P Smith, eds. Pediatric Hematology, 3rd
of plasminogen activator inhibitor-1 (PAI-1) and tissue edn. USA:Blackwell Publishing Ltd. 2006.pp.624-42.
plasminogen activator (TPA) are significantly higher as 4. Ignjatovic V, Mertyn E, Monagle P. The coagulation
compared to adults due to enhanced release of these system in children: Developmental and pathophysiologic
two factors from the endothelium shortly after birth. The considerations. Semin Thromb Hemost. 2011;37(7):723-9.
5. Lippi G, Franchini M, Montagnana M, Guidi GC. 8. Parmar N, Albisetti M, Berry LR, Chan AK. The fibrinolytic
Coagulation testing in pediatric patients: the young system in newborns and children. Clin Lab. 2006;52(3-
are not just miniature adults. Semin Thromb Hemost. 4):115-24. Review.
2007;33(8):816-20. 9. Poralla C, Traut C, Hertfelder HJ, Oldenburg J, Bartmann
6. Lippi G, Salvagno GL, Rugolotto S, Chiaffoni GP, Padovani P, Heep A. The coagulation system of extremely preterm
EM, Franchini M, Guidi GC. Routine coagulation tests infants: influence of perinatal risk factors on coagulation. J
in newborn and young infants. J Thromb Thrombolysis. Perinatol. 2012;32(11):869-73.
2007;24(2):153-5. 10. Sentilhes L, Leroux P, Radi S, Ricbourg-Schneider A,
7. Monagle P, Ignjatovic V, Savoia H. Hemostasis in Laudenbach V, Marpeau L, Bénichou J, Vasse M, Marret
neonates and children: pitfalls and dilemmas. Blood Rev. S. Influence of gestational age on fibrinolysis from birth to
2010;24(2):63-8. postnatal day 10. J Pediatr. 2011;158(3):377-82.
C H A P T E R 8
Anemia in the Newborn
Jayashree Mondkar, Shilpa Sanjay Borse, MR Lokeshwar
INTRODUCTION Beyond the first weeks of life many factors influence

what is considered as normal hematological parameters
Pediatricians caring for sick/full term/premature newborn in newborn period. Hb concentrations decrease in both
infants are often confronted with a variety of routine as normal term and preterm infants after birth to reach
well as life-threatening hematological problems. Anemia minimal levels of 9.4 to 14.5 g/dL in term infants by 7 to 9
in neonatal period remains a cause for concern due to weeks of age.1,4,5
likelihood of rapid decompensation in this vulnerable
group. Numerous physiological changes occur in
succession and rapidity in fetus and neonate, as the
Physiological Anemia
erythrocyte system in utero undergoes serial adaptation This “physiological” anemia1,3-6 occurs because of a decline
to meet progressively changing demand for oxygen from in erythrocyte mass due to the following reasons:
embryo stage to term. Thus, there is rapid change in • During intrauterine period the fetal oxygen saturation
normal hematological parameters from fetal period to is low at around 45 percent, erythropoietin levels are
immediately after birth and throughout neonatal period high and RBC production is rapid. Reticulocyte counts
even hours, days and weeks after birth.1-5 are 3 to 7 percent reflecting ongoing erythropoiesis.
An understanding about the basic physiology of With improved oxygen saturation to 95 percent after
hematopoiesis and appreciation of normal hematologic birth, the erythropoietin levels become undetectable
and laboratory values at birth is important because they hence RBC production stops, reticulocyte counts are
form the basis for the diagnosis, treatment and prevention, low and the hemoglobin level falls.
of many diseases that afflict these neonates. Interpretation • This factor coupled with a reduced lifespan of fetal
of laboratory findings and institution of appropriate RBCs, results in anemia that is not a functional one as
therapy requires understanding of maturational process oxygen delivery to the tissue is adequate.
and normal physiological variations that takes place At 8 to 12 weeks, hemoglobin levels reach their nadir,
during this period.1-5 oxygen delivery to the tissues is impaired, erythropoietin
production is stimulated and hemoglobin starts increasing.
ANEMIA Hemoglobin values rise from 8 to 10 g/dL at 12 weeks of
gestation to 13.7 to 20.1 g/dL (mean of 16.8) at term.1,5
Anemia in the term infants is defined as a hemoglobin The hemoglobin and RBC count fall earlier and to a
value of less than 13.5 g/dL during the first week of greater extent in preterm infants leading to “Anemia of
life. Values for umbilical artery blood tend to be about Prematurity”.
0.5 g/dL higher than sample obtained from umbilical
vein. Preterm infants have lower baseline values. Capillary
specimens obtained by heel stick have higher hemoglobin
Anemia of Prematurity
and hematocrit values than samples obtained from the Anemia of prematurity (AOP)6,8-12 is an exaggeration of
umbilical vein or peripheral blood.4-9 the physiologic anemia of infancy. The hemoglobin and
RBC count fall earlier and to a greater extent as low as • Rupture of normal umbilical cord, rupture of anomalous
7.8 to 9.6 g/dL in preterm infants leading to “Anemia of insertion of the cord and hematoma of the cord
Prematurity”. Shortened survival of RBCs to an average of containing large amount of blood, aneurysm of cord.
60 days (120 days life span in adult RBCs) and rapid body • Malformation of placenta and cord velamentous
growth with relative hemodilution are the contributory insertion, vasa previa.
factors. Besides, iatrogenic blood losses may be higher. The most common cause of anemia at or around the
Premature infants may require additional folate and B12 time of birth is due to fetal blood loss associated with
to reduce severity of anemia of prematurity.9,10 Vitamin E conditions like placenta previa and accidental hemorrhage
deficiencies is more common in small preterm infants.11 due to abruptio placentae.14,15 When there is partial
separation of the placenta, blood loss is predominantly
Nonphysiologic Anemia in Neonate maternal, however some fetal sinuses in the placenta
may rupture causing fetal blood loss and anemia. Also,
Pathological anemia in neonate may be caused by wide maternal blood loss and hypotension causes vasodilatation
spectrum of diseases and could be due to in placental vessels causing placental pooling of blood,
• Hemorrhage12-25 thereby aggravating fetal hypovolemia. This abnormality
• Hemolysis, increased RBC destruction26-36 is more common in multiple pregnancies and incidence
• Failure of red cell production.37-42 of hemorrhage is seen between 1 and 2 percent.
Nutritional anemia is not a cause of anemia during • Umbilical cord anomalies like venous tortuosity or
neonatal period (unlike in older children) even when the arterial aneurysm may lead to bleeding if injured.
mother is severely iron deficient, as fetus is an effective • Unattended precipitous delivery may lead to rupture
parasite of the mother. Anemia in the newborn often of normal umbilical cord. When cord ruptures, the tear
accompanies and is complicated by conditions like asphy generally occur in fetal third and bleeding is immediate
xia, shock, jaundice which make the situation even worse. and profuse.12,15
ETIOLOGY Other Causes of Hemorrhage Includes

• Fetomaternal hemorrhage15-19,21,22
Hemorrhage12-25
• Fetoplacental hemorrhage14,15
Profound anemia appearing at birth or during first • Fetofetal hemorrhage23,24
24 hours of life is most often due to hemorrhage or
isoimmune hemolytic disorder. Bleeding in the newborn Fetomaternal Hemorrhage15-19,21,22
though is often visible and evident, but if it occurs inside
the body—gastrointestinal tract and in the body cavity, The passage of fetal erythrocytes in maternal circulation
may not be recognized and may go unnoticed initially. occurs commonly during pregnancy.
• In 50 percent of pregnancies some fetal cells are passed
High index of suspicion in an anemic neonate without
in maternal circulation at some times during gestation
jaundice and with negative direct Coomb’s test will help in
or during birth process.
suspecting diagnosis of acute hemorrhage.
• In about 8 to 10 percent of pregnancies transplacental
Degree of anemia depends upon whether acute or
blood loss ranges from 0.5 cc to 40 mL of blood.
chronic.
• In about 1 percent of cases the loss may be even greater
as much as 100 mL.
Incidence of Hemorrhage in Newborn12-25 • It may be acute or chronic in nature.
• Twenty-five percent of all infants admitted to neonatal • Fetal hemorrhage may also occur in substances of
intensive care. placenta or may result in retroplacental hemorrhage.
• Five to ten percent of all severe neonatal anemias are More common type of fetomaternal hemorrhage
due to hemorrhage. occurs when infant is held above placenta as during
• One percent of all newborn nursery admissions. cesarean delivery. Anemia has been reported when infant
Anemia due to hemorrhage may occur in utero, during is held above the placenta before clamping the cord. Blood
labor and delivery or after birth. is continuously returned through the umbilical arteries to
the placenta, while hydrostatic pressure prevents continued
venous return to the infant.
Obstetric Causes of Blood Loss12,13,15,16 When infant is held above the introitus the placental
• Abruptio placenta22/placenta previa transfusion is either markedly reduced or completely
• Incision of placenta at cesarean section prevented.
Chapter-8 Anemia in the Newborn 47
In infants born by cesarean section, it is advisable

to keep the baby at least 20 cm below the placenta for
approximately 30 seconds before clamping the cord.
Other causes of fetomaternal hemorrhage

• Diagnostic amniocentesis—10 to 32%.16,17
• When infant is held above placenta during delivery—
(particularly after cesarean section) before cord is
clamped
• Traumatic injury to mother during pregnancy second
ary to
– Vehicular accidents
– Fall
– Abdominal trauma
• Application of fundal pressure during 2nd stage of
labor. Fig. 1 Kleihauer Betke’s test: Dark pink color fetal cells in mother’s
• Maternal toxemia. smear in a case of fetomaternal transfusion
• Erythroblastosis fetalis.
• Placental chorioangioma and choriocarcinoma seen Fetal RBC × 240
Cc of fetal blood =
in 1 percent of placenta. Maternal RBCs

Chronic causes of fetomaternal hemorrhage:18,19 Anemia Or 1 fetal RBC in 1000 maternal RBCs indicates 2 cc of feto-
in fetus occurs slowly if hemorrhage occurs repeatedly maternal hemorrhage.
during course of pregnancy. Fetus tries to compensate Causes of fetoplacental hemorrhage:14-19,21 Multi-lobed
and adjust hemodynamically and infant when born may placenta may be associated with vasa previa—(anomalous
present only with pallor, unexplained anemia and mild vessels crossing the os) vessels may be well compressed
hepatosplenomegaly. Chronic blood loss may lead to iron as well as lacerated during the 2nd stage of labor. The
deficiency anemia with hemoglobin values ranging from prenatal mortality rate range varies 50 to 80 percent.
4 to 6 gm/dL.18,19,22
High index of suspicion in an anemic neonate without Fetofetal hemorrhage (Twin-to-twin transfusion):23,24
jaundice and with negative direct Coomb’s test will help Simultaneous occurrence of anemia in one of the twins
in suspecting diagnosis of acute hemorrhage. Degree of and polycythemia in other should always arouse a
anemia depends upon whether acute or chronic. suspicion of twin-to-twin transfusion. Fetal transfusion is
seen in monozygotic twins with monochorial placentae.
Diagnosis of fetomaternal hemorrhage:16-19 It is Seventy percent of monozygotic twins have monochorial
confirmed by demonstrating the presence of fetal blood in placentae. Fifteen to thirty-three percent of such
maternal circulation by pregnancies have feto-fetal transfusion.
• Kleihauer Betke’s test (Fig. 1)
• Differential hemagglutination
Accidental Incision of Placenta or Umbilical
• Mixed agglutination
• Fluorescent antibody technique. Cord During Cesarean Section
All tests are sensitive and are capable of detecting as Lower segment cesarean section with anterior placenta
little as 0.1 cc of fetal blood in mother’s circulation. can result in direct placental injury. The placenta and
Kleihauer Betke’s test may be false positive in membrane should always be examined for the damage
• Maternal thalassemia minor from the fetal side following cesarean section. Hemoglobin
• Sickle cell anemia of the infant should be estimated at birth and again
• Hereditary persistence of fetal hemoglobinopathy. 12 to 24 hours later. Hemoglobin should also be estimated
If ABO blood group incompatibility is associated in all neonates born to mothers with unusual vaginal
diagnosis can be missed as infant’s A or B cells are rapidly bleeding, placenta previa or abruptio placenta. Anemia
cleared from maternal circulation by maternal anti-A and following obstetric accidents may not be realized if proper
anti-B antibodies. The amount of blood lost in a feto- attempt is not made to examine the placenta and cord in
maternal hemorrhage, can be calculated by the following time before disposing off the placenta and evidence of
formula: cause of anemia may be lost.
Anemia in the newborn may follow hematoma of Therefore anemia in neonates from intensive care
the cord containing large amount of blood.25 Rupture of unit may be caused by frequent blood sampling. It should
umbilical cord, or of aneurysm of cord or aberrant vessels be remembered that removal of 8 to 10 cc of blood from
and due to velamentous insertion. This abnormality is 1500 gm neonate constitute 8 percent of the blood volume
more common in multiple pregnancies and incidence of which is equivalent to about 400 cc blood from the adult.
hemorrhage is seen between 1 and 2 percent. Umbilical
cord anomalies like venous tortuosity or arterial aneurysm Vitamin K deficiency bleeding: Vitamin K is necessary
may lead to bleeding if injured. The condition is 10 times for synthesis of coagulation factors II (prothrombin), VII,
more common in twin than in single term pregnancy. IX and X in the liver. Newborns are relatively vitamin K
Perinatal death rates in such situations are about 50 to 80 deficient. Poor placental transfer of vitamin K, low vitamin
percent. Many are stillborn. K stores at birth, low levels of vitamin K in breast milk, and
sterility of the gut are contributory factors. Bleeding can
Hemorrhage in the postnatal period25 occur from different sites, though GI bleeding is the most
• Unattended precipitous delivery may lead to rupture common manifestation as hematochezia, hematemesis.
of normal umbilical cord. When cord ruptures Hematuria, oozing around the umbilical cord, bleeding
tear generally occurs in fetal third and bleeding is from circumcision, and venipunctures may occasionally
immediate and profuse. Severe bleeding may result in occur. Hematomas at sites of trauma, such as large cephal-
stillbirth; may manifest with severe respiratory distress hematomas and bruising, are also common findings.
and asphyxia. Intracranial bleeding may occasionally occur. Significant
• Hemorrhage may be due to birth trauma resulting in bleeding due to vitamin K deficiency bleeding and other
intracranial bleeding cephalhematoma, subgaleal inherited bleeding disorders may result in severe anemia
hemorrhage, retroperitoneal hemorrhage. in the newborn.
Other common causes
Diagnostic tools for complete blood count (CBC) for
• Slipped ligature.
• Bleeding disorder in the neonate. diagnosing anemia
• Hemorrhagic disease of newborn, now called vitamin K • Manual counting techniques. Not reliable, not
deficiency bleeding. reproducible
• Sepsis with DIC. • Electronic impedance principles/light scanner
• Intrauterine TORCH infections. principles.
• Iatrogenic anemia due to excessive blood sampling.
Various studies have shown that blood withdrawal for Advantages of electronic methods
investigations during first few weeks can range from 5 to
45 percent of total blood volume or 50.5 mL/kg per 28 days • Accurate
period hospitalization. • Reproducible
• 1 mL of blood represents 1 percent of total blood • Number of indices available in a short time.
volume particularly in premature neonates
Peripheral smear examination: In acute hemorrhage,
• Blood lost during the sampling may be estimated by
peripheral smear (PS) may show normochromic,
recording the amount of blood collected in mL and by
normocytic RBCs, whereas in chronic blood loss hypo
weighing the cotton swab on an electronic weighing
chromic, microcytic anemia.
machine, as two drop of blood in the cotton used
to stop bleeding represents 100 m of blood loss. Ten Increased retic count and number of nucleated RBCs
percent of blood loss during sampling for laboratory are seen in both acute and chronic hemorrhage.
monitoring is “hidden” and represents blood on cotton In chronic hemorrhage, the serum iron values are
swabs, in the dead space of syringe or tubing of the decreased and in acute may be normal.
butterfly needle. • Hyperbilirubinemia is differentiating feature in anemia
• Blanchette and Alvin Zupersky7 in their study of 52 of hemolysis versus hemorrhage. However, when there is
premature infants studied during first 6 weeks of life bleeding in tissues or body cavities, hyperbilirubinemia
reported mean blood loss through sampling 22.9 ± 10 may also occur in the latter situation.
mL of packed cells. Forty-six percent of infants studied • Bone marrow aspiration if done may show no stainable
had cumulative losses that exceeded their circulating iron. However, bone marrow aspiration is not indicated
red cell masses at birth. for diagnosis.
CLINICAL FEATURES Presence of the free fluid can be demonstrated by clinical

examination as well as an ultrasound evaluation. Jaundice
Acute Blood Loss may appear due to breakdown of RBCs from these
Clinical features depend on the rapidity of blood loss entrapped hemorrhages.
and amount of loss of blood. Degree of anemia depends Splenic hemorrhage: Splenic hemorrhage should be
upon whether blood loss is acute or chronic. Complete kept in mind in large babies with pallor, abdominal
obstetrical history gives clue to diagnosis. distension, scrotal swelling and radiographic evidence of
In history of vaginal spotting during last trimester or peritoneal effusion following difficult delivery or in babies
prior to delivery, suspect placenta previa. with erythroblastosis fetalis. Umbilical venous pressure is
• Following acute hemorrhage Hb may not drop in decreased rather than increased.
first 6 to 24 hours. Several hours may elapse before Hemorrhage may be subcapsular or free blood may be
profound anemia is documented. Even if Hb is
present in the peritoneal cavity.
initially normal, neonate should be repeatedly
An infant with ruptured liver is generally normal for first
followed up closely during next 12 to 24 hours and
24 to 48 hours and suddenly goes into shock, as hematoma
falling Hb may be noticed after some time due to
ruptures the capsule, causing hemoperitoneum. Mass in
hemodilution that accompanies. If the neonate is in
the right hypochondrium may be palpable.
shock, Hb determination should be performed on
Severe fetal bleeding may result in stillbirth; or may
venous blood as capillary Hb may be misleadingly
manifest with severe respiratory distress and asphyxia
high. The infant looks pale, sluggish, gasping and
in the newborn. It is important to differentiate pallor
with features of circulatory shock. If 20 percent or
resulting from severe anemia from perinatal asphyxia.
more blood is lost acutely, the signs and symptoms
of shock are present.
• Jaundice is absent and bilirubin levels do not increase. ETIOLOGY
• Mother may present with shaking chills as consequence
Anemia Due to Increased RBC Destruction
of transfusion reactions when there is blood group
incompatibility. This may result in stillbirth. (Hemolytic Anemia)
• Examination of placenta and cord should be performed Anemia as a consequence of hemolytic process is common
before it is thrown to ascertain the site of blood loss. in the newborn period. A hemolytic process is defined as a
Traumatic deliveries result in: pathologic shortening of the life span of the red blood cell.
The normal life span of adult RBC is 120 days. However,
• Subdural hemorrhage red cell life survival in term infants may be 60 to 80 days
• Subarachnoid hemorrhage and in 32 to 36 weeks gestation preterm babies cells may
• Cephalhematoma survive only 35 to 70 days. Since destruction of 1 g/dL of
• Blood loss in subaponeurotic area of the scalp. hemoglobin results in production of 35 mg of bilirubin,
Subaponeurotic hemorrhage usually extends hemolytic anemia in the newborn is always associated
throughout the soft tissue of the scalp and covers entire with significant hyperbilirubinemia.
calvaria and this blood loss can lead to exsanguination
and death. Boggy swelling of the head extending from Causes of Hemolytic Anemia of Newborn27-37
frontal region to nape of the neck may be present and
may be associated with the swelling of the eyelids. Hb Immune Hemolysis26-31
may drop as low as 2.2 g/dL at 48 hours of age and
infant may be in shock. It can be estimated that for • Allo/isoimmunization: Rh, ABO, minor group
each centimeter of increase in head circumference • Autoimmune: Autoimmune anemia due to passive
above that expected, 38 mL loss of blood and may also transplacental transfer of maternal antibodies.
develop hyperbilirubinemia.12,13
Nonimmune Hemolysis32-36
Other Hemorrhages12,13,25 • RBC membrane defects32,33 (Hereditary spherocytosis,
elliptocytosis, stomatocytosis, etc.)
Adrenal hemorrhage: Clinical picture including sudden • Enzyme defects (G6PD deficiency, pyruvate kinase
collapse, cyanosis, limpness, irregular respiration, presence deficiency, glucose phosphate isomerase deficiency)34,35
of flank mass accompanied by bluish discolora tion of • Hemoglobinopathies36 (alpha thalassemia/structural
overlying skin should suspect of adrenal hemorrh age. defects); beta thalassemia.
Hemolytic Disease of Newborn in the mother. Hence, the second and the subsequent
pregnancies have a higher chance of being affected.
Hemolytic anemia of newborn should be suspected In severely sensitized pregnancies, fetal marrow cannot
when there is keep up with red cell destruction and extra-medullary
• A rapid fall of hemoglobin concentration in the absence erythropoiesis resulting in hepatosplenomegaly occurs,
of evidence of hemorrhage in early neonatal period nucleated cells are poured into the circulation, giving this
• Evidence of increased red cell production: Reticulocytosis disorder the name “Erythroblastosis Fetalis”.
and increased normoblasts in prognostic signs Though Rh isoimmunization was the most common
• Jaundice during first 24 to 48 hours of life cause of hemolytic anemia in the newborn in the past,
• Abnormal erythrocyte morphology this condition is fast disappearing due to adequate
• Hemoglobinuria immunization of Rh negative mothers with anti-D.
• Positive direct Coomb’s test.
ABO incompatibility:26-31 ABO incompatibility is
common and occurs in approximately in 20 percent of all
Causes of Hemolytic Disease of Newborn pregnancies. In only 1:1000 births, severe disease occurs.
• Isoimmunization, ABO/Rh (minor) blood group ABO incompatibility has a similar pathophysiology but
incompatibility26-31 is a relatively milder disease. Group O mothers have
• Congenital or acquired defects of RBCs32-36 a predilection for producing IgG antibodies against
antigens A and B as against the normally produced IgM
Isoimmunization:26-31 Hemolytic disease of newborn as type. Antibody titers are usually >1:64. This is predilection
a consequence of isoimmunization is caused by passage may run in families. Mothers may be produced as an
of fetal red cells into the maternal circulation where they immune response to A and B antigens contained in food,
stimulate the production of antibodies. Placental transfer bacteria and vaccines. The presence of naturally occurring
of these maternal antibodies directed against fetal red cell antibodies in the maternal serum explains the frequent
antigens is the most common cause of neonatal hemolysis. occurrence of ABO hemolytic disease in first born infants
These antibodies of IgG class return to fetal circulation, in contrast to Rh D disease in subsequent gestations.
attach to the antigenic site on the fetal red cells leading to • A direct Coomb’s test usually positive in Rh
hemolysis of these cells. incompatibility and may be weakly positive in ABO
In this group of disorders, fetal and/or neonatal red incompatibility.
blood cell hemolysis results from the presence of maternal
• Peripheral blood smear shows polychromasia,
antibodies to the red cell antigens in fetal circulation.
increased number of erythroblasts.
Rh isoimmunization: When an Rh negative mother • In ABO incompatibility increased number of micro-
conceives an Rh positive baby, fetal red cells may cross spherocytosis may be seen on peripheral blood smear
the placenta and sensitize the maternal immune system. examination.
On subsequent exposure to Rh positive cells across • Laboratory investigations will reveal blood group
the placenta, the antibodies produced by the maternal incompatibility.
immune system may cross the placenta and cause • Levels of IgG, anti-A, or anti-B antibodies in the
hemolysis of fetal red cells. As few as 0.2 mL of fetal cells mothers of babies with ABO hemolytic disease are
are sufficient to cause maternal anti-D sensitization.26,27,29 significantly higher than in the mother whose infants
The initial IgM response is slow and weak. As IgM does do not have the disease increased.
not cross placenta, no fetal effects are seen. On second • Alloimmunization due to Minor Group Incompatibi
exposure to RhD, IgG antibodies formed readily cross lity.29,31 Alloimmunization can occur due to a variety of
the placenta, into fetal circulation and binds to the RhD other fetal red blood cell antigens.
antigen on fetal RBC membrane. Antibody coated fetal • Rh blood group system, cc, ee, ec, ce, of the among
red cells adhere to macrophages and lead to eventual the antigens Rh group alloimmunization with E and C
destruction of the cell. This hemolytic process can take occurs most frequently after anti-D sensitization
place starting in utero, resulting in marked compensatory • The principal antibodies found are anti-E, anti-C, anti-
over production of nucleated red cells. During pregnancy Kell, Duffy, Kidd, Fu, MNSs, etc.
fetomaternal transfusion may occur spontaneously in Alloimmunization to the accounts for up to 10 percent
about 7 percent of cases in the first trimester,16 percent of severely affected fetuses. As the Kell antigen is expressed
in the second trimester and 29 percent in the third on the surface of erythroid progenitors, whereas D antigen
trimester and in as much as 50 percent in the peripartum is not, Kell sensitization in addition to causing hemolysis,
period, leading to the formation of anti-D IgG antibodies results in suppression of erythropoiesis.
While in utero, excess of bilirubin produced by toxoplasmosis, mycoplasma pneumoniae, bacterial

hemolysis is removed by the placenta hence at the time of and viral infections may cause hemolytic anemia.
birth, child may not be jaundiced. Jaundice usually appears • IgM antibodies can cause disease and usually are
in the first 24 to 48 hours of life as neonatal immature active between 0 and 30 degrees celsius, hence are
liver is not in a position to conjugate excessive bilirubin referred to as cold agglutinins. These antibodies with
load. Significant hyperbilirubinemia is evident. It may be complement coat RBCs and cause hemolysis.
accompanied by anemia and hepatosplenomegaly. • Rapid onset of anemia, hyperbilirubinemia with
splenomegaly and hepatomegaly occur. Occasionally a
severe anemia requires treatment with corticosteroids,
Hemolytic disease due to minor blood group incompatibility
should be suspected when Coomb’s test is positive and there IV IgG or and immunosuppressive agent.31
is no evidence of major blood group incompatibility in mother
and child. The principal antibodies found are anti-E, anti-C,
Management of affected fetus
anti-Kell, Duffy, Kidd, Fu MNS systems. • Antibodies from the mother cross the placenta
and result in fetal red cell hemolysis. Presence of
Management:28,29 Management of severely affected iso autoimmune hemolytic anemia in mother may result
immunized fetus consists of early delivery with or without in hemolytic anemia in the newborn infant. Anemia
fetal transfusions depending upon gestation of fetus. may be associated with bacterial and viral infections
Management of neonate depends on degree of hemolysis in the newborn period and is often associated with
and level of indirect hyperbilirubinemia. jaundice (both conjugated and unconjugated fraction)
All Rh negative mothers with Rh positive fetus, and hepatosplenomegaly.
should be given Rh immunoglobulin at 28 to 30 weeks • Intrauterine, viral infections like congenital CMV,
of gestations and within 72 hours after delivery and after toxoplasmosis, congenital syphilis, herpes, all may
spontaneous or therapeutic abortion. Hyperbilirubinemia be associated with hemolytic anemia in the neonatal
must be aggressively treated to prevent kernicterus with period.
phototherapy, exchange transfusion. Whenever required
top-up transfusion with packed red blood cells may be Nonimmune hemolytic anemia in the
given for symptomatic anemia. newborn may occur due to
Autoimmune hemolytic anemia Conditions associated with defects of

• Red cell membrane32,33
• Anemia may be due to ‘warm’ or ‘cold’ antibodies • Red cell metabolism34,35
which signify the temperature at which antibodies • Hemoglobin synthesis37
become active. The rare combination of autoimmune • Red cell under production38-43
hemolytic anemia (AIHA) and pregnancy carries great Other causes include
risks to both the woman herself and the fetus. • Disseminated intravascular coagulation
• The degree of hemolysis in the fetus depends mainly • Congenital infections.
on the amount and avidity of the transferred antibody
Abnormalities of red cell membrane
for the fetal red cells. Fifty percent of women with this
condition are reported to improve during pregnancy,
especially in the third trimester. The diagnosis rests on Hereditary spherocytosis (Fig. 2)32,33
the demonstration of auto-antibodies directed against In approximately 50 percent of patients with hereditary
red cell surface antigens. In practice, this is detected by a spherocytosis, history of neonatal jaundice can be obtained and
positive direct antiglobulin Coombs’ test. Most of these may be of a sufficient magnitude so as to need phototherapy
and exchange transfusion and may lead to kernicterus if left
antibodies detected at 37°C are of the IgG subclass.
untreated. A family history of chronic anemia, splenectomy,
• Administration of prednisone, 2 mg/kg/day, to
cholecystectomy, pain in abdomen, unhealed ulcers may be
the mother with prenatally active AIHA may both present. Physical examination of parents may reveal mild to
reduce maternal hemolysis and reduce neonatal moderate splenomegaly.
morbidity. Management is to monitor carefully for Examination of peripheral blood smear (Fig. 3) reveals
hemolysis, hyperbilirubinemia, jaundice, to prevent characteristic microspherocytes and the osmotic fragility of
kernicterus.28,29 erythrocytes may be increased.
• Acquired hemolytic anemias in the newborn can Spherocytes however may be seen in the newborn period in
be caused by infection, drugs or toxins. Congenital other conditions like ABO incompatibility, septicemia, red cell
infections like syphilis, cytomegalovirus, rubella, 20 enzyme deficiency, etc.
Defects of red cell metabolism include Vit E, newborn infants with G6PD deficiency are at a
greater risk of developing hemolytic anemia than are
G6PD (Glucose 6 phosphate dehydrogenase) defi
adults. This particularly is the case in more severe types
ciency:34,35
affecting Asians and Mediterranean group. In this group
With G6PD enzyme deficiency, oxidation of membrane
of patients, jaundice in the newborn period may be severe
protein leads to precipitation of denatured hemoglobin
leading to kernicterus. Jaundice that occurs, appears to be
(Heinz bodies) thus shortening the RBC life span.
due to accentuation of physiologic jaundice of newborn,
• X-linked recessive disorder. History of anemia,
although jaundice may appear in some during first 24
jaundice may be elicited in maternal cousins, maternal
hours.
uncles, maternal grandfather and grand uncles.
• Abnormal RBC morphology with evidence of “Heinz
• Hyperbilirubinemia in G6PD deficient males can be
bodies” in the RBC (Fig. 4).
very severe.
• Intravascular hemolysis may be associated.
• In India, G6PD deficiency is most commonly seen
• Normal G6PD levels during acute hemolysis may not
in Parsis, Bhanushalis, Sindhis, Punjabi, Khoja
rule out G6PD deficiency as younger RBCs contain
communities.
high level of enzymes. Hence, the test may have to be
Because of the diminished capacity of neonatal RBCs
repeated after 6 weeks.
to deal with oxidative stress, as a result of lower glutathione
peroxidase, catalases as well as relative deficiency of Pyruvate kinase deficiency:35 The other common red
cell enzyme deficiency leading to hemolytic anemia in
newborn during first week of life is “pyruvate kinase
deficiency”.35 This disorder is generally characterized by
evidence of hemolysis with increased retic count, few or
no spherocytes on the peripheral smear, no blood group
incompatibility and a negative Coomb’s test. In some
instances abnormal RBC morphology with evidence of
Heinz bodies, intravascular hemolysis may be associated.
Furthermore, there is a tendency for jaundice to occur
more frequently in particular families and communities,
indicating that the genetic and environmental factors
may influence the presentation. In this group of patients,
jaundice may be severe leading to kernicterus.
Characterized by
• Evidence of hemolysis
• Increased retic count
Fig. 2 Hereditary spherocytosis showing jaundice in • Few or no spherocytes on the peripheral smear
mother and child
Fig. 3 Spherocytes on peripheral smear Fig. 4 Heinz bodies in RBCs in G6PD deficiency
• No blood group incompatibility epicanthal folds, hypertelorism, ptosis, short or

• Negative Coomb’s test webbed neck, congenital heart diseases and short
• Enzyme analysis is not easily available stature.
• It may need treatment like phototherapy/exchange Infections that cause anemia due to reduced red cell
transfusion. production include parvovirus B1, CMV, toxoplasmosis,
congenital syphilis, rubella, herpes simplex. Maternal
Hemoglobinopathies: Alpha thalassemia syndrome.36 infection with parvovirus B19 causes fetal anemia which is
Of the 4 varieties of alpha thalassemia the homozygous severe enough to lead to intrauterine death due to hydrops
alpha thalassemia which results from the absence of In addition to anemia, parvovirus infections cause marked
all four genetic loci for alpha chain synthesis, presents reticulocytopenia. Thrombocytopenia may also occur.41
in the neonatal period. In the absence of alpha chains,
Laboratory abnormalities include49
cord blood contains Bart’s hemoglobin (Gamma 4) and
Hemoglobin H (Beta 4). Most affected infants are stillborn, • Macrocytic anemia
although some may live for a few hours after birth. These • Absent/reduced reticulocytes
infants are hydropic at birth and thus are similar in • Elevated HbF
appearance to neonates with severe erythroblastosis due • Absence of erythroid precursors in the bone marrow
to Rh incompatibility. • Erythroid—myeloid ratio ranges from 1:6 to 1: 240
• Pancytopenia, accompanied by reticulocytopenia,
Gamma thalassemia syndrome: The production of
leukopenia and thrombocytopenia may be seen in
gamma polypeptides is regulated by four genes. The
severe septicemia and TORCH group of infections.
complete absence of gamma chains is incompatible with
Transplacental transmission of parvovirus B19 cause
fetal life. Intermediate reduction of gamma-polypeptide
hypoplastic anemia, which when severe may lead to
synthesis may produce a mild to moderate neonatal
intrauterine death. Those that survive intrauterine
anemia characterized by a reduced percentage of fetal
infection may be born with hydrops fetalis.
hemoglobin. This type of anemia resolves when significant
Osteopetrosis/marble bone disease may present with
beta chain synthesis begins. It is important to note that
anemia in the newborn period, which could be due to
beta thalassemia does not present in the neonatal period.
hemolysis or non-production. The disease is associated
Other unusual causes of neonatal anemia include
with hydrocephalus, hepatosplenomegaly and marked
congenital dyserythropoietic anemia, leukemia, microan
increase in the density of the bones particularly of long
giopathic, hemolytic anemia, DIC, etc.
bones, ribs and base of the skull.
Anemia due to red cell under production:37-42 Impaired • Other rare causes of anemia include transcobalamin
red cell production is an unusual cause of anemia in II Fanconi’s anemia usually does not manifest in the
the newborn period. Congenital pure red cell anemia newborn period and often presents with anemia
(Diamond Blackfan syndrome) is an uncommon disorder around the age of 5 to 8 years.
in which red cell precursors in the bone marrow are
markedly reduced or virtually absent while white blood MANAGEMENT OF NEONATAL ANEMIA49
cell and platelet production remains normal. The treatment of a neonate with anemia due to blood loss
It occurs either due to a lack of erythroid stem cells or depends on the degree of hypovolemia or anemia. Whether
immune suppression of stem cell differentiation. the blood loss has been acute or chronic. Baby born pale at
• Inheritance seems to follow an autosomal recessive birth should be differentiated from an asphyxiated baby.
inheritance. The disorder should be suspected in any Besides transfusion for hemorrhagic shock, adoption of
newborn with anemia and reticulocytopenia, normal transfusion protocols take a variety of factors into account,
platelets and leukocytes. including hemoglobin levels, degree of cardiorespiratory
• Musculoskeletal abnormalities—triphalangeal thumbs disease and traditional signs and symptoms of pathologic
may occur in a third of patients. The diagnosis anemia (Flow chart 1).
is confirmed by examination of bone marrow
aspirate which reveals a virtual absence of erythroid
Anemia with Shock
precursors. Mothers of affected children may have
increased incidence of miscarriages/abortion and Pale babies usually will have tachycardia with minimal or
premature birth. Associated physical abnormalities no cyanosis, decreased central venous pressure (CVP) and
are triphalangeal or duplicated thumb, cleft palate, a rapid fall in hemoglobin with circulatory collapse.
Flow chart 1 Diagnostic approach to neonatal anemia49
Abbreviations: DIC: Disseminated intravascular coagulation; G6PD: Glucose 6-phosphate dehydrogenase deficiency;
Hb: Hemoglobin; MCV: Mean corpuscular volume.
Guidelines for management In severely anemic babies with CHF, a partial exchange
transfusion with packed red cells may be carried out to
• Clear the airway
reduce circulatory overload.
• Administer oxygen and intubate if necessary
• Insert the catheter in the umbilical vein and measure
CVP Anemia due to Chronic
• Obtain blood specimen for investigation Hemorrhage/Hemolysis
• Rapid expansion of the vascular space with 20 mL/kg
of isotonic saline or ringer’s lactate, followed by either • Iron therapy is initiated for the neonates with anemia
type specific cross matched whole blood or packed red who are stable without any signs and symptoms of
cells resuspended with saline. failure or hyperbilirubinemia even though Hb is low
In infants with severe anemia or hypoxia, O, Rh negative and do not require blood transfusion.
RBCs are an acceptable alternative. • Phototherapy and double volume exchange
• In infants with anemia with congestive heart failure. transfusion for hyperbilirubinemia followed by a top
Furosemide 1 mg/kg followed by a packed cell up packed cell transfusion if required is the main stay
transfusion of 10 to 15 cc/kg may be given (3 mL/kg of treatment for neonates with isoimmune hemolytic
of packed cells or 6 mL/kg of whole blood rises the anemia depending upon levels of bilirubin and Hb
hemoglobin by 1 g/dL). levels and day of life (age).
• In severely anemic babies with CHF, a partial exchange 3. Bifano EM, Ehrenkranz Z. Perinatal hematology. Clin
transfusion with packed red cells may be carried out to Perinatol. 1995;23(3).
reduce circulatory overload. 4. Lokeshwar MR, Dalal R, Manglani M, Shah N. Anemia in
• Even though neonate has received the blood newborn. Ind J Pediatr. 1998;65:651-61.
transfusion, iron therapy is also needed because the 5. Dochain J, Lamaje L, Arabrechts L. Cited in Oski and
transfusions are not sufficient to totally replace the Nathan normal blood values in new born period
Hematologic problems in new born. WB saunders,
iron lost due to hemorrhage.
Philadelphia. 1982;7:3.
• Elemental iron in the dose of 2 mg/kg body weight
6. Stockman JA III, Oski FA. Physiological anemia of infancy
daily for three months is required to replenish iron
and the anemia of prematurity. Clin Hematol. 1978;7:3.
stores and return the hemoglobin to normal. 7. Blanchette VS, Zipursky A. Assessment of anemia in
• Recombinant erythropoietin treatment:43-48 Premature newborn infants. Symposium on perinatal hematology. In:
infants respond to exogenously administered Clinics in Perinatology, WB Saunders & Co, Philadelphia.
recombinant human EPO with reticulocytosis, modest 1984;11:489-510.
decreases in the frequency of PRBC transfusions have 8. Walker JL, Turnbull EPN. Hemoglobin and red cell in
been documented primarily in premature infants. human foetus and their relation to the oxygen content
The cost-benefit ratio for EPO has yet to be clearly of the blood in the vessels of umbilical cord. Lancet.
established. 1953;2:312.
• Babies on erythropoietin therapy should receive oral 9. Wardrop CAJ, Holland BM, Veale KEA, et al. Non-
iron therapy. physiologic anemia of prematurity. Arch Dis Child.
• Provision of adequate amounts of vitamin E, vitamin 1978;53:855.
B12, folate, and iron are important in the management 10. Worthington-white DA, Behnke M, Gross S. Premature
of anemia in premature infant.10,11 infants require additional folate and vitamin B12 to reduce
the severity of the anaemia of prematurity. Am J Clin Nutri.
• Diamond Blackfan syndrome can be managed by
1994;60:930.
corticosteroids and repeated blood transfusions and
11. Oski FA, Barness LA. Vitamin E deficiency: A previously
supportive line of treatment.38-42
unrecognized cause of hemolytic anaemia in the
• Anemia of transcobalamin II deficiency requires premature infant. J Pediatr. 1967;70:211.
weekly intramuscular injections of 100 micro grams of 12. Kirkman HN, Riley HD Jr. Posthemorrhagic anemia and
vitamin B12. shock in the newborn. A review. Pediatrics. 1959;24:97.
13. Norak F. Post hemorrhagic shock in newborns during
PREVENTION OF ANEMIA labor and after delivery. Acta Med Iugosl. 1953;7:280.
Newborn, particularly in premature infants reducing 14. Usher R, Shepard M, Lind J. The blood volume of newborn
the amount of blood taken for investigation purposes infant and placental transfusion. Aeta Paediatric Scand.
1963;52:497-512.
diminishes the need to replace blood. The use of
15. Golditch LM, Boyce NE. Management of abruptio
noninvasive monitoring devices, such as transcutaneous
placentae. JAMA. 1970;21:288.
oxygen saturation, partial pressure of oxygen, and partial
16. Chown B, Mclarey DC, Fish SA. Anemia from bleeding of
pressure of carbon dioxide, may allow decreased blood
the fetus into the maternal circulation. Lancet. 1954;1:219-
drawing. 20.
Iron prophylaxis to adolescent girls, antenatal iron 17. Woo Wang MYF, mccutcheon E, and Desforges JF. Feto-
administration during pregnancy, oral iron therapy 2 to 6 maternal hemorrhage from diagnostic transabdominal
mg/kg/day in preterm babies, starting by 4 to 6 weeks and amniocentesis. Am J Obstet Gynaecol. 1967;97:1123.
continued till weaning has been adequately achieved is an 18. Sims DG, Barron SL, Waldehra V, et al. Massive chronic
important preventive measure to prevent anemia in new feto-maternal bleeding associated with placental
born period and early infancy. chorioangiomas. Acta Paediatr Scand. 1976;65:271.
19. Blackburn GK. Massive feto-maternal hemorrhage due to
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C H A P T E R 9
Polycythemia and
Hyperviscosity Syndrome
Polycythemia is defined as an abnormal increase in the red blood cell mass. In neonates the hematocrit rises soon after birth,
peaks at around 2 hours of age and falls to about 57 percent in next 12 to 18 hours of age. The most widely accepted definition of
neonatal polycythemia is a venous hematocrit greater than 65 percent (capillary hematocrit >70 percent) or a venous hemoglobin
concentration in excess of 22.0 g/dL.1, 2 This cut off value has been chosen based on the observation that blood viscosity exponentially
increases above a hematocrit of 65 percent.3 Other definitions of polycythemia include a venous hematocrit of 64 percent or more at
2 hours of age,4 or an umbilical venous or arterial hematocrit of 63 percent or more.5
Hyperviscosity is defined as thickness of the blood greater than 14.6 centipoise at a shear rate of 11.5 sec–1. The normal blood
viscosity (mean ± SD) in the neonate is 1.18 ± 0.17 centipoise at a shear rate of 11.5 sec–1.2-6 Hyperviscosity and polycythemia have a
linear relationship up till a hematocrit of 60 percent. Hyperviscosity starts rising exponentially after a hematocrit of 65 percent and
markedly increases at a hematocrit of 70 percent or more.7 Hyperviscosity is influenced not only by the red cell mass, but also by other
factors such as plasma fibrinogen and local blood flow.
The terms polycythemia and hyperviscosity are often INCIDENCE OF NEONATAL POLYCYTHEMIA
used interchangeably although they are not equivalent as AND HYPERVISCOSITY
is evident from the description. Polycythemia is significant
only because it increases the risk of hyperviscosity The true incidence of polycythemia and hyperviscosity
syndrome. Hyperviscosity syndrome comprises symp is not known since majority of infants are likely to be
toms and signs caused by tardy flow and sludging of blood asymptomatic normal newborns. Hematocrit is not
within the blood vessels especially in the smaller arterioles routinely recommended or drawn in this population, most
and capillaries. Sludging of blood occurs because likely due to the controversy surrounding the treatment
increased red blood cell mass causes a relative decrease in of asymptomatic infant.8 The reported incidence of
the plasma volume and a relative increase in the proteins polycythemia ranges between 0.4 and 12 percent; in
and platelets. The sludging of blood within blood vessels USA 1 to 5 percent newborns reportedly suffer from
can also lead to thrombosis and infarcts in the territory polycythemia.2,5,7,9 This wide variation may be due to
supplied by them. different screening techniques, sampling sites (capillary
Viscosity is difficult to measure. It is usually measured versus peripheral, central venous or arterial), varied
by Wells-Brookfield cone-plate microviscometer. Since patient population, mode of delivery, method of measuring
instruments to measure viscosity are not readily available (Coulter counter or centrifuged capillary blood), and
in most neonatal intensive care units, hyperviscosity is sampling time. Sampling time is the most important source
usually suspected on the basis of clinical symptoms and of this variation. The hematocrit normally rises after birth,
signs in the presence of abnormally high hematocrit reaching a peak at two hours postpartum and then slowly
(polycythemia). decreases over the next 12 hours. At two hours of life the
upper limit (2 SD) of a normal capillary hematocrit is Microthrombi formation may occur in the blood vessels
71 percent while it is 64 percent for a venous hematocrit.5 supplying cerebral cortex, kidneys, intestines and adrenal
Hyperviscosity occurs in 6.7 percent of neonates. glands, with potential long lasting sequelae.
• Only 47 percent of infants with polycythemia
exhibit hyperviscosity and 24 percent infant with INCREASED FETAL ERYTHROPOIESIS
hyperviscosity have polycythemia.4,10,11 (PRIMARY POLYCYTHEMIA)
• It is more common in infants.
• Small-for-gestational age (SGA). Neonatal polycythemia can occur by two mechanisms.
• Large-for-gestational age (LGA). 1. Active form: Fetus/newborn produces excess number
• Infants born to diabetic mothers have predilection of red blood cells due to physiological triggers like
for developing polycythemia. Thirty percent and forty perinatal hypoxia, uncontrolled maternal diabetes and
percent infants suffer from polycythemia if mothers chromosomal defects in the fetus. It is also referred as
have diabetes or suffer from gestational diabetes primary polycythemia.
respectively. 2. Passive form: Fetus/newborn can acquire large
blood volume as a result of delayed cord clamping or
PATHOPHYSIOLOGY maternofetal transfusion. Increase in the red blood
cells by hypertransfusion is also called secondary
The mean venous hematocrit in term infants is 52 percent polycythemia.
at 12 to 18 hours of age5 though values up to 71 percent The causes of neonatal polycythemia can be divided
have also been described as normal at 2 hours of age by according to temporal happenings like:
many authors.12,13 • Before birth (antenatal)
The initial rise in the hematocrit is related to • During birth (intranatal)
transudation of fluid out of the intravascular space. As the • After birth (neonatal).
venous hematocrit increases, the viscosity rises. When These are summarized in Table 1.
the hematocrit increases to more than 65 percent, there is It can be secondary to pre-eclampsia, primary
exponential elevation in the blood viscosity that, coupled renovascular disease, chronic or recurrent abruptio
with decreased deformability of the fetal erythrocytes, placentae, maternal cyanotic congenital heart disease
in comparison to adult red blood cells, leads to sluggish including Eisenmenger syndrome14 postdated pregnancy
peripheral circulation, formation of microthrombi, fall in and maternal smoking. Awonusonu et al. (2002) have
the oxygen transport and tissue hypoxia (Flow chart 1). reported that the risk of symptomatic polycythemia
Tissue hypoxia leads to increased glucose metabolism, requiring partial exchange blood transfusion is 2.5 times
to generate adequate amounts of ATP by anaerobic respi more in mothers who smoke (1.59%) during pregnancy
ration, hypoglycemia and metabolic acidosis. Hypoxia than those who do not (0.64%).15 Most of these maternal
and acidosis further aggravate hyperviscosity. Increased conditions may also be associated with intrauterine
viscosity of blood, in general, mimics symptoms and growth restriction (IUGR).
signs of hypoperfusion. Polycythemia, therefore, creates The incidence of polycythemia increases with
a pathophysiological situation analogous to shock. increasing severity of fetal growth retardation. In severely
growth retarded fetuses, a hematological syndrome
Flow chart 1 Pathophysiology of polycythemia-hyperviscosity of polycythemia, thrombocytopenia, leukopenia and
increased numbers of nucleated red blood cells has been
described.16,17
• Endocrine abnormalities: Conditions associated with
increased fetal oxygen consumption may result in fetal
hypoxia and subsequent polycythemia like congenital
thyrotoxicosis and Beckwith-Wiedemann syndrome or
infants of diabetic mother (IDM) with poor glycemic
control. Polycythemia in IDMs correlates with
macrosomia and neonatal hypoglycemia.
• Chromosomal disorders: Trisomy 13, trisomy 18 and
trisomy 21 may be associated with polycythemia. The
exact mechanism is not well understood. However,
it has been observed that in trisomy 13, 18 and
21, placentae show trophoblastic hypoplasia and
Chapter-9 Polycythemia and Hyperviscosity Syndrome 59
Table 1 Etiology of polycythemia hypovascularity, which has been attributed to low

levels of vascular endothelial growth factor (VEGF)
Antenatal Intranatal Neonatal
and placental growth factor (PLGF).18,19 VEGF and
Hypoxia Hypoxia Genetic causes
• Trisomy13, 18, 21
PLGF are considered to play important roles in angio
• Beckwith- genesis and vascular permeability during placental
Wiedemann development. Therefore, low levels of these factors
syndrome could cause chronic fetal hypoxia, resulting in inc
IUGR, SGA infants Intrapartum Endocrine causes reased erythropoietin levels and polycythemia.20
asphyxia due to • Infants of diabetic • High altitude: Babies born at high altitude are found to
etiologies such as mothers have polycythemia due to relative paucity of oxygen.
• Obstructed labor • Neonatal
• Prolonged labor thyrotoxicosis
HYPERTRANSFUSION (SECONDARY
• Abruptio • Congenital
placentae hypothyroidism POLYCYTHEMIA)
• Congenital • Delayed cord clamping: It allows increased blood
adrenal volume to be delivered to the infant. When cord
hyperplasia
clamping is delayed to more than 3 minutes after
Infants of diabetic Delayed clamping
birth, neonatal blood volume increases by 30 percent.
mothers of umbilical cord
Gravity may facilitate transfer of large blood volume to
Placental Perinatal asphyxia
the newborn because of the position of the delivered
insufficiency
infant in relation to the maternal introitus before cord
Pre-eclampsia Hypertransfusions
clamping. Oxytocin may enhance blood flow to the
Maternal smoking Oxytocin use during
labor infant via umbilical vessels in the event of delayed cord
High altitude Holding the baby clamping.
below the introitus • Twin-to-twin transfusion syndrome: It occurs in
at the time of approximately 10 percent of monozygotic twin pregnan
delivery cies due to a vascular communications between twin
Postmaturity Milking of the babies. The recipient twin suffers from polycythemia
umbilical cord and hypervolemia.
Infants born • Maternofetal transfusion: It has been recognized as
to mothers a cause of blood transfer from the uterine vessels via
with chronic placenta to the fetal circulation due to placental vascular
cardiopulmonary malformations leading to hypervolemia, polycythemia
conditions and hyperviscosity syndrome in the newborn infant.21
Hypertransfusions • Intrapartum asphyxia: Perinatal asphyxia during
Primary delivery, due to any cause, may shift blood volume from
renovascular the placenta to the fetus to maintain cerebral perfusion.
disease This may lead to increased blood volume in the perinate
In utero asphyxia followed by polycythemia and hyperviscosity.
Twin-to-twin
transfusion
Maternofetal CLINICAL FEATURES
transfusions
Majority of appropriately grown term polycythemic
Genetic syndromes
newborn infants have no symptoms, particularly if the
Trisomy
Beckwith-
polycythemia is found on routine neonatal screening.
Wiedemann Symptoms, when present, are usually attributable to
syndrome hyperviscosity and poor tissue perfusion or to associated
Miscellaneous metabolic derangements such as hypoglycemia. About
congenital 50 percent of polycythemic infants develop one or more
hypothyroidism, symptoms. Clinically the baby may manifest skin color
Congenital adrenal from red (polycythemia), blue (cyanosis resulting from
hyperplasia peripheral stasis), yellow (jaundice due to breakdown of
Maternal use of large amount of RBCs; 34.5 mg bilirubin is produced from
propranolol 1g of hemoglobin) to pale when shock sets in.
• Common early symptoms include plethora, lethargy, infants or presence of risk factors (enlisted in Table 1)
hypotonia, poor suck and feeding, and tremulous should be screened for polycythemia.2
ness. Serious complications include cardiorespira • Screening is usually done in high-risk neonates at 2
tory distress (with or without congestive heart failure), hours of age. A normal value at 2 hours of age (venous
seizures, peripheral gangrene, necrotizing enterocolit hematocrit <65%) does not warrant further screening
is, renal failure (occasionally resulting from renal vein unless the neonate is symptomatic.
thrombosis), hyperbilirubinemia and priapism. Most • Hematocrit values >65 percent at 2 hours of age merit
of these symptoms are non-specific and may be relat repeat screening at 12 and 24 hours.
ed to the underlying causes rather than polycythemia • Polycythemia is diagnosed when venous hematocrit is
per se. >65 percent.2
• Central nervous system: It is the most common system • In the presence of clinical features suggestive of
to be affected. Early effects include lethargy, poor poor circulation with plethora and/or cyanosis and
feeding, easy startle, hypotonia, difficult arousal, tachypnea, a venous hematocrit measurement is used
tremors, irritability, jitteriness and seizures. Long term as a surrogate for diagnosing hyperviscosity because
sequelae include developmental delay and poor fine former can be readily done and the latter exponentially
motor control. increases after a hematocrit of >65 percent.
• Metabolic derangement: Hypoglycemia is the most • Capillary or venous hematocrit—which one to
common metabolic abnormality in the infant, reagent measure? Capillary hematocrit measurements depend
glucose strips frequently give falsely low values upon the blood flow and are significantly higher than
Therefore, blood sugar should always be reconfirmed by venous hematocrits and are therefore, unreliable.
a laboratory test. Hypocalcemia and hypomagnesemia However, capillary samples may be used for screening
are also known to occur in polycythemia. The elevated but all high values should be confirmed by a venous
red blood cell mass increases catabolism of the hematocrit for the diagnosis of polycythemia.
hemoglobin so hyperbilirubinemia is common and
even gall stones occasionally occur. Hematocrit Measurement
• Cardiopulmonary system: Tachycardia, tachypnea,
congestive cardiac failure and cyanosis may be found. Two methods are available:
Rarely persistent fetal circulation may develop with 1. Automated hematology analyzer: This calculates hem
poor prognosis. Polycythemia should be considered atocrit from a direct measurement of mean cell volume
as a differential diagnosis for transient tachypnea and the hemoglobin. Hematocrit (%) is approximately
of the newborn. Chest radiography may reveal three times the hemoglobin concentration in g/dL.
cardiomegaly, pulmonary plethora, hyperaeration and 2. Microcentrifuge: Blood is collected in heparinized
pleural effusion. Echocardiographic findings include microcapillaries (110 mm length and 1–2 mm internal
increased pulmonary resistance, bidirectional shunt diameter) and centrifuged at 10,000 to 15,000 revolu
and decreased cardiac output. tions per minute (rpm) for 3 to 5 minutes. Plasma
• Gastrointestinal system: Features of gastrointestinal separates and the packed cell volume is measured
affliction are poor suckling, vomiting, feed intolerance, to give the hematocrit. An automated analyzer gives
prefeed aspirates, abdominal distension, paralytic lower values as compared to hematocrits measured
ileus and necrotizing enterocolitis (NEC). by the centrifugation methods. Most of the reported
• Renal system: Hyperviscosity affects renal perfusion. literature on polycythemia is based on centrifuged
Oliguria, acute renal failure, renal vein thrombosis and hematocrits.8
decreased urinary sodium may occur in polycythemic Other laboratory tests to be done in a case of
infants. polycythemia:
• Miscellaneous: Thrombocytopenia, coagulation defects, • Kidney function tests: Renal functions should always
stroke, peripheral gangrene, thrombosis, priapism and be evaluated in a case of symptomatic polycythemia.
testicular infarction are well known complications of Blood urea (BUN) and serum creatinine may increase.
polycythemia. There may be dilutional hyponatremia and serum
potassium may rise. Judicious fluid and electrolytic
intake is warranted for good prognosis.
Laboratory Diagnosis
• Serum glucose and calcium levels should be deter
• Certain high-risk groups such as small for gestational mined in all symptomatic polycythemia and infants
age (SGA) infants, infants of diabetic mothers (IDMs), and vigorously treated if the patient has abnormal
monochorionic twins, large for gestational age (LGA) levels.
• Serum bilirubin rises rapidly in babies who have 20 mL/kg may be added to the daily requirements.2
polycythemia due to increased RBC destruction Extra fluid intake may be ensured either by enteral route
much beyond the neonatal hepatic conjugation (supervised feeding) or by parenteral route (IV fluids).
capacity. Serum bilirubin must be checked serially. If The rationale for this therapy is hemodilution and
nomograms are available then transcutaneous biliru resultant decrease in viscosity. However, liberal and
binometry can be a very useful modality to screen for extra fluid therapy may be associated with problems
hyperbilirubinemia as it is a noninvasive technique. especially in preterm babies. Hence conservative
However, when transcutaneous bilirubin index management by using extra fluids should be reserved
suggests a higher serum bilirubin level, it should always for hemodynamically stable babies with asymptomatic
be confirmed by laboratory method before instituting polycythemia.
therapy for hyperbilirubinemia. • Partial exchange transfusion: Partial exchange trans
• Arterial blood gases (ABG): Consider measuring ABG fusion (PET) is traditionally used as the method of
values to assess oxygenation in the symptomatic infant choice for the treatment of symptomatic polycythemia
with respiratory distress and cyanosis. to lower hematocrit as well as hyperviscosity. This
• Platelet count: Platelet count must be checked at base method is also employed to treat asymptomatic
line. Thrombocytopenia is present if thrombosis or polycythemia if hematocrit is >75 percent. PET aims to
disseminated intravascular coagulation (DIC) has set decrease the hematocrit to a target packed cell volume
in. Thrombocytopenia may also be found in babies of 55 percent. PET is performed with either crystalloid
born to pre-eclamptic mothers, who are prone to (normal saline or Ringer’s lactate) or colloid (5%
develop symptomatic polycythemia. albumin or fresh frozen plasma) solutions. Crystalloids
• Urinalysis: Proteinuria and casts may be present. are preferred because they are economical, easily
available, produce similar reduction in the hematocrit
as colloids22,23 and do not have the risk of transfusion
MANAGEMENT
associated infections (e.g. HIV, hepatitis B, hepatitis C,
• Possible ways of avoiding polycythemia include early CMV). Additionally, adult plasma may potentially
cord clamping and holding the baby at the level increase the blood viscosity when mixed with fetal
of the introitus at the time of delivery to minimize erythrocytes.
hypertransfusion. The blood volume to be partially exchanged is calculated
• A good glycemic control and management of by the following formula:
growth retardation in the antenatal period may Volume to be exchanged (V mL) = infant’s blood
prevent development of polycythemia and in turn volume × (observed hematocrit—desired hematocrit)/
hyperviscosity after birth. observed hematocrit.
• It is essential to exclude dehydration before a diagnosis The desired hematocrit is kept as 55 percent. Blood
of polycythemia is made. A clue to dehydration could volume is estimated to be 80 to 90 mL/kg in term and
be excessive weight loss. If this is present, increasing 90 to 100 mL/kg in preterm babies. As a rough guide, the
the fluid intake would be the appropriate therapeutic volume of blood to be exchanged is usually 20 mL/kg.
measure. The hematocrit should be measured again PET normalizes cerebral hemodynamics and imp
after correction of dehydration. roves clinical status of the infants with polycythemia.1
• Associated metabolic problems like hypoglycemia, PET has also been shown to reduce pulmonary vascular
hypocalcemia and acidosis should be treated resistance24 and increase cerebral blood flow velocity.25, 26
simultaneously. PET is a relatively simple procedure, but has numerous
The principles of management of neonatal polycythemia potential complications. Unfortunately, there are no
are: data regarding the incidence of complications of PET;
• To decrease red cell mass below threshold level. one can only extrapolate from the data on full exchange
• To remove excess blood volume. transfusions performed for neonatal hyperbilirubinemia.8
• To maintain metabolic and blood gas homeostasis till Reported complications in whole blood exchange include
the condition reverts back to normal. apnea, cardiac arrhythmia, decreased PR interval,27
The following modes of therapy have been employed embolism, vessel perforation, accidental hemorrhage,
for the treatment of polycythemia. hypothermia, reduction in blood pressure, cerebral
• Conservative management with additional fluid blood flow fluctuations, sepsis, necrotizing enterocolitis
intake: This mode of therapy may be tried in cases of and portal vein thrombosis. Hypernatremia and raised
asymptomatic polycythemia when the hematocrit osmolality following whole blood exchange transfusion
reaches 70 to 75 percent. An extra fluid aliquot of have also been reported by Jain, Puri and Faridi (1997).28
However, whole blood exchange transfusion is expected long-term (neurodevelopmental) outcome in patients
to have a higher incidence of complications than PET, with polycythemia. Studies by Black et al.16 and Goldberg
since the amount of blood to be exchanged is almost nine et al.17 did not demonstrate improvement in the long-term
times higher and the product utilized for the exchange is outcome with the use of PET in symptomatic polycythemia.
donor’s blood. Similarly PET has not shown any beneficial effect on
The statement of the committee of the fetus and long-term outcome in neonates with asymptomatic
newborn, American Academy of Pediatrics8 regarding the polycythemia.1
treatment of neonatal polycythemia with PET reflects both
The recent systematic review by Dempsey et al.31 also
the concern and uncertainty—“The accepted treatment supports these findings. In this review, randomized or
of polycythemia is partial exchange transfusion. However quasi-randomized trials in term infants with polycythemia
there is no evidence that exchange transfusion affects the and/or documented hyperviscosity were considered.
long term outcome. Universal screening for polycythemia Clinically relevant outcomes included were short-term
fails to meet the methodology and treatment criteria and (resolution of symptoms, neurobehavioral scores, major
also, possibly the natural history criterion”. Despite this complications) and long-term neurodevelopmental
ambivalent statement, the standard practice in most outcome. There was no evidence of an improvement in the
nurseries is to perform PET in symptomatic babies with long-term neurological outcome (Mental Developmental
a hematocrit greater than 65 percent or in asymptomatic Index, incidence of mental delay and incidence of
babies with a hematocrit greater than 70 percent .29,30 neurological diagnoses) following PET in symptomatic
or asymptomatic infants. Also, there was no evidence of
PHLEBOTOMY improvement in early neurobehavioral assessment scores
(Brazelton Neonatal Behavioral Assessment Scale). It
Michael and Mauer21 have described phlebotomy as a was concluded that PET may be associated with an early
successful treatment modality in cases suffering from improvement in symptoms, but there are insufficient data
maternofetal transfusion. It seems logical to reduce to calculate the size of the effect.
hypervolemia in cases of hypertransfusion state. Therefore, It is probable that the underlying etiology of
phlebotomy can be employed in cases where polycythemia polycythemia is a more important determinant of the
is a result of passive or secondary polycythemia. ultimate outcome. However, definitive data on long-term
outcome with treatment is still unavailable in infants
Routes for Partial Exchange Transfusion with symptomatic polycythemia and in asymptomatic
PET may be done through a infants with hematocrit >70 percent. Therefore, it may be
• Peripheral or central route: A peripheral route avoids advisable to perform a PET or consider plasma expansion
umbilical vessel cannulation and is done by using a with additional fluids based on the presence or absence of
peripheral arterial and venous line. Blood is withdrawn symptoms in these polycythemic neonates.
from the arterial line and replaced simultaneously
through the venous line. REFERENCES
• A central route requires umbilical vein cannulation. 1. Bada HS, Korones SB, Pourcyrous M, Wong SP, Wilson
The umbilical venous catheter may be used for WM 3rd, Kolni HW, et al. Asymptomatic syndrome of
withdrawing blood while the same amount of saline is polycythemic hyperviscosity: effect of partial exchange
replaced through a peripheral vein. Alternatively the transfusion. J Pediatr. 1992;120:579-85.
umbilical venous catheter may be used both for the 2. Jeevasankar M, Agarwal R, Chawla D, Paul VK, Deorari
withdrawal of blood and replacement with saline. AK. Polycythemia in the newborn. Indian J Pediatr. 2008;
• Dempsey et al.,31 in a recent systematic review 75:68-72.
have shown that PET through umbilical route may 3. Nelson NM. Respiration and circulation before birth. In:
be associated with increased risk of necrotizing Smith CA, Nelson NM, eds. Physiology of the Newborn
enterocolitis (Relative risk 8.68, 95 percent CI 1.06, Infant, 4th edn. Springfield: Charles C Thomas. 1976.
pp.17-25.
71.1).
4. Drew JH, Guaran RL, Cichello M, Hobbs JB. Neonatal
Short-term and long term outcome with polycythe whole blood hyperviscosity: The import factor influencing
mia: PET reverses the short term pathophysio logical later neurologic function is viscosity and not polycythemia.
abnormalities associated with polycythemia hyper Clinical Hemorheology and Microcirculation. 1997;17:
viscosity syndrome. It improves capillary perfusion, 67-72.
cerebral blood flow and cardiac function.1,24-26 However, 5. Wexner EJ. Neonatal polycythemia and hyperviscosity.
there is very little data to suggest that PET improves Clin Perinatol. 1995;22:693-4.
6. Riopel L, Fouron JC, Bard H. Blood viscosity during the 20. Widness JA, Pueschel SM, Pezzullo JC, Clemons GK.
neonatal period: the role of plasma and red blood cell type. Elevated erythropoietin levels in cord blood of newborns
J Pediatr. 1982;100:449-53. with Down’s syndrome. Biol Neonate. 1994;66:50-5.
7. Ramamurthy RS, Berlanga M. Postnatal alteration in 21. Michael AFJ, Mauer AM. Maternal-fetal transfusion as
hematocrit and viscosity in normal and polycythemic a cause of plethora in the neonatal period. Pediatrics.
infants. J Pediatr. 1987;110:929-34. 1961;28:458-61.
8. American Academy of Pediatrics Committee on Fetus 22. Deorari AK, Paul VK, Shreshta L, Singh M. Symptomatic
and Newborn Routine evaluation of blood pressure, neonatal polycythemia: Comparison of partial exchange
hematocrit, and glucose in newborns. Pediatrics. 1993;92: transfusion with saline versus plasma. Indian Pediatr.
474-6. 1995;32:1167-71.
9. Gordon EA. Polycythemia and Hyperviscosity of the 23. de Waal KA, Baerts W, Offringa M. Systematic review of
Newborn. J Perinat Neonat Nursing. 2003;17:209-21. the optimal fluid for dilutional exchange transfusion in
10. Wiswell TE, Cornish JD, Northam RS. Neonatal polycy neonatal polycythemia. Arch Dis Child Fetal Neonatal Ed.
themia: frequency of clinical manifestations and other 2006;91:F7-F10.
associated findings. Pediatrics. 1986;78:26-30. 24. Murphy DJ Jr, Reller MD, Meyer RA, Kaplan S. Effects of
11. Wirth FH, Goldberg KE, Lubchenco LO. Neonatal neonatal polycythemia and partial exchange transfusion
hyperviscosity: I. Incidence. Pediatrics. 1979;63:833-6. on cardiac function: an echocardiographic study.
12. Shohat M, Merlob P, Reisner SH. Neonatal Polycythemia. I. Pediatrics. 1985;76:909-13.
Early diagnosis and incidence relating to time of sampling. 25. Rosenkrantz TS, OhW. Cerebral blood flow velocity in
Pediatrics. 1984;73:7-10. infants with polycythemia and hyperviscosity: effects of
13. Shohat M, Reisner SH, Mimouni F, Merlob P. Neonatal partial exchange transfusion with Plasmanate. J Pediatr.
polycythemia II. Definition related to time of sampling. 1982;101:94-8.
Pediatrics. 1984;73:11-3. 26. Maertzdorf WJ, Tangelder GJ, Slaaf DW, Blanco CE. Effects
14. Mukhtar AI, Halliday HL. Eisenmenger syndrome in preg of partial plasma exchange transfusion on cerebral blood
nancy is a possible cause of neonatal polycythemia and flow velocity in polycythemic preterm, term and small for
persistent fetal circulation. Obst Gynecol. 1982;60:651-2. date newborn infants. Eur J Pediatr. 1989;148:774-8.
15. Awonusonu FO, Pauly TH, Hutchison AA. Maternal 27. Patil R. Hemodynamic and capillary blood gas changes
smoking and partial exchange transfusion for neonatal during exchange blood transfusion in early neonatal period.
polycythemia. Am J Perinatol. 2002;19:349-54. Thesis submitted to National Board of Examinations for the
16. Black VD, Lubchenco LO, Luckey DW, Koops BL, award of Diplomate of National Board in Pediatrics, 2007.
McGuinness GA, Powell DP, Tomlinson AL. Developmental 28. Jain A, Puri D, Faridi MMA. Biochemical changes during
and neurologic sequelae of neonatal hyperviscosity exchange transfusion in hyperbilirubinemia in term
syndrome. Pediatrics. 1982;69:426-31. newborn babies. Indian J Clin Biochem. 1997;12:119-24.
17. Goldberg K, Wirth FH, Hathaway WE, Guggenheim MA, 29. Roithmaier A, Arlettaz R, Bucher HU, Krieger M, Duc G,
Murphy JR, Braithwaite WR, Lubchenco LO. Neonatal Versmold HT. Randomized controlled trail of Ringer
hyperviscosity II. Effect of partial exchange transfusion. solution versus serumfor partial exchange transfusion in
Pediatrics. 1982;69:419-25. neonatal polycythemia. Eur J Pediatr. 1995;154:53-6.
18. Debieve F, Moiset A, Thomas K, Pampfer S, Hubinont C. 30. Acunas B, Celtik C, Vatansever U, Karasalihoglu S.
Vascular endothelial growth factor and placenta growth Thrombocytopenia: an important indicator for the
factor concentrations in Down’s syndrome and control application of partial exchange transfusion in polycythemic
pregnancies. Mol Hum Reprod. 2001;7:765-70. newborn infants? Pediatr International. 2000;42:343-7.
19. Bdolah Y, Palomaki GE, Yaron Y, Bdolah-Abram T, 31. Dempsey EM, Barrington K. Short and long term outcomes
Goldman M, Levine RJ, et al. Circulating angiogenic following partial exchange transfusion in the polycythemic
proteins in trisomy 13. Am J Obstet Gynecol. 2006;194: newborn: a systematic review. Arch Dis Child Fetal
239-45. Neonatal Ed. 2006;91:F2-F6.
C H A P T E R 10
Vitamin K Deficiency:
Bleeding in Newborns
Vitamin K deficiency bleeding (VKDB) refers to bleeding that occurs as a consequence of vitamin K deficiency during first six months
of life. Previously known as the hemorrhagic disease of the newborns, it has been renamed to emphasize that bleeding problems
during the neonatal period are not confined to those arising from vitamin K deficiency alone and that bleeding secondary to vitamin K
deficiency may occur beyond the first month of life.
DEVELOPMENT OF HEMOSTATIC SYSTEM Biology of vitamin K: Vitamin K is a generic name for several
molecules sharing a 2 methyl‐1,4 naphthoquinone ring
Hemostasis develops in an orderly way during intrauterine but differing regarding the side chain at the 3‐position.
life. The most basic reaction‐contraction of blood vessels Phylloquinone or vitamin K1 is from plant origin and has a
in response to injury‐is present from eight weeks, though phytyl side chain. The group of menaquinones or vitamin
its strength does not become normal until much later. K2 differs in the number of isoprenyl units in the side chain
Platelets appear in the circulation by 11 weeks and can and are synthesized by the bacteria in humans and animal
form aggregates by 12 to 15 weeks; they approximate intestine. Menadione or vitamin K3 is a synthetic and water
to adult numbers by 30 weeks. Clotting and fibrinolytic soluble vitamin K without a side chain. This preparation is not
plasma proteins are found from 10 to 11 weeks; the preferred as it has been shown to cause hemolytic anemia,
concentrations of some clotting factors reach adult values indirect hyperbilirubinemia and kernicterus. In our country,
in utero, but of others, mainly those dependent on vitamin only vitamin K3 preparations are available. Vitamin K acts as
K (II, VII, IX and X) are still low at term. a cofactor for gamma glutamyl carboxylase (GGCX) serving
Normal neonatal hemostasis reflects a highly complex as an electron donor for the post-translational conversion of
process dependent on interactions between endothelial protein bound glutamate into Gamma-carboxyglutamate.
cells, platelets and hemostatic proteins. It is now recog During this process, it is oxidized to vitamin K2, 3‐epoxide.
nized that the traditional extrinsic pathway involving Gla residues are calcium binding groups which are essential
tissue factors and factor VIIa, is the major pathway where for the biological activities of proteins in which they are
by coagulation is initiated and the thrombin plays a crucial found. Gla containing proteins are the coagulation factors
role in coagulation as well as platelet activation. II, VII, IX and X but also protein C, protein S, protein Z,
At birth concentrations of vitamin K dependent osteocalcin, etc.
factors (II, VII, IX and X) and contact factors (XI and XII) Vitamin K deficiency leads to the synthesis of under
are reduced to about 50 percent of normal adult values. carboxylated proteins unable to bind calcium and hence
Similarly, concentration of the naturally occurring anti inactive. In vitamin K deficient individuals, under
coagulants, antithrombin, protein C and protein S are low carboxylated forms of vitamin K dependent coagulation
at birth and as a consequence, both thrombin generation proteins (proteins induced by vitamin K absence
and thrombin inhibition are reduced in the newborn PIVKA) are released from the liver into the blood. PIVKA
period. are inactive in the coagulation cascade. PIVKA II or
Chapter-10 Vitamin K Deficiency: Bleeding in Newborns 65
undercarboxylated prothrombin is a marker of subclinical are at particular risk. Often, VKDB is the first sign of this
vitamin K deficiency. underlying condition. Vitamin K epoxide is recycled to
vitamin K by vitamin K epoxide reductase (VKOR). This
CHEMICAL STRUCTURE OF VITAMIN K recycling process is inhibited by coumarin and warfarin.
Vitamin K Cycle CLINICAL FEATURES

Diagnosis
The diagnosis of vitamin deficiency may be suspected from
the results of coagulation screening where initially, there is
isolated prolongation of the prothrombin time followed by
prolongation of the APTT, in association with the normal
concentrations of fibrinogen and normal platelet count.
Confirmation of the diagnosis requires measurements of
PIVKA II.
Treatment
Once the diagnosis is confirmed, intravenous vitamin K
should be administered to correct the existing deficiency.
Vitamin K deficiency bleeding (VKDB): As a consequence In suspected cases, vitamin K can be given while factor
of limited stores at birth, neonates are prone to concentrations are pending. In the presence of major
vitamin K deficiency if no sufficient intake is provided. bleeding, factor replacement therapy may also be required
Vitamin K deficiency has been traditionally classified as early, with fresh frozen plasma, prothrombin complex concen
classical and late depending on timing of the presentation. trate (FII, FIX, FX), or a four factor concentrate containing
all the vitamin K dependent factors.
Early VKDB: Presents within 24 hours of birth and is No formal studies were ever performed to establish
almost exclusively seen in infants of mothers taking what dose might be appropriate before it became standard
drugs which inhibit vitamin K. These drugs include practice to give every infant a 1‐mg dose at birth and to give
anticonvulsants (carbamazepine, phenytoin and bar it intramuscularly simply because that was the only product
biturates but not valproic acid), antitubercular drugs available. In 1990, an epidemiological study described an
(isoniazid, rifampicin), some antibiotics (cephalosporins) association between intramuscular (IM) vitamin K at birth
and vitamin K antagonists (coumarin, warfarin). and childhood cancer and leukemia. In response to these
Clinical presentation is often severe with cephalic findings, several European countries, Australia and New
hematoma, intracranial and intra‐abdominal hemorrhage. Zealand changed their policy to oral prophylaxis. In the
The incidence in an at‐risk group without vitamin K following years, new studies failed to confirm the association
supplementation is 6 to 12 percent. between IM vitamin K at birth and childhood cancer. A risk
Classical VKDB: Occurs between 24 hours and 7 days of solid tumors can now almost definitely be ruled out, but
of life and is associated with delayed or insufficient a small risk of leukemia cannot be excluded. When cases of
feeding. Clinical presentation is often mild, with bruises, late VKDB started to reappear, Denmark, Canada, Australia
gastrointestinal blood loss or bleeding from the umbilicus and New Zealand responded by reintroducing universal IM
and puncture sites. Blood loss, however, can be significant, prophylaxis, offering oral prophylaxis with repeated doses
and intracranial hemorrhage, although rare, has been to those parents refusing the IM injection at birth. Oral
described. Without vitamin K supplementation, incidence prophylaxis with repeated doses has remained the policy in
estimated is 0.01 to 0.44 percent. the Netherlands and in Germany, using different products
and dosing schemes. The American Academy of Pediatrics
Late VKDB: It is associated with exclusive breastfeeding. has always endorsed the IM route.
It occurs between the ages of 2 and 12 weeks. Clinical
presentation is severe, with a mortality rate of 20 percent
and intracranial hemorrhage occurring in 50 percent.
ORAL VITAMIN K
Persistent neurological damage is frequent in survivors. Oral vitamin K administration would appear to offer
In fully breastfed infants who did not receive vitamin K several advantages for routine VKDB prophylaxis. In
at birth, the incidence is between 1/15,000 and 1/20,000. addition to the concerns raised about a link with childhood
Infants with cholestasis or malabsorption syndromes cancer, other disadvantages with IM administration
include the trauma and complications associated with this given to all newborn as a single, intramuscular dose of 0.5
route of administration (hematoma, vessel or nerve injury, to 1 mg. (2) Additional research should be conducted on
abscess, or osteomyelitis) and the higher cost of therapy. the efficacy, safety, and bioavailability of oral formulations
While no oral liquid preparation is available, the injectable and optimal dosing regimens of vitamin K to prevent
product has been found to be safe and effective when given late VKDB. (3) Health care professionals should promote
by the oral route. Unfortunately, the rise in the use of oral awareness among families of the risk of late VKDB
vitamin K prophylaxis has led to an increase in reports of associated with inadequate vitamin K prophylaxis from
late VKDB. Several countries currently use an alternative current oral dosage regimens, particularly for newborns
mixed micellar preparation of vitamin K (Konakion MM®; who are breastfed exclusively.
Roche) for multidose oral prophylaxis. This formulation
is expected to provide greater absorption than traditional Vitamin K and Preterm Newborn
preparations and may make oral administration more
effective. Unfortunately this preparation is not available in Ever since the discovery of vitamin K, it has been clear that
most of the countries including India. premature infants are at particular risk of VKDB. Although
there is consensus on the fact that all premature infants
should receive vitamin K, neonatology units use a variety
The Cochrane Review of doses, dosing schedules, routes and formulations.
All trials using random or quasi‐random patient alloca Reports have shown very high plasma vitamin K levels in
tion, in which methods of vitamin K prophylaxis in infants preterm infants receiving 0.5 to 1 mg at birth. Although
were compared to each other, placebo or no treatment, no toxic effects of these excessively high serum levels
were included. Two eligible randomized trials comparing have been recognized, caution is warranted because the
a single dose of intramuscular vitamin K with placebo or functions of some Gla proteins are not fully understood. A
nothing, assessed effect on clinical bleeding. One dose recent randomized trial shows adequate serum vitamin K
of vitamin K reduced clinical bleeding at 1 to 7 days, levels in preterm infants receiving 0.2 mg at birth. In this
including bleeding after circumcision, and improved trial, preterm infants receiving 0.5 mg have elevated levels
biochemical indices of coagulation status. Eleven addi of vitamin K epoxide, suggesting inefficient recycling of
tional eligible randomized trials compared either a single vitamin K by VKOR in the immature liver. These findings
oral dose of vitamin K with placebo or nothing, a single support current empirical dosage recommendations for
oral with a single intramuscular dose of vitamin K, or preterm infants advising a reduced dose of 0.3 mg for birth
three oral doses with a single intramuscular dose. None weights <1,000 g and 0.5 mg for those >1,000 g and <1,500 g.
of these trials assessed clinical bleeding. Oral vitamin
K improved biochemial indices of coagulation status at Vitamin K and Cholestatic Disorders
1 to 7 days. There was no evidence of a difference between
Due to fat malabsorption and inadequate intake, infants
the oral and intramuscular route in effects on biochemical
with cholestatic liver disease are especially at risk for vitamin
indices of coagulation status. A single oral compared with
K deficiency. Some of the current standard regimens
a single intramuscular dose resulted in lower plasma
of oral vitamin K prophylaxis are mostly insufficient in
vitamin K levels at two weeks and one month, whereas a
cholestatic patients making them extremely vulnerable
3‐dose oral schedule resulted in higher plasma vitamin K
for VKDB. More than 80 percent of breastfed infants with
levels at two weeks and at two months than did a single
biliary atresia who received oral vitamin K prophylaxis
intramuscular dose. It was concluded that a single dose
(1 mg oral vitamin K at birth followed by 25 microgram
(1.0 mg) of intramuscular vitamin K after birth is effective
daily) developed a VKDB at the time of diagnosis. Forty
in the prevention of classic HDN. Either intramuscular or
three percent presented with an intracranial hemorrhage.
oral (1.0 mg) vitamin K prophylaxis improves biochemical
The empirical dosing guideline for oral vitamin K1 in
indices of coagulation status at 1 to 7 days. Neither
infants and children with chronic cholestasis is 2.5 to 5 mg
intramuscular nor oral vitamin K has been tested in
given two to seven times per week. Nevertheless, with this
randomized trials with respect to effect on late HDN. Oral
regimen, subclinical vitamin K deficiency seems prevalent
vitamin K, either single or multiple doses, has not been
despite normal prothrombin time (PT). In a group of
tested in randomized trials for its effect on either classic
43 cholestatic children supplemented following this
or late HDN.
schedule, 23 (54%) had elevated plasma PIVKA II levels
(>3 ng/mL) with normal PT. Vitamin K doses sufficient
The American Academy Recommendation to maintain normal coagulation may not be sufficient to
The vitamin K Ad Hoc Task Force of the American Academy maximize carboxylation of coagulation factors. Based on
of Pediatrics (AAP) recommends: (1) Vitamin K, should be the above‐mentioned data, it is thus of utmost importance
Chapter-10 Vitamin K Deficiency: Bleeding in Newborns 67
that, as soon as the diagnosis of cholestasis is made in an CONCLUSION

infant, extra vitamin K supplementation should be given
to prevent VKDB with its serious consequences. However, There is no doubt that all newborns need vitamin K. Classic
the best strategy for vitamin K supplementation in chronic VKDB is prevented by the administration of 0.3 to 1 mg
childhood cholestasis still remains a critical issue. Current vitamin K at birth; IM administration is the preferred route
regimens may be underestimating the optimal dosage of in at‐risk groups. IM administration of vitamin K at birth
vitamin K. is effective in preventing both classic and late VKDB. In
exclusively breastfed infants, oral vitamin K administration
Current International Scenario should be continued. Weekly oral administration of 1 mg
vitamin K is more effective in preventing late VKDB than
The various schemes for vitamin K administration being
daily administration of 25 μg. Infantile cholestasis needs
followed world over with risk of late HDN has been
extra vitamin K supplementation. Current regimens may
summarized in Table 1.
be underestimating the optimal dosage of vitamin K.
Table 1 Various schemes for vitamin K administration
BIBLIOGRAPHY
Administration Incidence per
scheme 1,00000 1. Chalmers EA. Neonatal coagulation problems. Arch Dis
Child Fetal Neonatal Ed. 2004;89:F475-8.
The Netherlands 1mg oral at birth 3.2
2. Controversies Concerning Vitamin K and the Newborn
followed by 25 µg
Committee on Fetus and Newborn Pediatrics. 2003;
daily for 2 weeks
112;191‐2.
Germany 2 mg oral at birth 0.44 3. Fear NT, Roman E, Ansell P, Simpson J, Day N, Eden OB.
followed by 2 mg United Kingdom Childhood Cancer Study Vitamin K and
at 1 and 4 weeks childhood cancer: a report from the United Kingdom
Denmark 2 mg oral at birth 0 Childhood Cancer Study. Br J Cancer. 2003;89:1228-31.
followed by 1 mg 4. Golding J, Paterson M, Kinlen LJ. Factors associated with
weekly till 12 weeks childhood cancer in a national cohort study. Br J Cancer.
1990;62:304-8.
Great Britain 1 mg IM 0.1
5. Hey E. Vitamin K what, why and when? Arch Dis Child
1 mg oral, 0.43 Fetal Neonatal Ed. 2003;88:F80-3.
continuing after 1 6. Puckett RM, Offringa M. Prophylactic vitamin K for vitamin
week K deficiency bleeding in neonates. Cochrane Database
1 mg oral, not 2.9 Syst Rev. 2000: issue 4.
beyond 1 week 7. Winckel MV, De Bruyne R, De Velde SV, Biervliet SV.
Vitamin K an update for the Pediatrician. Eur J Pediatr.
Nil 6.2
2009;168:127-34.
C H A P T E R 11
Bleeding Neonate:
Approach and Management
Normal hemostasis, the process that arrests bleeding after blood vessel injury, is achieved through normal functioning of platelets
and coagulation proteins along with vascular integrity. These functions are delicately balanced so that blood may freely circulate
within the intact vessels and if bleeding occurs, the site of bleeding can be effectively sealed. Disruption of one or more of these
factors results in bleeding. Blood is in a dynamic equilibrium between fluidity and coagulation. This is maintained by balance between
coagulation mechanism on one hand and fibrinolysis as well as anticoagulation on the other hand. Failure of this balance makes the
neonate susceptible for both hemorrhagic as well as thrombotic tendencies. Hemorrhage and thrombosis may result from variety of
pathological processes.1-10
Hemostatic functions in infants and newborns differ from • Hemostatic proteins (coagulation factors) resulting in
those in children and adults. However, abnormalities a stable clot. Disruptions of one or more of these factors
in some of these functions predispose the neonate to results in bleeding. This is in balance with the natural
bleeding, especially the preterm and sick neonates in inhibitors of coagulation factors present in blood like
the neonatal intensive care units. Bleeding in a neonate anti-thrombin III, protein C and protien S.
can be one of the important causes of morbidity and
mortality and can be a life threatening situation due to the MECHANISM OF HEMOSTASIS3-8
small blood volume of the neonate. Hence, any bleeding
neonate requires prompt attention and a rapid diagnosis Hemostasis can be considered in two phases—primary
and immediate institution of therapy. and secondary.
INCIDENCE Primary Hemostasis

It is estimated that 1 percent of all nursery admissions Vessel Wall Contractions and Platelet
and 25 to 30 percent of neonatal intensive care unit Plug Formation
admissions are complicated by disorder of bleeding. This • It is characterized by vessel wall contractions and
problem is accentuated in preterms and low birth weight platelet plug formation in smaller vessels. Following
babies and with their increasing survival the incidence of vascular endothelial disruption, a complex series of
encountering bleeding disorder has risen particularly in biochemical reaction set into motion.
neonatal intensive care units.1-13 • The exposed subendothelial structures attract platelets
and they adhere to the exposed collagen with the help
NORMAL NEONATAL HEMOSTASIS of von Willebrand factor and Fibronectin.
Normal neonatal hemostasis is a highly complex process • Following the platelet adhesion substances like ADP,
dependent on interactions between: Thrombaxane A2 and platelet factor III are released.
• Endothelial cells of the blood vessels • This leads to primary platelet aggregation which
• Platelets attracts more platelets to aggregate and to release
Chapter-11 Bleeding Neonate: Approach and Management 69
ADP and Thrombaxane A2 from its dense granules, are partly responsible for prolongation of activated
ultimately expanding the hemostatic plug. partial thromboplastin time (APTT) that is observed in
low birth weight infants.
Secondary Phase of Hemostasis3,12,14 • Factor VII levels reaches adult range by 5 days, while
other factors increase gradually over the 1st 6 months
It involves sequential activation of circulating coagulation
of life.
factors by intrinsic and extrinsic pathways ultimately
• von Willebrand factor (vWF) levels are increased at
to form a secondary stable fibrin clot. This controls
birth and although they decline slightly, they remain
hemostasis in large vessels. high. Neonatal VWF is made up of multimers which
have increased platelet aggregation in response to
Fibrinolytic Activity Ristocetin.
Under normal hemostatic mechanisms, where fibrin is • Factor XIII level in cord blood is 50 percent of that
deposited upon the vessel wall or in the tissues, fibrinolytic in adults, but as only small amount of factor XIII is
processes are simultaneously stimulated so that fibrin is required for its activation or clot stabilization, this low
slowly broken down into fibrin split products by plasmin values in newborns have no clinical significance.5,16,17
which is activated from its precursor plasminogen. • The plasma level, molecular weight, amino acid compo
Normally fibrinolytic mechanism is also balanced by its sition and immunological properties of fibrinogen in the
inhibitors present in the blood.8-10 newborn is comparable to that of adult. But it is found to
coagulase more slowly and has different chromatogram
Hemostasis in Newborn: Salient Features on DEAE cellulose. The term fetal fibrinogen is applied
to this factor and hence often newborn babies have
Due to physiological immaturity there are both quantitative prolonged thrombin time.5,18
as well as qualitative differences in hemostatic functions
in newborn as compared to older children. Inherited Permanent Abnormality of
Coagulation Factors
Primary Phase of Hemostasis
• Hemophilia A (Factor VIII def.)
• Though capillary fragility is normal in term infants it • Hemophilia B (Factor IX def.)
is increased in preterm. Vasoconstriction following • von Willebrand disease, etc.
injury is therefore incomplete in preterms2,3,6,8,12 and • Other rare deficiency of coagulation factors
hence intracranial hemorrhage is more common in • Coagulation factors are synthesized in the fetus from
premature babies. around the tenth week of gestation,16 and are not
• Platelet count in both term and preterm babies are transferred transplacentally from mother to the baby.
similar to that in older children. However platelet Hence, the values of coagulation factors estimated in
function such as adhesion, aggregation and release of newborn reflect the synthesis of the various factors in
factors like ADP and Thrombaxane A2 are abnormal. them.16
• At term, levels of factor V, VIII are equivalent to older
Secondary Phase of Hemostasis5,6,8,10,13,15 children and adults and hence if deficiency of these
• Extrinsic pathway involving tissue factor V and factor factors is present during newborn period then it
VIIa, is the major pathway where by coagulation is suggests inherited factor deficiency in them.5
initiated and the thrombin plays a crucial role in In healthy neonates this transient deficiency of clotting
factors does not play an important role as levels of 20 to
coagulation as well as platelet activation.
30 percent of coagulant activities are adequate for clot
• The concentrations of some clotting factors reach
formation. However, stresses like prematurity, sepsis,
adult values in utero, but concentrations of vitamin
asphyxia, apneic spells, hypoxia, acidosis, RDS, etc. can
K dependent factors (II, VII, IX and X) and contact
tilt the balance leading to bleeding episodes.
factors (XI, XII, prekalliekrien, high molecular weight
kininogen) are reduced to about 50 percent of normal
Fibrinolytic Activity
adult values. Levels of factors II, VII, IX, X are decreased
more so in preterm babies due to hepatic immaturity In newborn fibrinolytic activity is transiently increased
and poor availability of vitamin K. Hence hemorrhagic as compared to adults or older children. It declines to
disease of newborn is more common in premature adult level by 6 hours in term neonate. Plasminogen
babies.5,17 More immature the infant, lower is the factor levels are only half that of an adult and FDP is normally
XII activity. Reduced concentrations of factor XII, XI absent in healthy preterm and term infants. This low level
of plasminogen along with physio logical deficiency of • Amegakaryocytic thrombocytopenic purpura`

circulating anticoagulants like antithrombin III, protein-C • Sepsis
promotes thrombotic tendencies in neonates. Deficiency • Increased platelet consumption
or low level of plasminogen (around 50% of adult value- • Immune thrombocytopenic purpura
reaches normal adult value by 6 months) along with • Auto immune—Child born to mother with SLE or ITP
physiological deficiency of circulating anticoagulants like • Neonatal alloimmune thrombocytopenia–PIA antigen
antithrombin III and protein C and S, promotes thrombotic +ve child born to PIA -ve mother
tendencies in neonates. • Asphyxia, shock
• In addition plasminogen is present in fetal form • Sepsis, polycythemia, hyperviscocity
with both reduced functional activity and decreased • Thrombosis due to catheter, hemangioma
binding to cellular receptors. • IUGR with toxemia of pregnancy
• C4b binding protein is absent in neonates and protein • Heparin induced thrombocytopenia.
S therefore circulates in active free form. Qualitative:
• Tissue factor pathway inhibitors (TFPI) or external Drugs like aspirin given to mother and inherited
pathway inhibitors are around 65 percent of adult disorders of platelet function like
values. Glanzmann’s thrombasthenia, Bernard Soulier synd
rome.
Antithrombin III, Protein C and Protein S • Exaggeration of transient deficiency of coagulation
factors: Hemorrhagic disease of newborn.
Similarly concentration of the naturally occurring
• Transitory disturbances of coagulation mechanism
anticoagulants, antithrombin III, protein C and protein
as a result of associated systemic disease process, e.g.
S are low at birth and as a consequence, both thrombin
sepsis, liver disease, DIC, etc.
generation and thrombin inhibition are reduced in the
• Inherited permanent abnormality of coagulation
newborn period. Antithrombin III (ATIII) heparin co-
factors: Hemophilia A and B, von Willebrand disease,
factor II (HCII), beta-2 macroglobulin, are increased at
etc.
birth, continue to rise till the age of 6 months and reach
• Trauma alone or often associated with other factor
the twice normal adult values at this time.
deficiencies Slipped ligature, cephalhematoma, etc.5-8
Neonates are thus susceptible to hemorrhage and
thrombotic tendencies. This paradox is due to a combined
deficiency of coagulation factors along with defective Approach to Bleeding Disorders in Neonate
platelet function on one hand and decreased levels of • Detail history—antenatal, perinatal, postnatal, family
natural inhibitors of coagulation and fibrinolysis on the history
other. • Complete physical examination
• Selected laboratory investigations
Local Pathological Lesion • Confirmatory tests.
• Trauma
• Slipped ligature History
• Cephalhematoma, etc.
Maternal History
Combined Factors Deficiencies • Presence of an underlying maternal systemic diseases
• Disseminated intravascular coagulation (DIC). like pre-eclampsia, cardiovascular diseases, viral infec
• Hepatic dysfunction. tion
• Recent drugs taken like aspirin, anticonvulsants like
Etiology of bleeding in neonate:11-13,15 phenobarbitone and phenytoin Na and anticoagulants
Bleeding in a neonate may be due to: • History of collagen vascular disorder, past history of
• Vascular abnormalities, e.g. in prematurity–intracranial ITP in mother.
hemorrhage.
• Platelets abnormalities
Detailed Birth History
Quantitative:
• Congenital infections (CMV, Rubella, HIV) • Type of delivery, birth asphyxia, trauma.
• Thrombocytopenia with absent radius (TAR syndrome) • Gestational age should be noted.
• Certain syndromes • History of vitamin K given to neonate, use of antibiotics
• Fanconi’s anemia and whether neonate is receiving only breastfeeds.
Fig. 1 Wiskott-Aldrich syndrome
Family History shock, etc. In such babies bleeding is likely to be sec-

ondary phenomenon such as DIC, consumption platelet
History should include: coagulopathy, liver dysfunction, etc.3-5,12
• Family history suggestive of bleeding disorder, e.g.
history of excessive bleeding after injury or history of Site of Bleeding
menorrhagia in female members.
• Proper pedigree charting of any affected memebers, • Bleeding from umbilicus in a healthy child without any
both living and expired will help to know the type of evidence of umbilical sepsis or slipped ligature, suspect
inheritance of the disorder. factor XIII deficiency or hypodysfibrinogenemia.
X-linked inheritance: Factor VIII, IX deficiency— • Bleeding from circumcision or hematoma at injection
enquire similar history of bleeding episodes in male site in a healthy child-suspect factor deficiency or
siblings, maternal cousins, maternal uncles, etc. hemorrhagic disease of newborn.
Autosomal dominant: von Willebrand’s disease, dysfi • Bleeding from GIT is probably due to swallowed
brinogenemia, hemorrhagic telangiectasia maternal blood or vitamin K deficiency.
Autosomal recessive: Other factor deficiencies— • In a sick child-suspect DIC.
enquire history of consanguinity. • Big cephalhematoma following normal delivery
(without prolonged or difficult labor) should lead to
Physical Examination suspicion of inherited bleeding disorders.
• Petechiae or ecchymosis on presenting part, secondary
A rapid and thorough physical assessment of the bleeding to congestion and birth trauma may be seen soon
neonate should include: after birth and they gradually disappear and are not
• General examination. associated with bleeding anywhere else.
• Skin bleeds like purpura or petechiae in a healthy child-
Well Baby—A ‘Healthy’ Baby with suspect immune thrombocytopenia and differentiate
Bleeding Indicates it from mosquito bites (Fig. 2).
• Hemorrhagic disease of newborn
• Inherited coagulation factor deficiency Associated Findings
• Isoimmune thrombocytopenia
If associated hepatosplenomegaly jaundice or chorio-
• Platelet function disorders
retinitis present, it may suggest:
• Vascular causes, slipped ligature, etc.
• Congenital/acquired infections
• Leukemia
Sick Baby with Bleeding Indicates
• Erythroblastosis fetalis
Sepsis, asphyxia, RDS, hypothermia, apnic spells, acido- If associated with eczema—Wiskott-Aldrich syndrome
sis, hypoglycemia, seizures, prematurity, hypovolemia, (Fig. 1):
laboratory investigations are required to identify the

precise nature of the underlying cause of bleeding disorder.
It is necessary to confirm whether it is bleeding disorder
or not, particularly in a newborn baby with GI bleeding
as maternal blood swallow syndrome is seen during early
newborn period due to swallowing of maternal blood by
baby during the delivery or from the cracked nipple of
mother while feeding. Simple bedside test like Apt test will
differentiate these two as fetal hemoglobin is resistant to
denaturation by alkali where as adult hemoglobin present
in mother’s RBCs denaturates.
Apt Test
1 part of vomitus is mixed with 5 parts of saline and centri
fuged at 2000 rpm for 10 minutes. Add 4 cc of 10 percent
Fig. 2 Mosquito bite NaOH to 1 cc of supernatant centrifuged fluid. Brown
color indicates maternal blood and pink color fetal blood.5
In a suspected case of bleeding disorder further labora
Associated Congenital Anomalies tory tests need to be carried out. They can be divided into:
• TAR syndrome—absent radius with thrombocyto • Screening tests
penia • Special tests.
• Large hemangioma with DIC suggest Kasabach–
Merritt syndrome Screening Tests (Table 2)
• Syndactyly with bleeding: Factor V deficiency
They are applied to know the presence and nature of
• Ehler-Danlos syndrome: Ecchymosis, bruises, purpura
bleeding disorder so that relevant special tests can be
with hyperelastic skin.
done to confirm the diagnosis thus avoiding unnecessary
battery of tests in each case. They include:
Laboratory Approach (Table 1) • CBC
Though thorough history and clinical evaluation help • Peripheral smear examination
in suspecting the nature and type of bleeding disorders, • PT, APTT, BT and clot retraction.
Table 1 Laboratory tests

Platelets PT PTT BT CR Likely diagnosis
Sick infants
Decreased Increased Increased Increased Decreased DIC
Decreased Normal Normal Increased Decreased Early sepsis
Normal Increased Increased Normal Normal Liver disease
Normal Normal/L Normal/L Normal Normal Compromised vascular intergrity associated hypoxia, increased
prematurity, acidosis hyperosmolality
Healthy infants
Decreased Normal Normal Increased Decreased Occult infection or immune thrombocytopenia; thrombosis
Normal Increased Increased Normal Normal Hemorrhagic disease of newborn (vitamin K deficiency) or
common pathway defect
Normal Normal Increased Normal Normal Hereditary intrinsic clotting factor deficiencies
Normal Increased Normal Normal Normal Factor VII deficiency
Normal Normal Normal Normal Normal Bleeding due to local factors (trauma, anatomic abnormalities)
Factor XIII deficiency
Normal Normal Normal Increased Decreased Platelet abnormalities (rare)
Abbreviations: PT: Prothrombin time; PTT: Partial thromboplastin time; BT: Bleeding time; CR: Clot retraction time.
Table 2 Screening tests for bleeding disorders18-20 the common pathways and hence do thrombin time
and serum fibrinogen estimation. Therapeutic trial
Test Adult Full term Preterm
with vitamin K will normalize PT/APTT in hemorrha
Prothrombin 12 ± 1 14 ± 1.3 14 ± 1.3 gic disease of newborn.
time (sec) • Low serum fibrinogen with prolonged thrombin
Partial 42 ± 4 51 ± 10 57 ± 10.5 time suggest hypofibrinogenemia. If fibrinogen level
thromboplastin is normal and thrombin time is prolonged then it
time suggests dysfibrinogenemia or presence of inhibitors,
Thrombin 25 ± 2 23 ± 2.9 23 ± 2.4 heparin, etc.
clotting time • In a sick child normal platelet count and prolonged PT
(2U) and APTT suggest liver disorder—liver function test
Factor II (%) 81 ± 17 50 ± 14.5 31 ± 8.6 are needed.
Factor V (%) 90 ± 19 79 ± 17 70 ± 22 • Normal platelet count with normal PT and increased
PTT in a healthy child suggest intrinsic pathway defect
Factor VII-X (%) 93 ± 20 54 ± 12.2 37 ± 11
like hemophilia A, B or factor XI deficiency. Correction
Factor VIII (%) 87 ± 27 126 ± 56 116 ± 73 studies are required and estimation of factor levels.
Factor IX (%) 99 ± 23 35 ± 12.6 28 ± 11 • Normal platelet count with normal APTT with
Factor X (%) 89 ± 23 45 ± 12 31 ± 9.0 increased PT suggest extrinsic pathway defect due to
Antithrombin 99 ± 10 58 ± 9.6 33 ± 9.0
deficiency of factor VII—correction studies and factor
III (%) assay are needed for establishing diagnosis.
If all screening tests are normal including normal
Fibrinogen 315 ± 60 215 ± 35 256 ± 20
platelet count, PT and APTT following conditions should
(mg/dL)
be kept in mind.
• Local factors: Slipped ligature, umbilical sepsis,
The following table shows the interpretation and compromised vascular integrity, etc.
the likely causes of bleeding in a given case and further • Qualitative platelet disorders: Bleeding time will be
confirmatory tests required to be done in such newborn. prolonged with poor clot retraction. Do aggregation
Laboratory screening tests in differential diagnosis of study.
the bleeding infant. • Factor XIII deficiency: Do urea solubility test.
• Ehler-Danlos syndrome: Clinical examination for skin
Confirmatory Tests elasticity.
• Hemorrhagic telangiectasia: See for telangiectasia in
Platelet Disorders
mucous membrane of nose, bulbar conjuctiva, tongue
• Thrombocytopenia with normal PT/APTT in a and tips of the fingers.
healthy neonate suggest allo- or autoimmune throm-
bocytopenia Vitamin ‘K’ and Neonatal Hemostasis
• If autoimmune thrombocytopenia mother’s platelet (Fig. 3)12-17
count study for thrombocytopenia to rule out chronic
ITP/Test for collagen disorder should be done.
Biology of Vitamin K
• If alloimmune thrombocytopenia mother’s platelet Vitamin K deficiency bleeding (VKDB) also known as the
count study is normal. Platelet study in the mother and hemorrhagic disease of the newborns refers to bleeding
child—mother will be PLA1 antigen negative and a that occurs as a consequence of vitamin K deficiency
child PLA1 positive. during first year of life.
• Low platelet count with normal PT/APTT in a sick
child suspect platelet consumption as in septicemia Vitamin K
and if associated with prolonged PT/APTT it suggest
• Phylloquinone or vitamin K1 is from plant origin.
DIC. Do peripheral smear examination for burr cells,
• The group of menaquinones or vitamin K2 differ in
broken RBCs, helmet cells, serum fibrinogen which is
the number of isoprenyl units in the side chain and
decreased and FDP is increased.
are synthesized by the bacteria in humans and animal
intestine.
Coagulation Factors Defects
• Menadione or vitamin K3 is a synthetic and water
• Normal platelet count with prolonged PT/APTT in a soluble vitamin K without a side chain. This preparation
healthy child suspect vitamin K deficiency or defect in is not preferred as it has been shown to cause hemolytic
carboxylase is therefore limited in preterm because

precursor proteins themselves are deficient, often
below 30 percent of adult value.
• Though colostrums contain adequate amount of
vitamin K, lesser colonization by bacterial flora of
the gut of exclusively breastfed infants contribute to
lower plasma level of vitamin K. Cow’s milk contains
6 ug/dL of vitamin K1 as compared to breast milk which
contains 1.5 ug/dL vitamin K deficiency is classified
based on timing of presentation as follows:
– Early VKDB
– Classical VKDB
– Late VKDB.
Fig. 3 Vitamin K deficiency
Clinical Presentation
anemia, indirect hyperbilirubinemia and kernicterus. Early VKDB: Hemorrhagic disease of newborn.
Vitamin K acts as a cofactor for gamma glutamyl Clinical presentation is often mild, with bruises,
carboxylase (GGCX) serving as an electron donor for the gastrointestinal bleeding. Severe manifestations include
post-translational conversion of protein bound glutamate cephalohematoma, intracranial and intra-abdominal
into gamma carboxyglutamate. During this process it hemorrhage (Fig. 4). The incidence in an at‐risk group
is oxidized to vitamin K2, 3‐epoxide. Gla residues are without vitamin K supplementation 6 to 12 percent.
calcium binding groups which are essential for the Occurs between 24 hours and 7 days of life.
biological activities of proteins in which they are found. Gla
Classical VKDB: Associated with delay or insufficient
containing proteins are the coagulation factors II, VII, IX
feeding.
and X and procoagulants like proteins C, protein S, protein
Clinical presentation—without vitamin K supple
Z, osteocalcin, etc.
mentation incidence estimated is 0.01 to 0.44 percent.
• Vitamin K deficiency leads to the synthesis of under‐
• Bruises, gastrointestinal blood loss or bleeding from
carboxylated proteins unable to bind calcium and
the umbilicus and puncture sites
hence inactive. In vitamin K deficient individuals,
• Blood loss, however, can be significant, and intracranial
undercarboxylated forms of vitamin K dependant
hemorrhage, although rare, has been described.
coagulation proteins (proteins induced by vitamin
K absence PIVKA) are released from the liver into Late VKDB: Occurs in breastfed infants after 2nd month
the blood. PIVKA are inactive in the coagulation of life.
cascade. PIVKA II or undercarboxylated prothrombin
is a marker of subclinical vitamin K deficiency. As a
consequence of limited stores at birth, neonates are
prone to vitamin K deficiency if no sufficient intake is
provided.
Early Vitamin K Deficiency Bleeding

• Presents within 24 hours of birth.
• Seen in infants of mothers taking drugs which inhibit
vitamin K
• Anticonvulsants (carbamazepine, phenytoin and
barbiturates)
• Anti‐tubercular drugs (isoniazid, rifampicin)
• Antibiotics (cephalosporins)
• Vitamin K—antagonists (coumadin, warfarin).
• In preterms the liver is immature and incapable of
optimal synthesis of many of precursor proteins. The
action of vitamin K, as cofactor for gamma glutamyl Fig. 4 Cephalohematoma
Predisposing Factors for multidose oral prophylaxis. Unfortunately this

preparation is not available in most of the countries
• Prolonged antibiotic use including India.
• Prolonged/recurrent diarrhea Single dose (1.0 mg) of intramuscular vitamin K
• Cholestasis after birth is effective in the prevention of classic HDN.
• Malabsorption syndromes
Vitamin K, should be given to all newborn as a single,
• Clinical presentation may be severe with mortality rate
intramuscular dose of 0.5 to 1 mg. All premature infants
as high as 20 percent. Intracranial hemorrhage is seen
should receive vitamin K. Reports have shown very high
in 50 percent of these infants with residual neurological
plasma vitamin K levels in preterm infants receiving 0.5
damage in survivors.
to 1 mg at birth and adequate levels in those receiving 0.2
• In fully breastfed infants who did not receive vitamin
mg at birth.
K at birth, the incidence is between 1/15,000 and
Current empirical dosage recommendations for
1/20,000.
preterm infants:
• Dose of 0.3 mg for birth weights <1,000 g
Diagnosis of HDN
• 0.5 mg for those >1,000 g and <1,500 g.
The diagnosis of vitamin K deficiency may be suspected
from the results of coagulation screening. Initially there is CONCLUSION
isolated prolongation of the prothrombin time followed by
prolongation of the APTT, in association with the normal As a child is not a miniature adult so also a neonate
concentrations of fibrinogen, normal CBC and platelet is not a miniature child. It is important to realize and
count. to keep in mind the normal physiological variations
• Confirmation of the diagnosis requires measurements of hematolog ical parameters in term and preterm
of PIVKA II. neonates. The causes of bleeding in neonates are much
different from that in adult or an older child and hence
Treatment the approach to bleeding neonate is different than that
in older children.
• Intravenous vitamin K should be administered to
correct the existing deficiency. REFERENCES
• Factor replacement therapy may also be required
with fresh frozen plasma or prothrombin complex 1. Bleyer WA, Hakami N, Shephard TH. The development
concentrate (FII, FIX, FX). of hemostasis in the human foetus and newborn infant. J
Pediatr. 1971;79:838.
2. Gross SJ, Stuart MJ. Hemostasis in the premature infant.
Prevention Clin Perinatol. 1977;4(2):259-304.
• Routine prophylaxis with 1 mg vitamin K at birth. 3. Oski FA, Naiman JJ. Hematological problems in the newborn.
(Daily requirement of 5 to 10 μg in infants) and given 2nd edn, WB Saunders and Co, Philadelphia. 1972.p.236.
intramuscularly simply because that was the only 4. Glader BE, Buchman GR. Bleeding neonate. Pediatr.
1976;58:548.
product available.
5. Rosenberg RD. Physiology of coagulation—The fluid
• In 1990 an epidemiological study described an asso
phase. In: Hematology of Infancy and Childhood, Nathan
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C H A P T E R 12
Approach to Neonatal
Thrombocytopenia
Nitin K Shah
After birth the platelets are produced by megakaryocytes in the bone marrow and have a life of 9 to 10 days. In the fetus they are
produced predominantly in the liver. Platelets appear in the circulation in a fetus by 5 to 6 weeks of gestational age and platelet count
steadily rises to 159 ± 34 × 109/L by 10 to 17 weeks of gestation and 240 ± 60 × 109/L by 18 weeks of gestation, remaining constant at
that level thereafter until birth and beyond. Hence the lower limit of the platelet count in a newborn, irrespective of the gestational
age, remains at the adult level, i.e. > 150 × 109/L.
Thrombocytopenia in a newborn is defined as platelet They secrete granules that contain the factors to help
count lower than 150 × 109/L, though clinical bleeding aggregate further platelets and coagulation factors that
is usually seen with platelet count less than 50 × 109/L or help form a clot locally. They secrete vasoactive amines
even lower. Spontaneous and severe bleeding is usually that lead to vasospasm to prevent further blood loss.
seen when the platelet count drops to <20 × 109/L. Platelet membrane provides the surface required for
Presence of significant bleeding at higher platelet counts the coagulation to proceed.
should arouse suspicion of associated platelet dysfunction There are several growth factors that are required
as is seen in cases with inherited thrombocytopenia, e.g. for the formation of platelets from megakaryocytes
Bernard-Soulier syndrome. in the bone marrow. These include IL3, IL6, IL11, stem
A minimum platelet count of 7 × 109/L is required for cell factor, leukemia inhibitory factor, erythropoietin
maintenance of the vascular integrity, and in that sense and the most important, thrombopoietin (Tpo). Tpo,
a normal platelet count of > 150 × 109/L provides a lot of discovered in 1994, is a polypeptide glycoprotein that
reserve functional capacity. Similarly, 30 percent of the binds to its receptor c-mpl (named after its cell of
circulating platelets are stored in the spleen which can discovery, i.e. acute myeloproliferative leukemic cell)
be brought in to the circulation during the need or stress. expressed on the megakaryocytes, megakaryocytic
Normal bone marrow contains 6 × 106 megakaryocytes/kg precursors, hematopoietic precursor cells and platelets.
body weight. Tpo acts to commit early precursor cells to lineage
specific differentiation and enhances production and
The normal daily platelet production is approximately 35,000
release of platelets in the circulation. It acts via further
± 4,300 platelets per microliter of blood to maintain steady
levels of about 2,50,000 ± 1,00,000 platelets/mL. Normal bone signal conduction pathway and prevents apoptosis in
marrow can respond by increasing the platelet production by 7 the target cells. It also appears to play a role as pan-
to 8 fold during periods of thrombocytopenia. hematopoietic growth factor which explains why defects
in its metabolism ultimately leads to total bone marrow
Platelets are required at each stage of hemostasis. failure, as seen in rare cases of congenital amegakaryocytic
With the help of von Willebrand factor, they adhere to thrombocytopenia. Tpo levels are inversely proportional
the exposed endothelial cells at the site of injury and help to the total megakaryocytic mass. High levels of Tpo
to form the primary platelet plug. indicate aregenerative type of thrombocytopenia.
The bone marrow in neonates is very sensitive to Table 1 Causes of neonatal thrombocytopenia
insults like hypoxia which leads to suppression of the
• Perinatal hypoxia:
megakaryocytes more than other precursor cells. This
explains why thrombocytopenia is more common than – Maternal diabetes
other cytopenias in neonate, especially following hypoxic – Maternal hypertension, pre-eclampsia
– Intrauterine growth restriction (IUGR)
stress.
• Immune causes:
Incidence – Alloimmune
– Autoimmune
There very few prospective studies which have specifically
• Infections:
looked at the incidence of thrombocytopenia in newborns.
– Perinatal infections: Bacterial, TORCH, HIV
Recent prospective studies have shown that 0.5 to 4.1 percent – Late onset sepsis/NEC
of the neonates have thrombocytopenia. 30 percent of the • Disseminated intravascular coagulation (DIC):
babies admitted to the NICU have platelet counts < 150 × 109/L
– Asphyxia, infections
and around 10 percent of the babies will have counts < 100 ×
– Mis-matched transfusions
109/L.
– Congenital thrombotic thrombocytopenic purpura (TTP)
• A
neuploidy: Trisomy 18, trisomy 13, trisomy 21, Turner’s
Fortunately most of the episodes of thrombocytopenia syndrome
in NICU are mild to moderate but 20 percent of them
• Inherited
(6 percent of the neonatal admissions) will have severe
thrombocytopenia with counts < 50 ×109/L who are at a Giant platelets: Bernard-Soulier syndrome, May-Hegglin
risk of severe bleeding including intracranial bleeds. 80 anomaly, Sebastian syndrome, Fechtner syndrome, Epstein
percent of these babies with severe bleeds are sick, pre- syndrome, Alport’s syndrome, Montreal platelet syndrome,
Quebec syndrome, Gray platelet syndrome
terms who have sepsis or necrotizing enterocolitis (NEC).
• Immunodeficiency: Wiskott-Aldrich syndrome
• X-linked thrombocytopenia
Causes of Neonatal Thrombocytopenia • Hemophagocytic lymphohistiocytosis
While traditionally one can classify the causes of neonatal • Bone marrow failure: Fanconi’s anemia
thrombocytopenia in to those caused by (Table 1). • Thrombocytopenia with absent radius (TAR) syndrome
• Congenital amegakaryocytic thrombocytopenia
• Premalignant: Monosomy 7
Reduced Production
• Others
• Increased destruction or consumption
• C
onsumption: Kasabach-Merritt syndrome, vascular
• Sequestration thrombosis, hepatic hemangioendothelioma
• Dilutional
– Metabolic: Propionic academia, methylmalonic academia
• A combination of these.
– Miscellaneous
It does not help while approaching a case of neonatal – Congenital leukemia
thrombocytopenia as most of the causes listed are rare. – Exchange transfusions
It is easy to group the cases as per their onset, the nadir (i) Rh disease of newborn
of counts, the type of recovery, their mechanism of disease, (ii) Subcutaneous fat necrosis of the neonate
their associated findings, presence of physical anomalies,
presence of immunodeficiency and syndromes which
helps to narrow down the differential diagnosis. maternal diabetes, placental insufficiency, maternal
hypertension or pre-eclampsia. The platelet counts
Patterns of Neonatal Thrombocytopenia in low normal in the first 2 to 3 days, falls to a nadir at
4 to 5 days and recovers by 7 to 8 days. The counts rarely
While there are many etiological causes of neonatal
drop below 50 × 109/L. If the pattern of recovery does not
thrombocytopenia, this helps us narrow down the etiology.
follow this path one has to think of other causes of early
Most of the patients fall into two types of patterns.
onset thrombocytopenia like chromosomal anomalies,
Early onset type 75 percent of the neonatal throm
congenital or perinatal infections, inherited causes or
bocytopenia. Most babies have low platelet at birth or soon
immune causes.
after birth, in the first 72 hours of life. The causes include
thrombocytopenia due to chronic or acute hypoxia like in Late onset type: This accounts for the remaining 25 percent
a preterm, IUGR baby risk factors like perinatal asphyxia, of the cases and usually associated with late onset sepsis
Chapter-12 Approach to Neonatal Thrombocytopenia 79
with or without NEC. The counts drop to a low level and methylprednisolone in the dose of 1 mg/kg 8 hourly.
there may be significant bleeding in these babies. The treatment is required for 1 to 6 weeks till the
platelet counts is in the safe range.
Important Causes of Neonatal The severity depends on the maternal antibody titers.
Thrombocytopenia The second born neonate is usually severely affected, if
the first baby was symptomatic or had intracranial bleeds
Neonatal Alloimmune Thrombocytopenia especially with intrauterine onset. In such cases, fetal
This is the platelet equivalent of the Rh disease where allo- monitoring of the platelet counts is advocated and the
antibodies are produced by the mother against the paternal mother is given IVIg in the dose of 1 to 2 gm/kg/week
platelets antigens inherited by the newborn from the with prednisolone in the dose of 0.5 to 1 mg/kg/day. The
father which are missing in the mother. These antibodies fetus can also be given in utero HPA compatible platelet
pass across the placenta and destroy the baby’s platelets transfusions if the maternal treatment alone dose not
resulting in often moderate to severe thrombocytopenia. help. Elective LSCS is also advocated to prevent trauma to
Unlike in Rh disease, in more than 50 percent of cases the head.
first born babies are also affected. In 80 percent of cases
the missing platelet antigen in the mother is HPA-1a Neonatal Autoimmune Thrombocytopenia
(also known as PlA1), in 10 to 15 percent it is HPA-5b and
in the rest it is HPA-3a, HPA-1b or some other unknown This occurs due to transplacental transfer of the anti-
and rare antigen. In Asian communities, it is usually the platelet autoantibodies. It is suspected because of the prior
HPA-4 antigen. Development of the alloantibodies in history of thrombocytopenia in the mother due to ITP, SLE
HLA-1A negative women is strongly associated with HLA or some such auto-immune disease. The platelet count
DRB3 0101 (odds ratio of 140). in the mother may be normal even when the antibodies
1:350 pregnancies are associated with maternal anti- persist, especially following splenectomy. The antibodies
platelet antibodies, but only 1:1000 livebirths are associated are formed against the common platelet antigens like
with neonatal alloimmune thrombocytopenia proving gp1b/IX or gpIIb/IIIa. The disease is generally milder than
that a good number of them are silent or unnoticed. The the alloantibody mediated neonatal thrombocytopenia.
affected neonates, who otherwise look well, present with The newborn is usually normal at birth with even normal
purpura, bruising, mucosal bleeds and thrombocytopenia platelet count which drops after few days. Less than 10 to 15
at birth or immediately after birth. The platelet count percent of the babies have platelet counts < 50 × 109/L and
is usually < 30 × 109/L. The 20 percent of the affected the bleeding is usually milder. Intracranial bleeds occur
neonates develop severe bleeding including intracranial in < 1 to 2 percent of babies. In most the platelet counts
bleeds. 20 to 50 percent of the intracranial bleeds start in recover in the next 2 to 3 weeks though rarely they may
the intrauterine life and 20 percent of the survivors develop take a longer time. There is no role of antenatal treatment
long-term neurodevelopmental sequelae. The diagnosis is or LSCS. After birth, treatment is required in presence of
done by laboratory testing for the antiplatelet antibodies. significant bleeding or platelet counts < 30 × 109/L. The
treatment consists of IVIg and/or steroids as in the case
Treatment of alloimmune thrombocytopenia. Like in ITP, there is no
role of platelet transfusions as the autoantibodies will react
• Treatment of the mildly affected babies without
with all the platelets as the target antigens are common to
mucosal bleeds or with platelet count above 30 × 109/L
all the donors. The prognosis is usually excellent.
is conservative.
• Those with significant mucosal bleeds, evidence of Thrombocytopenia following hypoxia: This is the most
intracranial bleeds or platelet count < 30 × 109/L need common cause of early onset pattern of thrombocytopenia.
specific treatment including platelet transfusions, The newborn is usually a preterm or an IUGR baby
IVIg and steroids. HPA compatible platelets need to who has risk factors like maternal diabetes, maternal
be transfused. While one can use washed mother’s hypertension or pre-eclampsia or perinatal asphyxia. The
platelets (which obviously will be negative for the counts are 100 × 109/L in first few days, fall to a nadir of
missing platelets), HPA-1a negative platelets are 50 × 109/L at 4 to 5 days and recover by 7 to 8 days. The
usually stored and easily available in major centers in counts often recover above the normal levels. They
the west. may also have neutropenia and polycythemia besides
• If severe thrombocytopenia persists in spite of the thrombocytopenia. Though platelet destruction is an
platelet transfusions, one need to give IVIg in the dose important cause, especially in acute hypoxia where DIC
of 2 gm/kg body weight over 2 to 5 days and or IV with thrombocytopenia is seen in 50 percent of such
babies, in most with chronic hypoxia there is decreased and 15 to 20 percent of the patients have significant
production too as the major contributing factor. Less bleeding. Platelet transfusions are required for the
than 10 percent of such babies have evidence of DIC symptomatic patients.
and the Tpo levels are high suggestive of aregenerative
type of thrombocytopenia. There is evidence of Consumption of Platelets
increased erythropoiesis with increased levels of EPO
and circulating normoblasts. This with increased Tpo • Disseminated intravascular coagulation (DIC):
levels suggests preferential differentiation towards Con sumption and destruction of platelets occur
erythropoiesis with depressed megakaryopoiesis. This is in DIC which usually occurs following perinatal
because the megakaryocytes are sensitive to hypoxia and asphyxia or neonatal infection. The newborn will
are suppressed with hypoxia temporarily. The bleeding have the signs of the primary disease and will have
is usually mild. Most do not need any specific treatment. severe thrombocytopenia with significant bleeding.
Those who are symptomatic need platelet transfusions. The patient may have the signs of thrombosis like
gangrene. The prothrombin time and activated
partial thromboplastin time will be prolonged and
Thrombocytopenia due to Neonatal Infections
D-dimers or the fibrinogen split products (FDP) will
• Perinatal sepsis: Perinatal bacterial sepsis can occur in be raised. The peripheral smear will show evidence of
1 to 2/1000 livebirths. The common organisms include microangiopathic hemolytic anemia along with low
group B sepsis or E. coli in the west and gram-negative platelet counts. Treatment will include use of platelets
organisms like Klebsiella or E. coli in our country. The 50 along with fresh-frozen plasma besides treatment of
percent of the sepsis cases develop thrombocytopenia the primary cause.
and DIC is an important cause of low platelet counts. • Kasabach-Merritt syndrome: Large hemangioma
The platelet counts are low early in life and recover can occur over extremities, trunk, neck or in internal
with the treatment of the primary cause. organs. Platelets can get trapped in the slow circulation
• Congenital infections: TORCH group of infection can within the hemangioma and can lead to local
lead to thrombocytopenia. Most, but not all, will have consumption or localized DIC with consumption of
other signs like jaundice, anemia, congenital defects, other coagulation factors too. There may be multiple
hepatosplenomegaly, etc. Acute cytomegalovirus afferent and feeders to the hemangioma. The mass
(CMV) is a common cause of such thrombocytopenia. may not be restricted to the defined anatomical layers
0.5 to 1 percent of all newborns develops congenital and usually infiltrates deep in to the tissues making
CMV infection and though only 10 to 15 percent of it difficult to excise the hemangioma. The diagnosis
them are otherwise symptomatic, 75 percent of them is obvious when the hemangioma is seen externally,
have low platelet counts. There may be associated whereas it can be difficult if the hemangioma is in
neutropenia and the thrombocytopenia may persist for some organ. Very low platelet counts, evidence of
several months. Similarly, 40 percent of those infected microangiopathy, raised D-dimers or FDP levels and
with toxoplasma can develop low platelet counts. high retic count will suggest the diagnosis. Imaging
Other common cause in our country is perinatal HIV and vascular studies will help define the extent of the
infection and rubella infection as MMR is still not a lesion which may be actually much widespread than
part of our national schedule. appearing visibly. Medical treatment includes use
• Late onset sepsis: A common cause of late onset of of FFP followed by anti-fibrinolytic agents hoping to
thrombocytopenia is late onset of sepsis with or induce thrombosis of the important feeding vessels.
without NEC. Thrombocytopenia may be the first sign Interferon therapy and vincristine can help shrink
of infection, though usually other signs of infection the lesion permanently; however, they have their own
are also present. The platelet counts falls after the first side effects. Surgery often is mutilating and may land
72 hours and may take around 7 to 8 days to recover up into amputation.
after the treatment for sepsis is started and effective.
Less than 10 percent of these patients have DIC. The
Congenital and Inherited Thrombocytopenia
Tpo levels are high suggesting aregenerative type of
thrombocytopenia. This is also evident by the fact This includes causes like chromosomal anomalies and
that thrombocytopenia persist even after the infection rare inherited causes of low platelet count. The counts
is under control. This suggests that the cause of low are low at or soon after the birth. In most, but not all,
platelet is less production due to suppression of the there are other features of the basic disease. The platelet
megakaryocytes. The counts are usually < 30 × 109/L count can be very low in some of them needing platelet
transfusions and other treatment. The counts take a transfusions for significant bleeding. Stem cell trans
long to recover and may be low for months. Those with plant will be curative in this condition.
associated thrombasthenia bleed a lot at relative higher • May-Hegglin anomaly: This is an autosomal dominant
platelet counts. Some of them will have typical platelet condition characterized by thrombocytopenia,
morphology on smear examination. giant platelets, large inclusions in granulocytes and
• Chromosomal anomalies: The incidence of throm monocytes known as Dohle bodies and presence of
bocytopenia in various aneuploidy cases varies from physical defects like hearing loss, renal failure and
86 percent in trisomy 18 to 31 percent in trisomy 13, cataract. The platelet count is moderately low in the
6 percent in trisomy 21 and 31 percent in Turner’s range of 80 to 1,00,000/cumm but with significant
syndrome. Usually the thrombocytopenia is not severe bleeding due to associated platelet dysfunction. The
and many a times, it is associated with neutropenia defect is in the MYH9 gene that codes for Myosin
and polycythemia as seen in placental insufficiency. II-A which is the only protein expressed in platelets
The mechanism of thrombocytopenia may also be and neutrophils, which explains the characteristic
similar to that seen in placental insufficiency. defects seen in platelets and neutrophils. DDAVP,
• Inherited thrombocytopenia: Inherited thrombo anti-fibrinolytic agents help control bleeding. Platelet
cytopenias are a rare but interesting group of disorders transfusions are rarely required.
which provided dome insight in to the molecular • Gray platelet syndrome: It is an autosomal recessive
mechanisms for megakaryopoiesis. For some of these disorder with mild thrombocytopenia and significant
disorders, the molecular defects have been identified, bleeding. The platelets are unable to store alpha granule
like mutation of the CMPL in congenital amegakaryocytic proteins. This leads to poor aggregation especially with
thrombocytopenia, defect in transduction pathway after thrombin, ADP and collagen. The platelets appear
binding of Tpo to CMPL in TAR syndrome, mutations large, gray and bland with vacuolations. The treatment
in CBFA2 transcription factor gene RUNX1 (AML1) is platelet transfusion for significant bleeding.
in familial platelet syndrome with predisposition to • Inherited thrombocytopenia with immunodeficiency:
AML, mutation in GATA-1 transcription factor (xp This includes Wiskott-Aldrich syndrome and hemoph
11) in X-linked thrombocytopenia with microcytosis, agocytic lymphohistiocytosis.
mutation in myosin heavy chain A gene (MYH9) in giant • Wiskott-Aldrich syndrome: This is an X-linked disorder
platelet syndromes including May-Hegglin anomaly characterized by thrombo cytopenia, eczema and
and mutations in the WASP gene in Wiskott-Aldrich immunodeficiency. It is caused by mutations in the
syndrome and X-linked thrombocytopenia. Most, but not WASP gene located at Xp11-12 band. The Wiskott-
all, have associated congenital anomalies to point to the Aldrich syndrome protein is known to be involved in
possible diagnosis. Many of them have abnormal platelet the signal transduction and it regulates actin filament
morphology like giant platelet on peripheral smear assembly in platelets as well as lymphocytes affecting
examination. Some have associated platelet dysfunction their cytoskeleton. This explains the association of
leading to more bleeding than expected for the platelet thrombocytopenia and immunodeficiency. Patients
count. present with thrombocytopenia early in life with more
• Thrombocytopenia with giant platelets: This group bleeding than expected based on the platelet counts.
includes the disorders like Bernard-Soulier syndrome; The platelets are small in volume and the mean
May-Hegglin syndrome and its variants like Sebastian platelet volume (MPV) is characteristically low (as is
syndrome, Fechtner syndrome, Epstein syndrome and also seen in patients with TORCH group of infection).
Alport syndrome; Montreal platelet syndrome; Quebec Patient usually has lower GI bleeding. Eczema and
syndrome and gray platelet syndrome. immunodeficiency develop as the child grows.
• Bernard-Soulier syndrome: It is an autosomal recessive Some patients have milder phenotype with minimal
disorder. The platelet count is moderately low but immunodeficiency as the defect is restricted only to
the bleeding is quite significant. The platelets can be the platelets even when they have the same WASP
as large as 20 microns in diameter with cytoplasmic gene mutations (though restricted only to the exon 2
vacuoles. The defect is in the demarcation membrane of the gene), these patients are grouped as X-linked
system. This results in defect in gpIb-V-IX complex thrombocytopenia. Often these patients are mistaken
leading to poor adhesion. Diagnosis is made by absent as ITP later in the life.
response to Ristocetin in platelet function study. A close The treatment of Wiskott-Aldrich syndrome includes
differential with similar results is von Willebrand disease control of bleeding with platelet transfusions and treatment
which can be differentiated by normal von Willebrand of infections. In those with difficulty, splenectomy will also
factor levels and pattern. Treatment includes platelet help as also IVIg at times. Bone marrow transplantation
is the only curative treatment possible at present. The 10 While platelet transfusions help controlling the bleeding,
percent of the patients can progress to develop lymphoma the only curative treatment is bone marrow transplant. The
later in their life. 50 percent of them will progress to aplastic anemia over
next 5 years. And 5 percent of them can develop leukemia.
Hemophagocytic Lymphohistiocytosis (HLH) IL3 and GM-CSF have limited role and Tpo has no role
what so ever in the treatment of these patients.
This is a rare but interesting disease caused by mutations
in the genes for perforin or granzymes involved in the
killing of phagocytosed material by the phagosomes of the
Fanconi’s Anemia and TAR
macrophage-monocyte series and NK cells. Absence of Both are autosomal recessive disorders characterized by
effective killing by the phagosomes leads to unnecessary inherited bone marrow failure. Both have characteristic
stimulation of the macrophages and monocytes leading to physical anomalies. Both are caused by known mutations.
production of cytokines by these cells leading to what is Mutations in HOXA10, HOXA11 and HOXD11 genes are
described as cytokine storm which leads to all the symptoms implicated for TAR whereas more than 3 different types
like hemophagocytosis, cytopenias, liver dysfunction, CNS of Fanconi’s anemia gene mutations are identified for
changes, fever, DIC, increased triglycerides levels, etc. It is Fanconi’s anemia.
an autosomal recessive disorder which can present even The TAR presents with thrombocytopenia, bleeding
at or soon after the birth with fever, thrombocytopenia, and characteristic forearm deformity due to absence of
convulsions, hyperbilirubinemia and cytopenias. radius. Isolated abence of radii is seen in only 10 percent
The diagnosis is made by high index of suspicion and of the patients and 50 percent of the patients also have
demonstrating hemophagocytes in the bone marrow associated defects in ulna, knees or humorous, besides
or organs like liver, spleen or lymph nodes. Sometimes, absent radii. The thumb on the affected side is always
hemophagocytes are also seen on the peripheral blood present in TAR, which differentiates it from Fanconi’s
smears. The treatment includes use of steroids and VP- anemia (which rarely can present in neonatal period) where
16 to induce remission. Bone marrow transplant will cure such radial deformities are seen but the thumb is always
those who do not respond to conservative management. absent from the affected side. Besides this, other anomalies
Inherited thrombocytopenia with bone marrow seen in Fanconi’s anemia seen include short stature,
failure: This includes three disorders that is: microcephaly, mental retardation, hyperpigmentation,
1. Congenital amegakaryocytic thrombocytopenia, hypogonadism, renal anomalies, other skeletal anomalies
2. Thrombocytopenia with absent radius syndrome (TAR like rudimentary thumb, absent thumb, triphalangeal
syndrome) thumb, etc. Fanconi’s anemia usually presents as anemia
3. Fanconi’s anemia. in later life though it can presents in neonatal period in
10 percent of the cases. While TAR tends to improve after
Congenital Amegakaryocytic Thrombocytopenia the age of one year, Fanconi’s anemia usually progresses
with time in to complete bone failure and has high chances
This is a rare autosomal recessive disorder presenting as of developing malignancies. The only curative treatment is
neonatal thrombocytopenia which progresses over the bone marrow transplant with proper conditioning regime
time to total bone marrow failure. It presents with bleeding avoiding radiation. Another syndrome of congenital
and thrombocytopenia in the first few days to weeks of thrombocytopenia with radioulnar synostosis (CTRUS)
life where it may be confused with many other common is described where the patient has absent pronation-
etiologies especially alloimmune thrombocytopenia. supination due to fusion of radius and ulna. It behaves
20 percent of the patients can develop intracranial similar to TAR with improved platelet counts after the age
hemorrhage. 10 to 30 percent of them can have associated of 1 year. Defect in HOX 11a is implicated in this condition.
orthopedic or CNS anomalies. It is only when the patient
dose not recover that a bone marrow is ordered which
will pick up absent or grossly reduced megakaryocytes
Miscellaneous Causes
with normal granulopoietic and erythropoietic elements. There are some rare but important and distinct causes of
It is caused by mutations in the CMPL genes which neonatal thrombocytopenia like osteopetrosis which leads
results in marked maturation of the megakaryocytes and to thrombocytopenia and later bone marrow failure due to
its precursors. As the action of Tpo via CMPL is vital for calcification of marrow spaces; congenital leukemia and
maturation of other hemopoietic cells, even the other series metastatic neuroblastoma which cause thrombocytopenia
will get affected with time including the stem cells due due to bone marrow infiltration by the malignant cells and
to lack of preservation of apoptosis induced by Tpo. This organic acidemias and other metabolic disorders which
explains why there is total bone marrow failure with time. all can lead to bone marrow suppression.
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of hemophagocytic lymphohistiocytosis with HLH-94 15. Vanden OS, de Hass M, Vanden BE. Screening for c-mpl
immunochemotherapy and bone marrow transplantation. mutations in patients with congenital amegakaryocytic
Blood. 2002;100:2367-73. thrombocytopenia identifies a polymorphism. Blood.
7. Hohlfeld P, Forestier F, Kaplan C, Tissot JD, Daffos F. Fetal 2001;17:3675-6.
thrombocytopenia: a retrospective survey of fetal blood 16. Webert KE, Mittal R, Sigouin C, Heddle NM, Kelton JG.
samplings. Blood. 1994;84:1851-6. A retrospective 11-year analysis of obstetric patients
8. Kaplan C. Alloimmune thrombocytopenia of the fetus and with idiopathic thrombocytopenic purpura. Blood. 2003;
the newborn. Blood Rev. 2002;16:69-72. 102:4306-11.
S E C T I O N 3
RBC and WBC Disorders
CHAPTERS OUTLINE
13. Introduction and Classification of Anemias in Children
14. Nutritional Anemia in Infancy, Childhood and Adolescents
15. Megaloblastic Anemia
16. Anemia of Chronic Disease
17. Thalassemia Syndromes
Mamta Vijay Manglani, Ambreen Pandrowala
Ratna Sharma, MR Lokeshwar
18. Sickle Cell Anemia in Children
19. Antenatal Diagnosis of Hemoglobinopathies
20. Red Cell Membrane Disorders (Spherocytosis, Elliptocytosis, Stomatocytosis)
21. Red Cell Enzymopathy
22. Autoimmune Hemolytic Anemia
Rajiv Kumar Bansal
23. Paroxysmal Nocturnal Hemoglobinuria
24. Diagnosis and Management of Acquired Aplastic Anemia in Children
Nitin K Shah
25. Inherited Bone Marrow Failure Syndromes
Revathi Raj
26. Benign Disorders of Neutrophils
Bharat R Agarwal
C H A P T E R 13
Introduction and Classification
of Anemias in Children
Anemia can be defined as a reduction in the hemoglobin concentration, hematocrit, or the number of red blood cells (RBC) per cubic
millimeter. Conventionally, the lower limit of the normal range is set at two standard deviations below the mean for the normal
population. Thus, 2.5 percent of the normal population will be mistakenly classified as anemic. The primary function of red blood
cells is to deliver adequate quantities of oxygen to meet the body’s metabolic demands. Thus, a measure of oxygen metabolism
and accompanying cardiovascular compensation should be considered in defining anemia. Children with cyanotic congenital heart
disease, respiratory insufficiency, or hemoglobinopathy that alters oxygen affinity can be functionally anemic with hemoglobin in the
normal range.1,2
Hemoglobin (Hb) concentration varies with age, with extrusion of the nucleus that causes loss of RBC synthetic
higher values being present in the newborn and adolescent ability. Normal RBCs survive an average of 120 days,
male. The high Hb level at birth occurs in response to the while abnormal RBCs can survive as little as 15 days. The
low fetal ambient oxygen tension (PO2 of 30 mm Hg). hemoglobin molecule is a heme-protein complex of two
Immediately after birth, the rise in arterial PO2 (around 90 pairs of similar polypeptide chains. There are six types
mm Hg) decreases erythropoietin production, producing of hemoglobin in developing humans: the embryonic,
the ‘‘physiologic anemia’’ seen in the first 2 months of Gower 1, Gower 2, Portland, fetal hemoglobin (HbF) and
life (i.e. Hb concentration of around 10 g/dL at 2 months normal adult hemoglobin (HbA and HbA2). HbF is the
of age). This period is followed by a steady rise in Hb, primary hemoglobin found in the fetus. It has a higher
reaching a maximum at about 14 years of age. The zenith
affinity for oxygen than adult hemoglobin, thus increasing
of hemoglobin concentration occurs earlier in preterm
the efficiency of oxygen transfer to the fetus. The relative
infants and may be more in terms of lower Hb levels as
quantities of HbF rapidly decrease to trace levels by the
well as prolonged anemia. Table 1 gives the approximate
values of some vital hematologic parameters in pediatric age of 6 to 12 months and are ultimately replaced by the
age group. adult forms, HbA and HbA2.
PHYSIOLOGY OF HEMOGLOBIN CLASSIFICATION OF ANEMIAS

PRODUCTION
Anemias can be classified according to their appearance
Erythropoietin is the primary hormone regulator of red in the microscope, which is the morphologic. The former
blood cell (RBC) production. In the fetus, erythropoietin methodology classifies anemias into:
comes from the monocyte/macrophage system of the Morphologic classification:
liver. Postnatally, erythropoietin is produced in the • Microcytic
peritubular cells of the kidneys. Key steps in red blood cell • Macrocytic
differentiation include condensation of red cell nuclear • Normocytic types.
material, production of hemoglobin until it amounts The latter classification categorizes anemias into three
to 90 percent of the total red blood cell mass and the main categories (Table 2)3:
88 Section-3 RBC and WBC Disorders
Table 1 Age-specific blood cell indexes

Age Hemoglobin g/dL Hematocrit (%) MCV, mm3 (fL) MCHC, g/dL (g/L) Reticulocytes
(g/L)
• 2
6–30 weeks’ 13.4 (134) 41.5 (0.42) 118.2 (118.2) 37.9 (379) –
gestation*
• 28 weeks’ gestation 14.5 (145) 45 (0.45) 120 (120) 31.0 (310) (5 to 10)
• 32 weeks’ gestation 15.0 (150) 47 (0.47) 118 (118) 32.0 (320) (3 to 10)
• Term (cord)
†
16.5 (165) 51 (0.51) 108 (108) 33.0 (330) (3 to 7)
• 1–3 days 18.5 (185) 56 (0.56) 108 (108) 33.0 (330) (1.8–4.6)
• 2 weeks 16.6 (166) 53 (0.53) 105 (105) 31.4 (314)
• 1 month 13.9 (139) 44 (0.44) 101 (101) 31.8 (318) (0.1–1.7)
• 2 months 11.2 (112) 35 (0.35) 95 (95) 31.8 (318)
• 6 months 12.6 (126) 36 (0.36) 76 (76) 35.0 (350) (0.7–2.3)
• 6 months–2 years 12.0 (120) 36 (0.36) 78 (78) 33.0 (330)
• 2–6 years 12.5 (125) 37 (0.37) 81 (81) 34.0 (340) (0.5–1.0)
• 6–12 years 13.5 (135) 40 (0.40) 86 (86) 34.0 (340) (0.5–1.0)
• 12–18 years
– Male 14.5 (145) 43 (0.43) 88 (88) 34.0 (340) (0.5–1.0)
– Female 14.0 (140) 41 (0.41) 90 (90) 34.0 (340) (0.5–1.0)
• Adult
– Male 15.5 (155) 47 (0.47) 90 (90) 34.0 (340) (0.8–2.5)
– Female 14.0 (140) 41 (0.41) 90 (90) 34.0 (340) (0.8–4.1)
Abbreviations:
MCV: Mean corpuscular volume; MCHC: Mean corpuscular hemoglobin concentration.
* Values are from fetal samplings.
† Less than one month, capillary hemoglobin exceeds venous: 1 hour—3.6 gm difference; 5 days—2.2 gm difference; 3 weeks—1.1
gm difference. Adapted with permission from Siberry GK, Lannone R, Eds. The Harriet Lane handbook: a manual for pediatric house
officers, 15th edn. St Louis: Mosby, 2000.
Table 2 Physiologic classification of anemia3

Classification of anemia according to underlying mechanism
Mechanism Specific examples
Blood loss
Acute blood loss Trauma
Chronic blood loss Gastrointestinal tract lesions, gynecologic disturbances
Increased red cell destruction (hemolysis)
Inherited genetic defects
Red cell membrane disorders Hereditary spherocytosis, hereditary elliptocytosis
Enzyme deficiencies
Hexose monophosphate shunt enzyme deficiencies G6PD deficiency, Glutathione synthetase deficiency
Glycolytic enzyme deficiencies Pyruvate kinase deficiency, Hexokinase deficiency
Hemoglobin abnormalities
Deficient globin synthesis Thalassemia syndromes
Structurally abnormal globins (hemoglobinopathies) Sickle cell disease, unstable hemoglobins
Acquired genetic defects
Deficiency of phosphatidylinositol-linked glycoproteins Paroxysmal nocturnal hemoglobinuria
Contd...
Chapter-13 Introduction and Classification of Anemias in Children 89
Contd...
Mechanism Specific examples
Antibody-mediated destruction Hemolytic disease of the newborn (Rh disease), transfusion
Mechanical trauma reactions, drug-induced, autoimmune disorders
Microangiopathic hemolytic anemias Hemolytic uremic syndrome, disseminated intravascular
coagulation, thrombotic thrombocytopenia purpura
Cardiac traumatic hemolysis Defective cardiac valves
Repetitive physical trauma Bongo drumming, marathon running, karate chopping
Infections of red cells Malaria, Babesiosis
Toxic or chemical injury Clostridial sepsis, snake venom, lead poisoning
Membrane lipid abnormalities Abetalipoproteinemia, severe hepatocellular liver disease
Sequestration Hypersplenism
Decreased red cell production
Inherited genetic defects
Defects leading to stem cell depletion Fanconi anemia, telomerase defects
Defects affecting erythroblast maturation Thalassemia syndromes
Nutritional deficiencies
Deficiencies affecting DNA synthesis B12 and folate deficiencies
Deficiencies affecting hemoglobin synthesis Iron deficiency anemia
Erythropoietin deficiency Renal failure, anemia of chronic disease
Immune-mediated injury of progenitors Aplastic anemia, pure red cell aplasia
Inflammation-mediated iron sequestration Anemia of chronic disease
Primary hematopoietic neoplasms Acute leukemia, myelodysplasia, myeloproliferative disorders
Space-occupying marrow lesions Metastatic neoplasms, granulomatous disease
Infections of red cell progenitors Parvovirus B19 infection
Unknown mechanisms Endocrine disorders, hepatocellular liver disease
• Classification and on the basis of the underlying The next step is to evaluate the red cell indices. Of
physiologic process. these, the MCV is the most useful. MCV is the mean
• Anemias associated with decreased red cell production volume of single red cell expressed in femtoliters. It is a
• Anemias due to increased red cell destruction red cell index directly measured by the electronic counter
• Anemias due to blood loss. and enables the classification of anemia by red blood cell
It is also important to realize that as in other issues the size as microcytic, normocytic, or macrocytic (Flow charts
diagnosis of anemia can be easily made from the history 1 and 2) (Figs 1 and 2).4
and physical examination. Some of the salient features in While this classification is arbitrary and categories are
the history as well as physical examination are given in not mutually exclusive, it provides a useful starting point
Tables 3 and 4. for directing further evaluation. In children less than age
10 years and above 2 years, the lower limit for the MCV is
Role of complete blood counts (CBC) and peripheral smear approximately 70 fL + age in years. After 6 months of age, the
(PS) for diagnosis of anemia: CBC, red cell indices and approximate upper limit for the MCV is 84 + 0.6 fL per year
PS play an immensely vital role in helping a clinician for until the upper limit of 96 fL in adults is reached. The mean
the differential diagnosis of anemia. It is imperative that corpuscular hemoglobin (mean Hb content of a single red
care is taken in obtaining the sample. Venous samples cell expressed in pictograms, MCH) and mean corpuscular
are preferred. When capillary samples are obtained, it hemoglobin concentration (hemoglobin concentration
is important that the extremity is warm and that a free within individual red cells expressed in %, MCHC) are
flow of blood is obtained. To ensure accurate results, an calculated values and generally less diagnostic. The MCH
adequate volume of blood should be obtained to avoid usually parallels the MCV. Both the MCV and MCH have
excessive dilution by the anticoagulant. Analysis of the small measurement errors and biological variations. The
hemoglobin concentration is preferred as it is determined MCHC is a measure of cellular hydration status. It remains
by direct spectrophotometry. In contrast, the hematocrit is relatively constant throughout development and in most
determined indirectly by calculations using the red count clinical settings. A high value (> 35 g/dL) is characteristic
and mean corpuscular volume (MCV). of spherocytosis, while a low value is most commonly
Table 3 History of a child with anemia

Symptom Implications
Maternal history Anemia, blood loss during pregnancy, pica may predispose for iron deficiency
Family history Anemia, splenomegaly, gallstones, splenectomy, leg ulcers, jaundice, transfusion dependency
indicates hemolytic anemia. Look at the ethnicity and race as sickle cell anemia and thalassemia are
more common in certain communities
Age Iron deficiency is unlikely in term infants before 6 months of age. Anemia which manifests in the
neonatal period is the result of recent blood loss, isoimmunization, or initial manifestation of
congenital hemolytic anemia or congenital infection
Gender G6PD and PK deficiency seen in males as inheritance is X linked
Neonatal history Anemia and jaundice may point to a congenital hemolytic anemia such as HS, prematurity
predisposes to an early iron deficiency state
Pica Indicates iron deficient state
Diet history Predominant cows milk leads to cows milk allergy and GI blood loss leading to iron deficiency
Infections Hepatitis induced aplasia, infection assorted hemolysis, parvovirus induced red cell aplasia, worm
infestation leads to blood loss and Fe deficiency
Drugs Oxidant induced hemolytic anemias, phenytoin induced megaloblastic anemia, drug induced
aplastic anemia
Hemoglobinuria Indicates an intravascular hemolysis like autoimmune or G6PD deficiency
Bleeding manifestations May indicate a second cell line being involved or a bleeding or clotting defect which may be causing
the anemia
Diarrhea Malabsorption or inflammatory bowel disease
Dactylitis or painful episodes Sickle cell anemia, bony pains may indicate a infiltrative disorder of marrow
Blood loss Acute or chronic
Table 4 Physical examination of a child with anemia

Physical findings Implications
Tachycardia, respiratory distress Acute anemia or decompensated state
Hemolytic facies Chronic hemolytic anemia
Platonychia, koilonychia Iron deficiency anemia
Glossitis Iron deficiency or cobalamin deficiency
Blue sclera Iron deficiency anemia
Vitiligo Pernicious anemia
Telangiectasias on body Similar lesions in the GIT
Splenomegaly, jaundice Hemolytic anemia
Lymphadenopathy Infiltrative disorder
Hepatomegaly CCF, infiltrative disorder
Cardiomegaly Chronic anemia
Bleeding manifestations Bleeding diathesis or secondary to marrow infiltration
Knuckle pigmentation, loss of position or B12 deficiency
vibration sense
Failure to thrive Chronic anemia, systemic illnesses
Facial or digital anomalies Fanconi’s anemia, Diamond-Blackfan syndrome
Rectal examination Rectal varices or polyp
associated with iron deficiency. The red cell volume The next step is to assess the white blood cell (WBC)
distribution width (RDW) reflects the variability in cell and platelet counts. Is the anemia isolated or are other
size and can be used as a measure of anisocytosis.2,4 Some cell lines affected? Pancytopenia results from disorders
kind of anemias can easily be diagnosed on distinctive that are distinct from those causing simple anemia and
abnormalities seen on the peripheral smear (Table 5 and generally mandate analysis of the bone marrow. Thus
Figs 1 to 19).1,5 on the basis of morphology we can divide the anemias
Flow chart 1 Classification of anemia based on morphology4
Flow chart 2 Approach to anemia6
into microcytic hypochromic and an approach to this is tear drop red cells) is characteristic of diseases in which
given in Flow chart 3.6 The anemias can be macrocytic the normal bone marrow is replaced by tumor or other
and an algorithmic approach to macrocytic anemias is diseases. Elevated WBC and/or platelet counts in children
given in Flow chart 4.6 The normocytic normochromic are most often due to reactive processes. While infection
anemias are associated with many systemic disorders is the most common cause, other etiologies including iron
and an approach to them is delineated in Flow chart 5.6 deficiency anemia, autoimmune disorders, or hemolytic
A leukoerythroblastic blood picture (nucleated red cells, anemia, vitamin E deficiency, and postoperative states
reticulocytosis, a shift to the left in the neutrophil cell line, are possible. Microscopic examination of the PBS can
Fig. 1 Dimensions of a normal RBC Fig. 2 Microcytic hypochromic anemia: RBC have a increased
central pallor and the size of RBC is smaller than the nucleus of a
lymphocyte
Table 5 Some peculiar red cell abnormalities on the smear1,5

Morphologic characteristic Basis of the abnormality Comment
Howell-Jolly bodies RBC nuclear remnants Increased with brisk hemolysis, increased following splenectomy, pernicious
anemia, CDA
Basophilic stippling RNA remnants/ Impaired globin chain
aggregated ribosomes production (thalassemia; lead
intoxication), unstable hemoglobinopathies and iron deficiency
Pappenheimer bodies Iron ferritin granules in Increased following splenectomy increased with transfusional iron overload
cytoplasm
Heinz bodies Hemoglobin aggregates Needs brilliant cresyl blue, crystal violet stains. Unstable Hb, enzymopathies,
hemoglobin H and thalassemia syndromes
Burr cells Membrane perturbance Chronic renal failure, common smear preparation artifact
Acanthocytes (spur cells) Membrane perturbance Hepatic insufficiency
Nucleated RBCs Normoblast nuclei High with brisk hemolysis present with myelophthisis
Sickle cells RBC distortion by Sickle cell disease
hemoglobin polymers
Target cells Low ratio of hemoglobin Prominent in thalassemia
to red cell membrane; Present with iron deficiency
RBC dehydration
Spherocytes Defective membrane Hereditary disorder
protein Immune hemolysis
aid in further focusing the differential. Assess the size, Presence of inclusions such as basophilic stippling (as
color, and shape of the red cells. The normal red blood seen in thalassemia, lead poisoning) should be noted.
cell is about the size of the nucleus of a small lymphocyte. Nucleated red blood cells are never normal, except in the
On a well-stained blood smear the area of central pallor newborn, and are indicative of a stressed marrow. The
in a normal erythrocyte has a diameter about one-third number and morphology of WBCs and platelets should
of that of the entire cell. Cells with excessive central also be assessed. Toxic granulation suggests an acute
pallor are hypochromic. Absence of central pallor is inflammatory state while hypersegmented neutrophils are
seen in spherocytosis. Polychromasia with large cells is characteristic of vitamin B12 and folate deficiency.
indicative of reticulocytosis. Distinctive abnormalities Reticulocytes are newly formed red cells with residual
in shape are suggestive of red cell membrane disorders strands of nuclear material called “reticulin” that remain
(e.g. spherocytosis, stomatocytosis, or elliptocytosis) or following extrusion of the nucleus from bone marrow
hemoglobinopathies (e.g. sickle cell disease, thalassemia). normoblasts. These new erythrocytes are 10 to 20 percent
Flow chart 3 Approach to microcytic hypochromic anemia6
Flow chart 4 Approach to macrocytic anemia6
Flow chart 5 Approach to normocytic normochromic anemia
Fig. 3 Macrocytic anemia with RBC larger than the size of Fig. 4 Spherocytes lack central pallor and may appear smaller
nucleus of lymphocyte than typical red
larger than red cells on average and have a faint bluish following release from the bone marrow. Thereafter, the
tint with Wright-Giemsa stain. Staining with a supravital erstwhile reticulocytes are identical to other red cells
dye such as brilliant cresol blue highlights the residual already in the circulation. The pathophysiology of anemia
nuclear material and definitively identifies reticulocytes. involves either the production of too few erythrocytes or
These staining characteristics persist for only 1 or 2 days the production of large numbers of red cells in concert
Fig. 5 Stomatocytes Fig. 8 Basophilic stippling: Numerous, small purple inclusions in

RBCs. Aggregates of ribosomal RNA. Most commonly seen in lead
poisoning
Fig. 6 Elliptocytes Fig. 9 Toxic granulation: Increased basophilic granules in

neutrophils. Seen in severe infections, burns, malignancies, and
pregnancy. Distinguish from basophils
Fig. 7 Sickle cells Fig. 10 Dohle bodies: Sky blue inclusions in cytoplasm of
neutrophils. Seen in infections, burns, myeloproliferative disorders,
and pregnancy. Composed of RER and glycogen granules
Fig. 11 Peripheral smear from a patient with infection shows Fig. 14 The RBC deformity (arrow) shown in this image is referred
granulocytes with Dohle bodies to as a “bite” cell: G6PD deficiency
Fig. 12 The open, sieve-like chromatin (salt and pepper) pattern Fig. 15 Note the fragmented schistocytes, burr cells, and helmet
in nucleus of megaloblastic cells is apparent cells
Fig. 13 Reticulocytes: Immature RBCs. Contain residual ribosomal Fig. 16 Howell-Jolly bodies are red cell inclusions which are
RNA. Reticulum stains blue using a supravital stain (new methylene residual nuclear fragments
blue). Counted and expressed as % of total red cells
with their rapid destruction or loss. The reticulocyte count the cause of the anemia. A terminological peculiarity
is the arbiter of the type of anemia, being low in the former sometimes confuses discussions of reticulocytes. The
instance and high in the latter. This basic distinction “reticulocyte count” reported by most clinical laboratories
allows the clinician to home dramatically the search for is given as a percentage of reticulocytes with respect to red
have fewer red cells than normal. Consequently, the use

of a percentage of red cells to determine reticulocyte
production is invalid in the very circumstance where it is
most needed. A normal “reticulocyte count” of 1 percent in
the face of a hemoglobin value of 5 g/dL and 2.0 × 106 red
cells/μL is patently abnormal. A number of formulae have
been promulgated that purport to correct for the anemia
and allow a clinician to assess whether the reticulocyte
percentage value is elevated or depressed. This semantic
and conceptual trap is best avoided by using the true
reticulocyte count, which is the number of reticulocytes per
microliter of blood. This value is also termed the “reticulocyte
number” to distinguish it from the percentage reticulocyte
Fig. 17 Pappenheimer bodies: Clusters of dark blue granules,
count. When the hemoglobin level is normal, blood contains
irregular in size and shape. Composed of iron and ribosomal RNA.
between 50,000 and 1,00,000 reticulocytes/μL. An anemia
Seen in sideroblastic and hemolytic anemias
with a reticulocyte number below this level is prima facie
evidence of a hypoproliferative anemia.2,5 The reticulocyte
percentage can be corrected to measure the magnitude of
marrow production in response to hemolysis as follows:
Reticulocyte index = reticulocyte % × observed
hematocrit/normal hematocrit × 1/μ where μ is a
maturation factor of 1 to 3 related to the severity of the
anemia. The duration of maturation as blood reticulocytes
is taken as μ. The value for μ for various hematocrit is as
follows: Hct 45%- μ 1.0, Hct 35%- μ 1.5, Hct 25%- μ 2.0, Hct
15%- μ 2.5. The normal reticulocyte index is 1.0; therefore,
the index measures the fold increase in erythropoiesis.
The usual marrow response in acute hemolytic anemia is
reflected by a reticulocyte index of 2 to 3, whereas in long
Fig. 18 Acanthocytes are red cells which retain a spherical shape standing chronic hemolysis, the increase in erythropoiesis
but have a spiculated appearance is approximately 6-fold.7
APPROACH TO HEMOLYTIC ANEMIA

Hemolysis is defined as an abnormally increased rate of
red blood cell destruction. It may be acute or chronic,
congenital or acquired, and intrinsic or extrinsic to the
RBC. With chronic hemolysis, anemia may or may not be
present depending on the rate of red cell destruction and
the degree of bone marrow compensation. Moreover, the
clinical signs and laboratory findings in hemolysis depend
on both the rate and site of red cell destruction. If red cells
are destroyed extravascularly in the reticuloendothelial
system, the normal site of red cell catabolism, Hb is
Fig. 19 Target cells degraded to iron, bilirubin metabolites, and amino acids.
Because hepatic clearance of bilirubin can increase
cells in the sample. The normal value is 1 to 2 percent. This substantially, a normal serum bilirubin does not exclude
reflects the practice of enumerating reticulocytes relative hemolysis. Unconjugated (indirect) bilirubin will be
to erythrocytes in the early days of hematology when this increased in more severe hemolysis resulting in jaundice.
work entailed manual counting of many microscopic When hemolysis occurs intravascularly, free hemoglobin is
fields. Expression of “reticulocyte count” as a percentage released into the plasma where it is bound by haptoglobin
of red cells is a reasonable practice as long as the number and subsequently cleared in the liver or lost in the urine.
of red cells is in the normal range. Anemias by definition An increase in plasma hemoglobin, a decrease in serum
haptoglobin, and the presence of hemoglobinuria suggest for sensitization has caused a dramatic decrease in the
intravascular hemolysis. incidence of this disease. Currently, immune-mediated
Congenital hemolytic anemias include disorders of hemolysis in the infant most often reflects a maternal
the red cell membrane (e.g. hereditary spherocytosis, response to other blood group antigens. Mechanical
stomatocytosis, or elliptocytosis), hemoglobinopathies damage is usually intravascular and associated with red
(e.g. hemoglobin SS), and red cell enzyme deficiencies (e.g. cell fragments on the PBS. The history and/or physical
glucose-6-phosphate dehydrogenase (G6PD) deficiency, examination can often identify the source of damage.2
pyruvate kinase deficiency). A family history positive for The following clinical situations warrant a specialist
anemia, splenomegaly, jaundice, and/or gallstones supports referral:
a congenital hemolytic anemia. Ethnic background can • Anemia requiring transfusion
also be helpful. Hereditary spherocytosis can occur in any • Acute or active bleeding resulting in anemia
ethnicity, but is most common in whites. An elevated MCHC • Moderate to severe anemia in a child or elderly or a
and spherocytes on the PBS supports this diagnosis. An pregnant lady
MCHC greater than 35.4 coupled with an RDW >14 is almost • Anemia showing no response or inadequate response
always diagnostic of hereditary spherocytosis. The osmotic to transfusions
fragility test or ektacytometry are confirmatory. Family • Anemia showing no response or poor response to
members should also be screened. G6PD deficiency is most medical treatment
common in those of African or Mediterranean descent (with
• A chronic or recurrent anemia
the latter tending to have more severe disease). Individuals
• Anemia of chronic renal failure
with G6PD deficiency often present with acute hemolysis
• Recurrent or long standing bleeding disorders
after an infection or after encountering an oxidant stress.
• Anemia of systemic disease, if disproportionate to
Signs of acute intravascular hemolysis with tachycardia,
disease or of moderate severe degree
jaundice, and hemoglobinuria will be observed. The PBS
• Anemia secondary to bleeding disorders, leukemia or
reveals schistocytes and spherocytes initially, then becomes
hematological malignancies
normal after the enzyme deficient cells are hemolyzed. The
sickle cell syndromes are usually detected by newborn • Where the underlying causative disease is likely to
screening in the United States and the pediatrician becomes result in progressive or persistent anemia
aware of the diagnosis before anemia or other clinical • Anemia where the underlying causative disease is
manifestations occur. More detailed information about undiagnosed or not clear
these syndromes can be found elsewhere in this volume. In • Where surgery or other intervention procedure is
any patient with congenital hemolytic anemia, an aplastic contemplated
episode is the exception to the rule that hemolytic anemias • When there are co-existent abnormalities of platelet or
are associated with reticulocytosis. While many viruses leukocyte
have been implicated, human parvovirus B19 is the most • When you are uncertain of your diagnosis or treatment.
frequent cause. Complete suppression of erythropoiesis
can last for 7 to 10 days after infection. Prompt intervention Key Points
with red blood cell transfusions as needed can be life saving.
Acquired hemolytic anemias can be immune-mediated • There are numerous ways of classifying the causes of
or secondary to factors that cause mechanical damage to anemia, and no one way is necessarily superior to another.
• Regardless of the specific algorithm followed in evaluating
the red cells such as toxins, mechanical or abnormal heart
severe anemia, it is essential that easily remediable causes
valves, and fibrin strands in disseminated intravascular
such as nutritional deficiencies, hemolysis, and anemia
coagulation (DIC) or hemolytic uremic syndrome (HUS).8,9 of renal insufficiency are identified early and treated
Immune-mediated destruction is confirmed by a positive appropriately.
direct and indirect Coombs’ test. Spherocytes may be seen • In general, the differential diagnosis of severe anemia
on the PBS. Antibody-mediated hemolytic anemia can can be substantially narrowed by subcategorization into
occur as part of a more generalized autoimmune process “microcytic,” “normocytic,” and “macrocytic” subtypes on
(such as lupus) or after exposure to a drug. However, in the basis of mean corpuscular volume.
children it is most often a self-limited disease following • However, such classification is a starting point and not
a viral illness. In neonates, immune-mediated hemolysis infallible. Each category then can be deciphered using a
is due to placentally-transferred maternal antibodies. stepwise approach that utilizes readily accessible laboratory
tests.
Historically, this was most commonly due to sensitization
• Hematology referral is always appropriate for complicated
to the Rh antigen in Rh negative mothers. The advent
or less defined cases.
of anti-D serum in women who are a potential set-up
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and Oski’s Hematology of Infancy and Childhood, 6th edn. JP, Foerster J, Lukens JN, et al. eds. Wintrobes Clinical
Philadelphia: WB Saunders. 2003.pp.409-18. Hematology, 11th edn. Philadelphia: Lippincott Williams
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C H A P T E R 14
Nutritional Anemia in Infancy,
Childhood and Adolescents
Anemia is diagnosed when Hb concentration and of food specific nutrients (like iron, folic acid, vitamin
HCT (number and size of RBCs) is lower than the level B12, protein, vitamin C, vitamin E, trace elements, etc.),
considered normal for the person’s age/sex group. poor health facilities, poor socioeconomic status, faulty
dietary patterns, the degree of urbanization, educational
Nutritional anemia (NA) is the most common cause of anemia background, prevalence of hook worm and other worms
in childhood. Nutritional anemia is a pathological condition in infestations, repeated bacterial infections, tuberculosis,
which hemoglobin and hematocrit levels becomes abnormally urinary tract infections (UTI), etc. vitamin A deficiency
low, because of deficiency of, one or more essential nutrients and other mineral deficiencies, etc. also influences the
needed for Hb formation, and for hemopoiesis, regardless of the
incidence of anemia particularly in children.
cause of these deficiencies. When anemia is due to nutritional
deficiency, increasing the person’s intake of deficient nutrient
However, many individuals with seemingly normal
will raise the Hb and HCT. Hb level respond to iron administration with a rise in Hb,
which implies that they were actually deficient in iron.
Hence assessing the frequency of iron deficiency anemia
World Health Organization (WHO) criteria for the
in a population by means of Hb measurement tends to
diagnosis of anemia (Table 1) are hemoglobin levels less
underestimate true prevalence.2
than 11 gm% in children between 6 months and 6 years
and below 12 gm% in children between 6 and 14 years.
In developing countries like ours, besides deficiency IRON DEFICIENCY ANEMIA
IN CHILDREN
Iron lacks the glitter of gold, and the sparkle of silver, but it
Table 1 Criteria for diagnosis of anemia outshines both in biologic importance. —Nancy C Andrews.3
Hemoglobin levels to diagnose anemia (g/dL)1
Age group No anemia Mild Moderate Severe Historical
Children 6–59 months >11 10–10.9 7–99 <7
Pallor known since Mahabharata. Father of Pandavas was
of age
known as Pandu as he was looking pale white. Therapeutic
Children 5–11 years >11.5 11–11.4 8–10.9 <8
of age use of iron was known in Greek mythology—Drinking
Children 12–14 years > 12 11–11.9 8-10.9 <8 wine in which sword rusted, was line of treatment. Loha
of age Bhasma and Mandura Bhasma being used in Ayurveda
Non-pregnant women > 12 11–11.9 8–10.9 <8 since 5000 years use of iron salt is the main therapy of
(15 years of age) modern medicine.4
Pregnant women > 11 10–10.9 7–9.9 <7
Men > 13 11–12.9 8–10.9 <8 Iron Deficiency Anemia
Source: Hemoglobin concentration for the diagnosis of anemia Iron deficiency anemia (IDA) is most widespread micro
and assessment of severity. WHO nutrient deficiency and affects infants, young children
Chapter-14 Nutritional Anemia in Infancy, Childhood and Adolescents 101
(6–24 months), preschool children, adolescents and parti 115,000 maternal death and 591,000 perinatal deaths
cularly women of childbearing age, and pregnant and globally per year. Almost 58 percent pregnant women in
lactating mother. India are anemic and it is estimated that anemia is the
Inspite of efforts of increasing the awareness, educa underlying cause for 20–40 percent of maternal deaths
tion, food fortification and multiple control programmes in India. Iron deficiency is by far the most common
nutritional anemia still remains widely prevalent with sig nutritional cause of anemia. The prevalence of anemia
nificant morbidity in developing countries and to a lesser has actually increased from NFHS-2 to NFHS-3. The
extent even in the developed countries.57 percentage of children with any anemia increased from
Iron is important for a number of iron-dependent 74.3 percent in NFHS-2 to 78.9 percent in NFHS-3 in the
enzymes including the catalaze, peroxidase, cytochromes, period between the two surveys, there was an increase
and ribonucleotide reductase. It plays a vital role in in the prevalence of mild anemia (from 23% to 26%) and
hemoglobin which is important for oxygen carriage in moderate anemia (from 46% to 49%) percent of pregnant
multicellular organisms like man. women and 24 percent of men. The prevalence of anemia
among ever married women increased from 52 percent in
An Estimated Global Prevalence1-30 NFHS-2 to 56 percent in NFHS-3.30,69 However, it may be
associated with folic acid deficiency and other nutritional
One-third (20–30%) of world’s population, i.e. 2150 deficiencies like vitamin B12, pyridoxine, copper, etc. The
million people are iron deficient, out of which 1200 condition is more prevalent in developing countries (36%)
million are anemic, of which 90 percent live in third world than in industrialized developed countries (8%).
countries. Recent World Health Organization (WHO)/ India falls in the category of high prevalence for nutritional
United Nations Children’s Fund estimates suggest that anemia.2,41
the number of children with iron-deficiency anemia (IDA)
is greater than 750 million.1 Although the prevalence of IDA among Pregnant and Lactating Women42-51
anemia is estimated at 9 percent in countries with high
development, in countries with low development, the The overall iron requirement increases from a pre-
prevalence is 43 percent in absolute numbers; anemia adolescent level of 0.7 to 0.9 mg Fe/day to as much as
affects 1.62 billion people globally with about 293 million 2.2 mg Fe/d or perhaps more, in heavily menstruating
children of preschool age, 56 million pregnant women young women. Hence, adolescent girls are unlikely to
estimated to be anemic. With global anemia prevalence acquire substantial iron stores during this period; intake
estimates of 47 percent in children younger than 5 years, may average as little as 10 to 11 mg Fe/day. The low
42 percent in pregnant women, and 30 percent in non- iron stores in these young women of reproductive age,
pregnant women 15–49 years. In the tropical countries, will make them susceptible to IDA during pregnancy.
the incidence is at times almost 100 percent. IDA is an Dietary intake alone is insufficient in most cases to
extremely serious public health problem in India, 1624 meet the requirement of pregnancy. Adequate iron
babies die everyday due to IDA (The World Health Report– supplementation of adolescent girls is essential towards
Preventing Risks, Promoting Life, 2002). lowering the incidence of anemia during pregnancy.
Young children and pregnant women are most Recent study showed that prevalence of IDA among
affected, with an estimated global prevalence of 43 percent pregnant and lactating women is over 75 percent and more
and 51 percent respectively. IDA is most common in the than half of pregnant women and a third lactating women
age group of six months to three years.21-30 Children aged are moderately or severely anemic. In some states, an
12 to 17 years (adolescence) and espe cially among anemia prevalence as high as 87 percent has been found
pregnant women and lactating mothers. among pregnant women from disadvantaged groups.47
The reported prevalence of nutritional anemia in In pregnant women, the incidence of nutritional
preschool children varies from 44 to 74 percent.22-30 anemia vary from developed countries:
In the adolescent period (10–19 years), it has been • Europe 18%
found that the incidence of nutritional anemia is about
11 to 90 percent and increases from 10 years onwards • South Asia 75%
and continues to remain high till 18 years of age.31-40 With • South-East Asia 63%
increasing age, the prevalence rate declines in males, and • India 88, 38–88%
not in the females.
At least half of all married women aged 15 to 49 years Each year out of 500,000 maternal deaths 100,000 are
and adolescent girls are believed to have some degree due to iron deficiency. Mortality attributable to anemia
of IDA. Anemia is estimated to contribute to more than was found to be 20 percent in Africa and 22.6 percent in
Asia. Iron requirements are higher during the second and Table 2 Incidence of anemia in developing countries
the third trimester and iron balance during this period like India1-25,27,30-36,40
depends more on the adequate intake of bioavailable iron
Amongst preschool children 70% (76–90%)
rather than the store at conception.
1–3 years 63–74%, (ICMR 1977,
Iron Requirements in Different Age Groups NFHS1998-99)
School age children 37%, (60–80%)
• Pregnant females 30 mg/day Children between the age group of 74
• Females 11–30 years 15 mg/day 6–35 months
• Adult males 10 mg/day 3–6 years ICMR in 19778 44%
– Up to 10 years 10 mg/day Adolescent period 10–19 years, 50%
• Full-term infants 1 mg/kg/day from 4 months of age and increases from 10 years
onwards and continues to remain
• Low birth weight babies 2 mg/kg/day from 2 months of age high till 18 years of age2
• Babies < 1000 g 4 mg/kg/day from 2 months of age Nonpregnant women 35%, (81–84%)
– 1000–1500 g 3 mg/kg/day from 2 months of age Pregnant women 50% (30–50%)
80–88%
Surprisingly, IDA is not just a problem in developing
Women from South Asia 60%
countries. According to CDC, although ID is more com
mon in developing countries, a significant prevalence was South-East Asia 50%
observed in US during early 1990s in toddlers and women Africa 40–50%
of childbearing age. In one study, 7 percent toddlers (1–2 Adult male 18% (48–56%)
years), 9–16 percent of adolescent and adult females
(12–49%) were found to have iron deficiency. Although
• Report of the NFHS-230,79 shows that the prevalence of
incidence of iron deficiency is uncommon, they are still
anemia has not much changed in 1998 to 1999 and is
above the healthy people 2010 objectives of 5 percent,
still 74 percent among children of 6 to 35 months of
1 percent and 7 percent for toddlers, preschoolers and
age. It has now been realized that iron deficient state
females 12 to 49 years respectively. Anemia is a major
without anemia is even much more common. Over the
contributory factor for increased maternal morbidity
last 50 years, the prevalence of iron deficiency anemia
and mortality and accounts for 19.1 percent maternal
has ranged between 68 and 97 percent in children.
death.44,45,48,50
Infants, toddlers, preschool and adolescents are at a
great risk of developing IDA.21-25,27,30-36,40
REPORTED INCIDENCE OF IDA IN CHILDREN Shahbuddin et al. from Bangladesh35 reported anemia
(TABLE 2) in 94 percent adolescent boys and 98 percent adolescent
girls. Who conceive during or shortly after adolescence are
• Nearly half of the world women with anemia live in
likely to enter pregnancy with low or absent iron stores or
South–Asia
IDA.
• In a recent study, in an urban slum of Delhi, nearly half
Classification of countries with respect to degree of
of the anemic young children had other nutritional
public health significance is:16
deficiencies notably vitamin B12 and folic acid as the
direct or associated cause52 High-risk 40 or > % of population anemic
• Gopalan37 in 1997 reported that only 38 percent from
Medium risk 15–40% population anemic
urban Delhi, 19 percent of rural Rajasthan only had
normal Hb level Low-risk Less than 15% population anemic
• Dr Currimbhoy17 reported anemia 70 to 80 percent in
children in 60s
• Dr Mamta Manglani et al. 60 to 70 percent in 90s SOURCES OF IRON
• Dr MR Lokeshwar reported in office practice — 50 to 60 Major sources of food iron and type of dietary iron
percent (unpublished data) available:
• Thirty to ninety percent of adolescent children in India • Ultimate absorption of iron into mucosal cells mainly
are anemic depending upon socioeconomic condition depends upon bioavailability of iron in the various
and whether from rural or urban area31-40 foodstuffs.
• Dietary iron is available as. of iron by one- third. Calcium in the form of milk, cheese or
• Heme iron. (calcium added to the bread), depresses iron absorption.
• Nonheme iron. Administration of 50 mg of vitamin C increases iron
absorption by two fold.
Heme Iron An average adult has about 3 to 5 g of iron and children
have 55 mg/kg/body weight. It is more in males as
• The nonvegetarian foods is available as hemoglobin compared to females. Seventy percent of iron in the body is
and myoglobin in meat, fish and poultry. It is the richest in the form of Hb. 3.9 percent is incorporated in myoglobin
source of iron. Heme iron is highly bioavailable, since and various other iron containing enzymes. Plasma iron
it is absorbed intact within the porphyrin ring and the forms only 0.1 percent of the body iron. Iron balance in the
absorption of this is not affected by any another food body is achieved mainly by control of absorption of iron
and is better absorbed than nonheme iron. It is the rather than its excretion, most of the iron is recirculated
richest source of iron containing 10 to 18 mg of iron in the body. Only 1.15 mg of irons is excreted daily. Thus a
per 100 gm. But in developing countries like ours, the daily requirement is minimum. Absorption of iron mainly
intake of these products is generally low. depends upon dietary content of iron.
• Nonheme iron is available in the form of ferric
complexes. Nonheme iron is markedly affected by
promotive and inhibitory iron binding ligands. Mucosal Cell Control
Foods rich in iron are cereals, pulses, legumes, Bajra,
Site of absorption is the 1st and 2nd part of the duodenum,
nuts, dates, jaggery, green leafy vegetables, and meat,
and at times jejunum. Maximum absorption of iron occurs
fish and liver preparations.
from the duodenum.
• Milk is a poor source of iron: Breast milk, the primary
source of infant nutrition is poor in iron, containing Two steps are involved in the absorption of iron:
0.28 to 0.73 mg/L. Whereas the bioavailability of iron 1. Entry of iron from the intestinal lumen into the mucosal
in cow’s milk is just 10 percent and that of breast milk cell.
is 50 percent (20–80%) making it a good source of iron. 2. Its passage from the mucosal cell into the plasma.
Hence, iron deficiency rarely occurs in exclusively Appropriate iron balance in the body is achieved by
breastfed infants till the age of 4 to 6 months. Breast mucosal cell control through transferrin and apoferritin
feeding does not protect against iron deficiency after receptors. The iron molecule that is taken into the
the age of 6 months, unless iron containing weaning mucosal cell across the brush border, can bind either to
foods are introduced. the apoferritin molecule or the ferroportin molecule in the
– Factors affecting iron absorption: Heme iron is not mucosa. Iron status of the body at the time of the formation
affected by presence of any factors in the gut. The of the mucosal lining cells determines the amount of iron
absorption of nonheme iron is retarded by alkaline that is absorbed.
pH, presence of phosphates, phytates, bran, starch, If iron in the plasma is adequate, or increased iron
tannins, calcium, antacids, other metals (Co, Pb), stores, there is increased messenger iron in the mucosal
etc. Phytates, which constitute 1 to 2 percent of cell. This messenger iron stimulates the production of
many cereals, nuts and legumes, play a major apoferritin. Iron binds to apoferritin, which remains in the
role in the causation of nutritional anemia in the mucosal cell. There is increased transferrin saturation.
developing world. Thus, whenever there is increased transferrin satu
It is enhanced by ascorbic acid, free hydrochloric acid, ration or, serum iron is normal and adequate, a larger
presence of sugars and amino acids in the diet, presence fraction of the iron entering the mucosal cell is held back
of heme iron (nonvegetarian source of iron) and EDTA.53-56 as ferritin and discarded, as the cell is desquamated and
The bioavailability of iron from a particular dietary ultimately excreted after 3 to 4 days, and gets denuded with
source affects the amount absorbed. Ferrous iron is the cell within 3 to 4 days. If iron is required in the body, it
better absorbed compared to ferric iron. It is estimated is bound to the ferroportin, which is then transferred to the
that in wheat based diet, iron absorption is around transferrin (produced in the liver), which carries it across
2 percent and in rice based diet, it is 5 to 13 percent.53,54 Poor the mucosa. It is then utilized in the bone marrow for
bioavailability of iron in largely cereal-based diet is major hemoglobin production, in the muscle tissue for myoglobin
cause of IDA in most developing countries. Fish, meat and in the body for various other enzymes. Any excess
and poultry are good sources of iron and bioavailability is iron is stored in the form of ferritin in the liver. The RBCs
around 20 to 30 percent. Increasing the dietary intake to circulate for their life span of approximately 120 days and
meet the caloric needs will also increase the dietary intake are then destroyed in the spleen, liberating the free iron,
which is then retransported to the bone marrow and other Causes of Iron Deficiency
tissues for its reutilization. Thus, most of the iron is cycled
continuously in the body, with only 1 to 1.5 mg/day of iron • Decreased iron stores: Preterms, small for date babies
being excreted through the intestinal epithelial cells after • Decreased intake/assimilation and reduced absorption
completion of their life span. Since 10 percent of ingested – Prolonged breastfeeding and delayed introduction
iron is absorbed and the daily loss is only 1 to 1.5 mg, one of iron rich, complementary feeds after the age of
needs to ingest about 10 mg of iron daily, except during 6 months adds fuel to the fire
periods of extra needs. Iron is stored in the body in the – Diet containing low iron or nonbioavailable dietary
form of ferritin and hemosiderin. iron: It is estimated that in the wheat millet based
In iron deficiency state, the mucosal cell transports diet, iron absorption is around 2 percent and in rice
the iron rapidly to the circulation, where it combines with based diet, iron absorption is around 5 to 8 percent.
transferrin and is transported to the site of utilization and – Low over all dietary intake
storage. Only 1/10th of the dietary iron is absorbed by the – Nonbreastfed infants on cow’s milk
GI mucosa. – Malabsorption of iron: Chronic diarrhea, celiac
disease, cow’s milk allergy, GI surgery, giardiasis,
etc.22
Hepicidin and its Role in Iron Metabolism57,58 – Rarely genetically determined absorptive defect
Hepicidin plays a key role in the regulation of iron specific for iron.
metabolism and iron deficiency. Hepatocytes produce • Increased requirement:
hormone peptide (cysteine rich) hepicidin. Hepicidin – During periods of growth—preterm infants, tod
controls the release of iron from variety of cell such as dlers, puberty
macrophages, hepatocytes enterocytes, etc. to plasma. It – During reproductive age in females
primarily controls iron absorption. The recycling of the – Pregnancy
iron from red cell lysis and release of iron from tissue – Lactation.
iron stores is carried by interaction of hepicidin with • Increased losses: Blood loss due to any cause/chronic
ferroprotein which is a cellular iron exporter. Release of blood loss. In gastrointestinal bleeding the chronic loss
ferroprotein is controlled by hepicidin. of few milliliters of blood daily is sufficient to deplete
iron stores and lead to iron deficiency.
– Gastrointestinal bleeding
Iron Transport and Storage
– Diverticulitis
Transferrin helps in transport of iron from the intestines to – Polyps
the site of its utilization. – Fetomaternal hemorrhage
– Repeated blood sampling
Transport of Iron Across the Placenta – Blood losses in the menstrual cycle (Menorrhagia).
Females loose 40 mL blood per month in the
The transport of iron across placenta occurs against a menstrual cycle, this increases the daily iron losses
gradient, thereby protecting fetus against iron deficiency. to 1.5 mg/day.
Babies with low iron stores may be born, to mothers – Intestinal-parasites–hookworm-infestation
who are severely iron deficient during pregnancy. Most giardiasis:59-65 Infants are exposed to intestinal
of the placental transfer of the iron occurs during the helminths from the time they crawl. Such infants
3rd trimester of pregnancy. As a consequence of this, suffer repeatedly from gastroenteritis, and other
all preterm babies invariably develop anemia unless infections further depleting their iron stores.51,52
supplemented by iron and iron deficiency in the mother Particularly for those from rural areas. Four
may cause preterm labor. hundred fifty million people all over the world
Normal infants at birth have about 75 mg of iron harbor this parasite and about 0.2 mL of blood/
per kg body weight, two-third of which is present in red worm of ankylostoma per day may be lost and with
blood cells. Once the iron is assimilated in the body it nector infestation each worm accounts for loss
is not excreted. Normal body losses of iron are about about 0.1 to 0.5 mL/day. Female subjects harboring
20 ug/kg/day. Most of these loss occur by shedding of cells more than 100 worms (5 mL/day blood loss) and
from intestinal mucosa. Average loss of iron per day in male subjects harboring more than 250 worms
children is 0.9 mg/day or 0.5 mg/m2/day. 0.6 mg/day is lost (12.5 mL/day blood loss) tend to become anemic.
in the GIT in the form of RBCs, bile or exfoliated mucosal Chakma T, et al.62 from Madhya Pradesh reported
cells. The rest is lost from the sweat, desquamated cells of intestinal parasite in 50 percent of adolescents
the skin and the urinary tract. (Hook worm 16.3% lumbricoid 18.5%).
• Adolescents: Adolescence, is a period of rapid growth, Iron deficiency stage: This refers to lack of iron of sufficient
weight gain and blood volume expansion. Iron severity to restrict production of hemoglobin.
requirement increases from preadolescent level of 1. First stage—storage iron depletion: Iron reserve is
0.7 mg to 0.9 mg iron per day to as much as 2.2 mg decreased or absent. It is characterized by reduced
iron per day or more particularly heavily menstruating serum ferritin, reduced iron concentration in marrow
women and liver tissue. Hb, serum iron, transferrin levels and
• In pregnancy, 4 mg of iron is lost per day in the last two saturation are within normal limits
trimesters. During lactation 0.5 to 1 mg of iron is lost 2. Second stage—iron limited erythropoiesis (Iron defici
per day ency without anemia): Refers to a milder form of iron
• False concern about the body figures, food fads: During deficiency where Hb has not fallen enough to meet the
adolescence false concern about the body figures, criteria of anemia. It may be transient and consists of
food fads, ignorance, particularly in girls lead to iron a decrease in the transportation of iron Hb level may
deficiency. Lack of knowledge of nutritional factors still be normal or in the low normal range. Serum iron
further adds to the problem particularly in adolescent is low and total iron binding capacity (TIBC) increased
girls with low transferrin saturation and low serum ferretin
• Poor iron absorption: levels
– Malnutrition/iron poor diet/malabsorption syndro 3. Third stage—iron deficiency anemia: It represents the
mes chronic infection/chronic diarrhea celiac more severe form of iron deficiency. The supply of
disease/giar diasis/helminthiasis, sprue,
��
hypopro iron to erythroid cells in marrow is impaired, causing
teinemia, cow’s milk allergy also contribute to a a reduction in Hb concentration, with progressive
high prevalence of anemia microcytic hypochromic anemia. Hb concentration has
– Gastrointestinal surgery: Polyps/Meckel’s diverti fallen and decreased serum iron, transferrin saturation
culum/hemorrhagic telangiectasia/peptic ulcer, and serum ferritin levels. There is an increase in the FEP
diverticulitis are other causes of bleeding diathesis (free erythrocyte protoporphyrin) detectable anemia,
and iron loss microcytosis, hypochromia on the peripheral smear
– Milk-induced enteropathy is the most common with low MCV and MCH and high RDW.
cause of occult GI bleeding seen in approximately
more than 50 percent of infants with IDA seen in Clinical Features of IDA
the western world Iron deficiency an isolated hematological condition
– Rarely genetically determined absorptive defect associated with anemia. It is a systemic disease involving
specific for iron. multiple systems. The appearance of symptoms depends
• Fetomaternal hemorrhage: Among the other causes upon:
of blood loss leading to anemia in newborn. In about • Hemoglobin level
50 percent of all pregnancies there is some degree of • The rate of fall of hemoglobin
fetomaternal hemorrhage. Eight percent are significant • Hemostatic adjustment of various organs systems
(0.5 – 40 mL fetal blood loss) • Age of the child
– Repeated venipunctures for investigations, hemo • Maturity of various organs
dialysis, and regular blood donations are important • Underlying cause.
iatrogenic causes of iron deficiency due to chronic If the fall of hemoglobin is gradual as in iron deficient
blood loss anemia, then the onset of symptoms is insidious and may
• Inadequate transport: go unnoticed till Hb drops to as low as 4 to 5 gm/dL. Child
– Atransferrinemia may not have any obvious symptoms. Gradual onset of
– Antitransferrin receptor antibodies. pallor may escape notice even when the hemoglobin falls
to 4 to 5 gm.
PATHOGENESIS However, if the drop of Hb is rapid, child may be
brought in serious conditions—tachycardia, signs of
Stages of Iron Deficiency
congestive cardiac failure and even gasping conditions as
Iron deficiency anemia (IDA) is the end stage of a relatively it some times occur in G6PD deficiency or autoimmune
long drawn process of deterioration in the iron status of an hemolytic anemia. Initial symptoms include pallor,
individual. tiredness, lassitude, easy fatigability, anorexia, weakness,
The spectrum of iron nutrition status can be divided lack of concentration, breathlessness, irritability, puffi
into three stages: ness, edema feet, etc.
However, all cases of anemia may not have pallor,

especially in mild cases of anemia, and associated with
icterus, cyanosis, dark pigmentation, may mask pallor.
Pallor can also be seen in nonanemic conditions as
color of the skin not only depends on the Hb content, but
on the state of blood vessels of the skin (Vasoconstriction
as seen in vasovagal syncope), presence of edema as seen
in nephrotic syndrome, myxedema, congestive cardiac
failure, even in absence of anemia. Hence, it is always
prudent to rely on Hb or HCT estimation to detect anemia.
• Later hyperdynamic circulation leads to palpitation,
shortness of breath, easy fatigability, reduce exercise
tolerance and heart failure
• Changes in epithelial cells: These include koilonychias,
platonychia, atrophic glossitis, angular cheilosis are
uncommon in children, however may be observed,
especially with long standing anemia and in adolescent
children (Fig. 2). The triad of dysphagia due to esophagal
webs, koilonychias (Fig. 1) and splenomegaly in a child
with IDA is known as Plummer–Vinson or Paterson– Fig. 2 Adolescent child with IDA
Kelly syndrome and are not common in children
• Mild hepatosplenomegaly is also not uncommon in
children with iron deficiency anemia
• Pica: Pica usually is the manifestation of iron deficiency – Amylophagia: Eating laundry starch—uncooked
and is relieved when condition is treated. Pica is a rice
habitual ingestion of unusual substances like: – Pagophagia-ice: It is unexplainable symptom.
– Geophagia:83 Mud or clay and can decrease absor Often seen in pregnant women.
• Other common causes of pica are lead poisoning and
ption of iron and aggravate IDA
psychological disturbances
• Clay84 can behave in the gut as an exchange resin and
can interfere with iron absorption.
Pica though may be a manifestation of iron deficiency,
is also considered to be a predisposing factor. It is both
effect and cause of IDA.
Clinical features may be due to anemia or in addition
due to underlying etiology of anemia28,29,66-70
In mild anemia: There may be no signs and symptoms but
a definite sense of well being and better exercise tolerance
is observed following treatment. All the symptoms of the
anemia like fatigue, breathlessness, irritability, anorexia,
etc. may be seen. Spleen is often enlarged slightly in
children, but is of normal consistency.
Patients with Mild to Moderate Anemia

(Hb 6–10 gm%)1,71
• May or may not have any symptoms
• They are usually diagnosed during routine laboratory
testing
• This is because of compensatory mechanisms like
Fig. 1 Koilonychia and puffiness of face and eyes and severe increase in 2,3 DGP levels and a shift of O2 dissociation
pallor in a case of IDA (Rare in children) curve.
Severe IDA < 5 gm% cases. However, oral iron therapy only minimally
changes saturation of transferrin and hence practically
• May present with fatigue, breathlessness, tachycardia, it does not have any adverse effect on incidence of
systolic murmur and later on, signs of CCF may develop infection. In several studies, results show that infants
• Anorexia, loss of appetite who receive iron supplementary formulae have fewer
• Increased infections episodes of respiratory and gastrointestinal infections
• Irritability than those who receive unsupplemented formula.
• Other features: Depletion of nonheme iron containing • Reduced myeloperoxidase can also lead to altered
tissue proteins is responsible for various other mani white cell function.
festations.
Growth Retardation
Diet History There is marked reduction in weight of iron deficient
A detailed diet history is very important, especially in children, though height seems to be unaffected.
infant with anemia.
• Exclusive breastfeeding for 4 to 6 months
Exercises Intolerance80,81
• Introduction of good home made weaning food con Maximum work capacity, work output and endurance are
taining iron thereafter impaired in iron deficiency state. This is due to reduction
• Iron deficiency develops where there is poor breast- in the mitochondrial enzyme a-glycophosphatase dehy
feeding, prolonged breastfeeding beyond 1 to 2 years drogenase besides due to anemia.
especially with introduction of improper weaning food
is also a cause of nutritional anemia Effects on Pregnancy Outcome42-46,48,50,51
Iron deficiency has a wide range of clinical and
Estimates from the World Health Organization report
functional consequences:
that from 35 to 75 percent (56% on average) of pregnant
• Behavioral changes: These changes occur due to
women in developing countries, and 18 percent of women
diminished activity of aldehyde oxidase, required for
from industrialized countries are anemic. However,
serotonin catabolism, thus leading to increased levels
many of these women were already anemic at the time
of serotonin and 6-hydroxy nidole compounds. MAO
of conception, with an estimated prevalence of anemia of
which is also required for catabolism of catecholamine
43 percent in nonpregnant women in developing countries
is also reduced.
and of 12 percent in women in wealthier regions. Even for
• Short attention span, irritability, stubbornness, dec
women who enter pregnancy with reasonable iron stores,
reased cognition and scholastic performance and
iron supplements improve iron status during pregnancy
conduct disorders are common in children with iron
and for a considerable length of time postpartum, thus
deficiency, leads to learning disabilities and scholastic
providing some protection against iron deficiency in the
backwardness. These neurological changes that
subsequent pregnancy.
occur due to iron deficiency may be long term or even
Maternal iron deficiency in pregnancy reduces fetal
irreversible. Anemic children have poorer endurance
iron stores, perhaps well into the first year of life. Iron
capacity and lack physical fitness.72-81
deficiency anemia in pregnancy is a risk factor for preterm
– Breath holding spasms in less than 3 years child.
delivery and subsequent low birth weight and possibly for
– Altered immune response: It is believed that IDA
inferior neonatal health.
children have increased susceptibility to infec
tions due to immunosuppression. Humoral, cell-
mediated and nonspecific immunity and the Regulation of Iron Transfer to the Fetus
activity of cytokines which have an important role Most iron transfer to the fetus occurs after week 30 of
in various steps of immunogenic mechanisms gestation. Serum ferritin usually falls markedly between
are influenced by iron deficiency anemia.82 Iron 12 and 25 weeks of gestation, probably as a result of iron
deficiency affects both cell mediated as well as utilization for expansion of the maternal red blood cell
humoral immunity, though phagocytic activity mass. Serum transferrin carries iron from the maternal
may be normal. circulation to transferrin receptors located on the apical
• Some studies state that immunity is enhanced in iron surface of the placental syncytiotrophoblast, holotrans
deficient state. This is due to increased unsaturated ferrin is endocytosed, iron is released, and apo-transferrin
transferrin which inhibits bacterial growth and hence is returned to the maternal circulation. The free iron then
high dose IV iron therapy could be harmful in such binds to ferritin in placental cells where it is transferred
to apotransferrin, which enters from the fetal side of It is desirable that anemia and its severity are then
the placenta and exits as holotransferrin into the fetal quantified through the accurate laboratory tests.
circulation. This placental iron transfer system regulates
iron transport to the fetus. Screening Tests85-91
When maternal iron status is poor, the number of
placental transferrin receptors increases so that more iron Manual determination of these red cell indices are time
is taken up by the placenta. Excessive iron transport to consuming and not very reliable and not reproducible.
the fetus may be prevented by the placental synthesis of With the availability of electronic particle counters
ferritin. There is a substantial amount of evidence showing estimation of RBC parameters—Hb, PCV, MCV, MCHC,
that maternal iron deficiency anemia early in pregnancy RBC count, RDW, has become easy to perform, accurate,
can result in low birth weight subsequent to preterm reproducible and practical.
delivery. Evaluation of peripheral smear examination is must.
The changes in these parameters include:
Diagnosis of Iron Deficiency Anemia • Red cell count, hemoglobin and hematocrit are all
decreased in IDA. MCV, MCH and MCHC are also
(Flow Chart 1)84-90
decreased.
Laboratory tests in iron deficiency anemia are required to • The peripheral blood film shows hypochromic,
diagnose IDA and to establish its cause. Iron deficiency microcytic red cells (Fig. 3).
anemia, should initially be suspected clinically by taking • If anemia is severe, morphological abnormalities such
proper detailed history, dietic history, socioeconomic as poikilocytosis and target cells. Pencil cells may be
status, and proper clinical detailed examination—looking seen.
at tongue, mucosa of inner side of lip and palate, conjunc • When iron deficiency is associated with deficiency of
tiva or nails, paleness around the crease of the palm and other hematinics like vitamin B12 or folate, there may
thorough systemic examination to detect underlying be a dimorphic picture with hypochromic, microcytic
cause. red cells along with macrocytic red cells. These routine
Flow chart 1 Approach to IDA-hypochromic microcytic anemia
Child is said to be anemic when the hemoglobin and/or

hematocrit is two standard deviation (SD) below the mean
for that particular age and sex.
Reticulocyte count is usually normal unless the
child has an acute or recent blood loss or has received
hematinics. However in mild iron deficiency, RBC
morphology and indices are not altered.
Confirmatory Tests (Table 3)

• Serum iron is reduced (N = 50–180 ug/dL)
• TIBC is increased, more than 470 mcg/dL (N = 250–450
ug/dL)
• Transferrin saturation is low—less than 16 percent
suggestive and less than 7 percent diagnostic of severe
IDA.
Fig. 3 Peripheral smear examination showing microcytic • Serum ferritin is less than 10 to 12 ng/mL. However,
hypochromic anemia when infectious or inflammatory diseases like rheuma
toid arthritis, collagen disorders, liver disorders,
chronic renal disease or malignancy are also present,
investigations may not be useful to diagnose early iron the serum ferritin level is usually higher, but less than
deficiency state. 50 to 60 ng/mL as ferritin is an acute phase reactant.
• Reticulocyte count is normal unless the patient has had The test still lacks sensitivity and normal value does not
a recent acute blood loss or the patient has received. reliably exclude iron deficiency.
Hematinics, in which case it may be increased.
Following iron therapy there is reticulocytosis which
peaks at 1 to 2 weeks.
Free Erythrocyte Protoporphyrin
• In severe IDA, reticulocyte count may be decreased. Free erythrocyte protoporphyrin (FEP) and protoporphy-
Low levels of Hb, MCV< 80 ug/dL, MCH < 27 Pg and rin—heme (PH) ratio: Erythrocyte protoporphyrin, the
high levels of RDW with low SI, TS, TIBC and FEP precursor of heme accumulates in red blood cells when
indicate IDA. it has insufficient iron to combine with the form of heme.
• Klee92 G in his study showed that more than half of the The blood can be conveniently tested by putting drop of
62 patients with IDA had a MCH value clearly within a blood on a cover slip and reading the result directly. The
normal range and nearly 70 percent of cases exhibited FEP can be measured by a simple fluorescence assay per
distinct microcytosis, suggesting that MCV is much formed directly on the thin film of blood. Both FEP and PH
more sensitive than MCH in determining changes of ratio is elevated in iron deficiency. Normal values of FEP
iron deficiency. MCHC < 33 percent and is the last of are 30 to 40 ucg/dL RBC and PH ratio 16 (+5.3). FEP values
the indices to be affected and is the least important in above 70 mcg/dL RBC and of PH ratio above 32 is consid
diagnosis of IDA. MCV is more sensitive than MCH in ered to represent iron deficiency.
diagnosis of IDA. RDW is the quantitative measure of • FEP/Hb ratio increases when iron reserve is exhausted,
anisocytosis. It is increased in IDA and normal or low even before anemia becomes apparent
RDW values are unlikely to be present with IDA.
• Microcytosis also may be seen in other conditions.
Table 3 Confirmatory tests
Differential Diagnosis of Microcytic Age in Serum ferritin Transferrin RBC/FEP ug/dL
Hypochromic Anemia (Table 4) years ng/dL saturation
percent
• Iron deficiency anemia 0.5–4 < 10 <12 >80
• Anemia of chronic infection and inflammation
• Thalassemias and abnormal hemoglobinopathies traits 5–10 < 10 <14 >70
• Lead poisoning 11–14 < 10 <16 >70
• Sideroblastic anemia. >14 < 12 <16 >70
Table 4 Differential diagnosis of microcytic/hypochromic anemia entry of transferrin-bound iron into cells by a process
of endocytosis. sTfR increases with enhanced red cell
Iron Thalassemia Chronic
production but iron deficiency is the only disorder in
deficiency minor infection
which there is increased serum receptor combined with a
Clinical low level of red cell production. Unlike the serum ferritin,
Age 6–24 months Any Any which only identifies iron deficiency, sTfR measures its
Community Any High risk Any severity. The normal reference value is 2.8 to 8.5 mg/L.
Pica ++++ — — Values above 9 mg/L are considered abnormal. Unlike
many other iron measurements, the level remains normal
Diet Milk/bottle — —
in patients with anemia of chronic inflammation or
feeding
infection.
Behavior Irritable/listless — — The most sensitive method available to distinguish
IDA from anemia of chronic disease is a combination of
Breath holding +++ — — plasma sTfR and the log of plasma ferritin concentration,
Epithelial/nails Koilo/ — — i.e. sTfR-ferritin complex. If it is high (>4) it indicates IDA;
Platonychia if <1 it indicates chronic disease.
Laboratory Molecular genetics of iron deficiency: Human transfer in
RBC count Low High Low gene has been reported that human transferrin G2775
MCV Low Low N-Low mutation is a risk factor for iron deficiency.
Depletion of stainable iron from bone marrow:
RDW High N High
(Routinely bone marrow examination not required) Bone
Hemolysis N ++ N marrow aspirates can be stained for hemosiderin by Perl’s
RBC count Low High Low reaction and iron content is graded from 0 to 4. Although
TIBC/FEP High N High is the most accurate technique to evaluate iron status, it is
Ferritin Low N High an invasive procedure and therefore impractical.
sTfR/serum ferritin High N Low Response to Therapy
ESR N N High
• In uncomplicated IDA, administration of iron shows a
HbA2 N High N predictable reticulocytosis and a rise in Hb. A positive
Iron trial response Good Poor Poor response to therapy can be defined as a daily increase
in Hb concentration of 0.1g/dL (0.3 or 1% rise in HCT)
from the fourth day onwards.
• If the serum ferritin is low but the hemoglobin is
• The ratio is normal in thalassemia trait and renal
normal, the individual is at risk of iron deficiency,
anemia
while if the hemoglobin is low but the serum ferritin is
• FEP/Hb ratio remains elevated during iron therapy and
normal further hematological assessment is required
returns to normal only after majority of cells containing
to identify the cause of anemia.
FEP formed during iron deficiency are replaced
• FEP/Hb ratio is not subject to daily fluctuations and Laboratory Tests in Iron Deficiency Anemia
sudden changes as in transferrin saturation
• The highest value of FEP is seen in lead intoxication—a • Low serum iron Less than 75 mg/dL
level of FEP greater than 160 ug/dL of RBC is taken as
• Increased total iron More than 470 mcg/dL
cut-off value for detection of lead intoxications. binding capacity
• FEP/Hb ratio in the range of 5.5 to 17.5 ug of Hb may
• Low transferrin saturation Less than 12% and 14% for
be attributed to either IDA or lead intoxication with infant and children
or without associated iron deficiency. This is not
• Serum ferritin < 10 µg/L in children and
used regularly due to the cost of the machine and 12 µg/L in adults
problems of standardization. Advantage of FEP is that • Increased red cell
unlike serum iron studies, FEP values are not altered protoporphyrin
immediately after iron therapy. concentration
Soluble transferrin receptor (sTfR) measures the severity • Depletion of stainable iron Graded from 0 to 4
of IDA and values more than 9 mg/L are considered from bone marrow
abnormal. Transferrin receptors (TfR) facilitate the
TESTS FOR THE STORAGE COMPARTMENT • Irritable, cranky child, breath holding spasm, history of
pica, worms’ infestation, chronic bleeding.
• Serum ferritin: A level of < 10 µg/L in children and • Microcytic, hypochromic anemia on PS, relative decrease
12 µg/L in adults is suggestive of iron depletion. Serum in RBC count and increased RDW and low MCV
ferritin is increased in infections, inflammation, liver • Leukocyte count is usually normal.
disease, parasitic infestations, enteric infections and
even upper respiratory tract infections Hypersegmented neutrophils may be seen due to
• Bone marrow evaluation: Morphologically bone concomitant B12 or folate deficiency or due to iron defi
marrow shows erythroid normoblastic hyperplasia ciency-induced interference with folate utilization, or due
though the normoblasts may be smaller than normal to IDA-induced impairment of jejunal function leading
(micronormoblasts) to poor absorption of B12 or folate. Thrombocytosis may
Bone marrow aspiration is not routinely indicated occur in patients with IDA, as a result of iron therapy per
in the diagnosis of IDA. The degree of cellularity and se or due to underlying condition such as malignancy or
the proportion of myeloid to erythroid cells on bone bleeding:
marrow examination vary depending on the severity • In infants and children the TS < 12 to 16 percent is
as well as the duration of IDA diagnostic. Ferritin value is < 12 to 16 ng/dL
• Prussian blue staining of the bone marrow is also used • Increase in FEP
for diagnosing IDA, though it is rarely indicated. The • Therapeutic test: Treat the child with 3 to 5 mg/kg of
iron content is graded from 0 to 4. elemental iron.
There is increase in retic count at 1 to 2 weeks and Hb
TESTS FOR PLASMA IRON COMPARTMENT level reaches to normal by 2 to 3 months.
It includes:
• Serum iron When to Suspect b-thalassemia Trait?
• Total iron binding capacity (TIBC), transferrin • Community: Kutchi, Lohana, Punjabi, Sindhi, Neo
saturation (SI). buddhist, Mahars and other high-risk communities
It is a major drawback is diurnal variation after 3 years • Microcytic/hypochromic anemia with target cells,
of age. Morning samples show higher levels. Hence if polychromasia, relative increase in RBC count and
morning sample shows a serum iron < 30 ug/dL, it is normal RDW. Normal iron study including ferritin
suggestive of IDA. level
• TIBC: It is less subject to biological variations. But • Poor and inadequate response to iron therapy.
normal range is 250 to 400 ug/dL. An increase in TIBC
indicative of IDA.
• Transferrin saturation: It is the ratio of above two Screening Tests
values and is consistent and hence is a very useful test • NESTROFT,93,94 discriminant functions.93,94 PS exami
for IDA. nation
Serum iron • Confirmatory tests: Increase in HbA2 more than 3.5
TS = × 100
TIBC percent
• b-chain synthesis (silent carrier).
In adults TS < 16 percent is suggestive of IDA. In infants
and children the diagnostic value is < 12 to 16 percent. TS < • Free erythrocyte protoporphyrin (FEP) and proto
6 percent is diagnostic of IDA at any age. porphyrin—heme (PH) ratio: Erythrocyte proto
It is important to realize that one should collect fasting porphyrin, the precursor of heme, accumulates in red
sample (nonlipemic) and all forms of iron supplements blood cells when it has insufficient iron to combine
should be stopped for 48 to 72 hours before collection with to form heme. Elevation of FEP mainly EZP level
while performing iron studies. is an early and sensitive indicator of iron deficiency.
FEP can be measured by a simple fluorescence
assay performed directly on a thin film of blood on
When to Suspect IDA? a glass slide. Both FEP and PH ratio are elevated
• Anemia during age of rapid growth, i.e. first 6 months in iron deficiency. Normal values of FEP are 30 to
to 3 years of life and adolescence. 40 mcg/dL RBC and PH ratio 16 (+5.3). FEP values
• Child not breastfed but fed with faulty diet, diluted above 70 mcg/dL RBC and of PH ratio above 32 is
artificial powder milk especially improper weaning, thought to represent iron deficiency. In uncomplicated
prolonged breastfeeding, bottle feeding. iron deficiency anemia, red cell FEP levels may range
from 100 to1000 µg/dL. EZP is also elevated in chronic Bone Marrow Examination
lead poisoning and sideroblastic anemia
• Serum iron: Normal serum iron level varies Bone marrow aspiration is not recommended for the
considerably. It has a diurnal variation with a peak diagnosis of IDA, as there are simpler, noninvasive
in the morning and trough in the evening. Serum and relatively inexpensive tests, which diagnose
iron concentration may also be affected by chronic IDA reasonably well. In contrast, bone marrow iron
infection, malignancies and chemotherapy as well staining, though a gold standard, is very painful,
as iron medication. Values below 40 mcg/dL (<12 expensive and cumbersome to perform. However, bone
mcg/dL in young children) are considered diagnostic marrow when done shows increased cellularity with
of iron deficiency (in absence of infection or other micronormoblastic erythroid hyperplasia. On staining
disorders which affect iron metabolism) with prussian blue (Perl’s reaction), there is little or no
• Total iron binding capacity (TIBC) and transferrin stainable iron seen.
saturation (TS): TIBC is the measure of plasma
transferrin, which is free, not bound to iron. The Response to Therapy
normal value of TIBC is 250 to 350 mcg/dL. Since
In uncomplicated IDA, administration of iron shows a
serum iron is about 100 mcg/dL, normally only one-
predictable reticulocytosis and a rise in hemoglobin. Hb
third of transferrin is utilized to bind iron, giving a
concentration remains the most dominant predictor of
normal transferrin saturation of 33 percent. In iron
response to therapy in uncomplicated iron deficiency.
deficiency states, TIBC is increased (> 350 mcg/dL)
A positive response to therapy can be defined as a daily
and transferrin saturation is reduced to below 16
increase in Hb concentration of 0.1 g/dL (0.3 or 1% rise in
percent (<12% for children)
HCT) from the fourth day onwards. Lower the initial Hb,
• TIBC below 200 mcg/dL is characteristic of inflam
greater is the response following iron therapy.
matory disease
• Factors that affect serum iron concentration do not
alter values of TIBC Treatment of Iron Deficiency Anemia
• Serum ferritin: The serum ferritin is a sensitive
Basic principles of management include:
laboratory index of iron status. It is the best non-
1. Correction of anemia
invasive test (gold standard with a high specificity
2. Treatment of underlying cause
and adequate sensitivity) for evaluating iron status
• Management of IDA: If the patient is severely pale
in the body. It is estimated that each ng/mL of serum
and sick looking, breathless, has tachycardia, raised
ferritin is equivalent to 8 to 10 mg of storage iron. A
JVP and tender hepatomegaly, it is suggestive of
serum ferritin value of less than 12 ng/mL is highly
congestive cardiac failure. Such a patient needs
specific for iron deficiency but gives no information
immediate attention and prompt treatment including
about its magnitude.91 Ferritin levels are estimated
admission, diuretics, anti-CCF measures and packed
by radioimmunoassay (RIA), or ELISA techniques.
cell transfusion. And other treatments depending on
Serum ferritin is increased in chronic disorders, e.g.
underlying cause
chronic infection and inflammation, malignancies,
• One should not waste time in lengthy diagnostic tests
chronic liver disorders. In presence of any of these, a
and do as minimum tests as required.
coexisting iron deficiency anemia can be missed.
• Even removing too much blood for various tests in
• Soluble plasma transferrin receptor (sTfR): Transferrin
small infants, can be hazardous as it can precipitate
receptors (TfR) facilitate the entry of transferrin bound
cardiac failure. Instead, one can arrange for packed
iron into cells by a process of endocytosis in iron
cell transfusion and remove blood for various tests just
deficiency anemia, transferrin receptors are increased
before starting transfusion.
probably due to an increased turnover associated
• Other way if the patient is pale but comfortable and
with ineffective erythropoiesis and, an increase in
not sick, there is neither need to give packed cell
cellular transferrin receptor expression produced
transfusion nor start ‘gun shot’ therapy without proper
by iron starvation. Unlike the serum ferritin, which
investigations and establishing the diagnosis.
only identifies iron deficiency, the serum transferrin
receptors measure its severity. Value above 9 mg/L The management of iron deficiency anemia is consi
are considered abnormal health level in healthy male dered in two parts:
and female are 56 mg/L and is normal in anemia of 1. Treatment of the individual patient
chronic infection and inflammation.95 2. Treatment at public health level.
The successful management requires: • It may be given at bed time as compliance is better
• Confirmation of the diagnosis in children as child goes to sleep and vomiting and
• Thorough investigations to find out the etiology and to pain abdomen is much less as intestinal activities are
treat the cause slow during sleep. Compliance in the first month of
• Supplementation of iron. therapy is important as majority of iron absorption
It is important to find out the etiological factor for iron occurs during this period. It is continued for at least
deficiency to prevent failure of therapy and recurrence of 2 to 3 months after hemoglobin becomes normal, to
deficiency after treatment is stopped, especially in older replenish stores.66,85
children who are likely to have a secondary IDA associated
with underlying cause. Treatment of worms, giardiasis, Dose
bleeding from any site, recurrent infections is must to treat • For infants and children: Iron store present at birth
the patient adequately besides iron. and the highly bioavailable iron in breast milk protect
Infants usually have poor dietary history. Lack of an infant from IDA up to 6 months. Supplementation
breastfeeding associated with bottle feeding or prolonged with medicinal iron has been recommended by WHO
breastfeeding and improper weaning and poor intake of for all children beyond 4 to 6 months of age and low
iron containing food as a cause of iron deficiency. birth weight babies from 2 months onwards, for
Promotion of exclusive breastfeeding for first 4 to preventive supplementation, iron dosage is 2 mg/kg
6 months. Thereafter introduction of proper and age per day for children of all age groups. Children of 6 to
appropriate food items after 6 months and continuing 35 months of age should receive a daily uniform dose
breastfeeding for as long as possible, along with prop of iron folic acid (IFA) supplement (20 mg elemental
hylactic iron supplementation will prevent iron defici iron + 100 mcg folic acid) in liquid from (3–6 mg/kg/
ency during infancy and early childhood. In older day of elemental iron). Although the desired Hb level
children diet modifications to improve total calorie is usually reached in 2 months, iron therapy should
intake and iron containing foods in diet will prevent iron continue for another 3 months to build up iron
deficiency. stores.
• For women (15 years +) with severe anemia (Hb < 7 g/
Management of IDA consists
• Iron therapy—including of
replenishment of stores. dL): National Nutritional Anemia Control Program
– Oral (NNACP)96 recommends two tablets of iron-folate tablet
– Parenteral. per day (each tablet containing 100 mg of elemental
• Treatment of underlying causative factor iron and 500 mcg of folic acid) for a minimum of 100
• To prevent recurrence of deficiency preventive measures: days. Prolonged duration of treatment is required to
– Diet counseling
correct the anemia and replenish iron stores.
– Iron supplementation
– Iron fortification.
Restoration of Hb to normal with ferrous salts requires
3 to 6 months of Rx. Replenishment of body iron stores
requires further therapy for additional 2 to 4 months.
• Risk of accidental iron poisoning in small children
IRON THERAPY
• In developed countries, tablet containing ferrous iron
Aim is to give iron in enough dose, for enough number of are the second most common cause (after aspirin) of
days so as to normalize the Hb levels and replenish stores, accidental poisoning among small children leading to
in a convenient way with least number of side effects. hospitalization and several death.
It can be given either: • Preparations of iron formulations.
• Orally All dietary iron has to be reduced to ferrous form to
• Parenterally. enter the mucosal cells. Various iron salts available include
ferrous fumerate, ferrous gluconate, ferrous sulphate-
hydrous anhydrous form, etc.
Oral Iron Therapy
Bivalent iron salts like ferrous sulfate, fumarate, gluco
Advantages of oral iron therapy are cheap, effective, nate, succinate, glutamate and lactate have been preferred
safe, convenient and well tolerated and preferred and over ferric salt preparations.
advocated route of therapy. • Type of iron salt:
• Best absorbed when given on an empty stomach or in – Ferrous sulfate (20% elemental iron) is commonly
between the meals in divided doses. used for tablet preparations.
– Ferrous fumarate (33% elemental iron) is environ are less and well tolerable if proper counseling is done
mentally more stable. and before starting therapy.
– When ferrous preparation is taken on empty • Rarely case of acute iron poisoning occurs by taking
stomach absorption increases, however side effect accidental or suicidal overdose.
also increases. • Once or twice a week oral iron therapy has been found
Many new iron preparations are not affected by food to be equally effective if not better with less side effects.
or milk enabling administrations without consideration of
the timing of feed. These are iron polymaltose complex and Daily versus Weekly Supplementation99-104
carbonyl iron (highly purified metallic iron with a particle
size less that 5 mm). However, most of these preparations In humans, the intestinal mucosal turnover time is 5 to
have not been found to be effective in clinical setting. 6 days and serves as the basis for the weekly preventive
Ferrous salt is the drug of choice as it is absorbed best supplementation regimen:
and is cheapest. Iron salts of carbonate, citrate, choline • Iron absorption from GI tract depends upon iron
citrate and pyrophosphate are not absorbed efficiently content in the mucosal cells. Less the iron contents
(Table 5). more the absorption
Other salts available are: • One of the major problem with daily supplementation
• Ferrous fructose regimen is that supplements must be taken for a long
• Ferrous succinate period of time for achieving desired improvement in
• Ferrous lactate iron status
• Ferrous carbonate • It has been seen that iron deficient and anemic women
• Ferrous glycine citrate can absorb as much as 30 to 40 mg dose of iron ingested
• Ferric ammonium citrate on an empty stomach
• Colloidal iron. • Iron administration every 3rd day (intestinal mucosal
turn over time in rat is 3–4 days) was more efficient
Various Forms of Iron Preparation in iron deficient rats than when administered daily.98
Indeed, in weekly dosing food iron absorption is also
• Uncoated tablets: Uncoated tablets are cheaper but have
better maintained. Weekly dose is considered as cost
more side effects. Enteric coated tablets have better
effective with requirement of lesser number of doses,
availability with less side effects but are expensive.
with fewer side effects and better compliance. Recent
Flavored chewable tablets are available which have
research in experimental animals and field studies
better compliance but again are costly. And overdose is
more common. among the preschool children in China and other
• Syrups and drops can lead to staining of tongue and countries have indicated that intermittent therapeutic
teeth and are costly, may not be stable. dosage can be as effective as daily dosage in correcting
• Iron in hemoglobin form is also available but has the mild to moderate anemia and iron deficiency and in
little advantage. Content of elemental iron is low and anemia prophylaxis.96,97 A number of studies in Indian
is expensive and often fails to provide the required setting have shown weekly iron supplementation is
amount of iron. effective
• Side effects of oral iron therapy: Nausea, vomiting, • With daily administration of iron, gastrointestinal
abdominal cramps, diarrhea, constipation, staining of complaints were more common in majority of the
tongue and teeth, blackish discoloration of stools, etc. studies and were rare with weekly dose. Serum
are common side effects. By and large the side effects ferritin levels were nonsignificantly higher in daily as
compared to weekly supplement
• In Bombay, Mehta et al.100 in a study of 1748 adoles
cent girls (10-18 years) from urban slums revealed
Table 5 Percentage and amount of iron in some commonly
prevalence rate of anemia in 63.8 percent. Iron supple
used iron tablets preparation of iron compounds97
mentation resulted in beneficial effect and prevalence
Per tab Per tab Elemental % of anemia decreased to 61.16 to 26 percent in daily
(mg) iron iron (mg) group and 65 to 33.9 percent in weekly group and no
Ferrous fumarate 200 66 33 changes 64.8 to 58.4 percent in control group and there
Ferrous gluconate 300 36 12 was statistically significant increase in Hb level—rise of
mean 0.939 g. Hb in daily group as compared to 1.54 g
Ferrous sulfate (7H2O) 300 60 20
in weekly group. Rise in ferritin level was 5.04 ng/mL
Ferrous sulfate 200 74 3 in weekly group as compared to 4.69 ng/mL in the dai
(anhydrous)
ly group. Sheshadri et al. from Baroda101 in her study
reported rise in Hb in daily group was 0.749 percent as Indications

compared to 0.44 gm% in the weekly group side effects
of iron supplementation were almost absent in weekly • Oral iron not sufficient to compensate the need for
regimen as compared to 15 percent in daily regimen increasing deficit as in persistent /significant bleeding-
• A WHO report based on 11 trials published involving like epistaxis (telangiectasia), gastrointestinal bleeding,
14000 subjects for a period of 4 to 7 months in school etc.
children, adult, nonpregnant women and pregnant • Decreased absorption as seen in various GI disorders–
women revealed weekly supplementation was as Chronic malabsorption syndromes: Chronic diarrhea,
effective as when given daily whereas in some studies cystic fibrosis, Crohn’s disease, surgery, gastrointesti
slightly less therapeutic effect was seen in weekly nal disease, etc.
supplementation as compared to the daily supple • True intolerance for oral iron therapy.
mentation. • Having severe side effects on oral therapy.
Based on these studies and recent multicentric study • For receiving recombinant erythropoietin therapy, or
in India has now recommended that adolescent girls on for use in treating functional iron deficiency.
attaining menarche should be given weekly dosage of the • Noncompliance may make oral iron treatment in some
IFA tablet containing 100 mg elemental iron and 500 ug patients inadequate
folic acid once a week accompanied by appropriate dietary
supplementation.97 Types of Parenteral Iron Preparations
• Intravenous (IV)
Response to Therapy • Intramuscular (IM)
The first response is the decreased irritability and a • Parenteral iron products available are:
subjective improvement – Iron dextran,
↓ – Ferric gluconate,
This is followed by a marrow response and reticulocytosis – Iron sucrose.
peaking at 5 to 7 days. Following parenteral administration of iron, the
↓ iron carbohydrate complex is separated by the reticu
Hb rises at a slower rate, i.e. 0.25 to 0.4 g/dL/day. It loendothelial system. Iron is gradually released into the
normalizes by 6 to 8 weeks but it may take little longer. circulation.
Failure of oral iron therapy: Iron Dextran Complex

• Wrong diagnosis
Iron dextran is a colloidal solution of ferric oxyhydroxide
• Inadequate dose, inadequate length of treatment
complexed with polymerized dextran.
• Poor compliance, etc. This can be easily tackled by proper
knowledge on part of physician and proper counseling of Total dose to be given as, divided intramuscular
parents injections or as full dose IV therapy.
• Having severe side effects The iron requirement can determine from the
• Decreased absorption as seen in various GI disorders equation:
• Gastrointestinal bleeding, which is aggravated by oral iron Iron (mg) = weight (kg) × Hb deficit (g/dL) × 80/100 ×
therapy 3.4 × 1.5 or
• Increased iron loss not met with oral therapy, e.g. ongoing weight (kg) × Hb deficit (g/dL) × 4.
GI bleeds, or true intolerance. Such cases can be treated Dose: Dose of iron (mg) = weight (lbs) × Desired
with parenteral iron therapy increment of Hb (g/dL) × 3.
• Chronic malabsorption syndromes—chronic diarrhea,
a. Intravenous route (IV): There are two methods:
cystic fibrosis, Crohn’s disease, etc.
• Infusion of iron dextran diluted in ratio of 5 mL of
Oral iron not sufficient to compensate the need for increasing
deficit as in persistent bleeding (Epistaxis). iron dextran complex in 100 mL of normal saline.
Initially flow rate should be kept at 20 drops/min
for 5 to 10 minutes and there are no reactions, then
rate can be increased to 40 to 60 drops/min.
Parenteral Iron Therapy • Bolus injection of iron dextran: Bolus dose of iron
dextran diluted in 20 mL of saline.105-107 Both these
Intravenous Iron
routes are however used after a prior sensitivity
Parenteral iron therapy should usually be avoided as testing where 1 mL of iron dextran solution is diluted in
having severe side effects. 20 cc of normal saline and injected slowly over 10 to
15 minutes following which one should observe for or a 1 percent per day rise in hematocrit which is more
reactions for 1/2 to 1 hour rapid than is anticipated in the adult.
• Sodium ferric gluconate intravenous injection or • Iron absorption is maximum during the initial phase of
infusion. therapy and declines from 14 percent in the 1st week to
Ferrlecit (sodium ferric gluconate complex in 7 percent in the 4th week to 2 percent after 4 months.
sucrose injection) is indicated for treatment of iron Blood transfusion may be needed in most severe cases
deficiency anemia in pediatric patients age 6 years of IDA when the hemoglobin level is below 3 to 4 g/dL or
and older. Standard dose of 125 mg in 100 mL of when superimposed infection may interfere with optimal
normal saline intravenously over 60 min. If the therapeutic response. Packed red cells may be slowly
patient’s serum ferritin is less than or equal to 100 given preferably 2 to 3 mL/kg at one time.
ng/mL and the transferrin saturation is less than
or equal to 20%, the dose can be repeated over 8
Side Effects
weeks.
• Iron sucrose injection Venofer Reactions can occur with both IM and IV therapy and can
– Iron sucrose cucrose injection (Venofer) is an be either immediate or delayed.
iron hydroxide sucrose complex in water. • Immediate reactions
– Iron sucrose is administered by intravenous – Pain at the injection site
injection or infusion. The recommended – Vomiting, nausea, headache, malaise, flushing,
schedule is to administer 100 mg intravenously metallic taste, such reactions are brief in duration
over 5 min, 1–3 times weekly upto 1,000 mg if and often are relieved by slowing the rate of
required. infusion.
The rate of administration should not exceed – Severe reactions like anaphylaxis, hypotension,
20 mg per minute. Side effect includes hypotension, cardiac arrest, etc. should be contraindicated to
nausea, and lower back pain. further doses.
• Delayed reactions
Intramuscular route (IM): This is very painful and
– Arthralgia, fever, myalgia, regional lymphadenitis
may lead to serious allergic reactions and hence not used
in children. Intramuscular injections are best given into
the upper outer quarter of gluteal region using Z tract Prevention of IDA
technique. A dose of 0.1 mL should be given as test dose The basic approaches to the prevention of IDA are:
intramuscularly and there are no reactions within 1 hour, • Supplementation with medicinal iron
full dose (to a maximum of 0.5 cc) can be given. • Dietary modification (Table 6)
Though most of the reaction are mild and tran • Fortification of foods with iron
sient, anaphylactic reactions may be life-threatening • Other measures, which could play an indirect role in
and hence one should always keep injection adrenaline, improving the iron status are control of viral, bacterial
hydrocortisone and resuscitative measures handy before and parasitic infections (hookworm infestation-correct
injecting. ed by regular deworming measures), malaria. Improve
ment in poor health facilities, poor socioeconomic
Response to Therapy status, faulty dietary patterns, the degree of urbaniza
• Rapid hematologic response can be confidently tion, educational background, provision of safe water,
predicted in iron deficiency. environmental sanitation, health education, vitamin A
• A positive response to therapy can be defined as a daily deficiency and immunization, etc.
increase in hemoglobin concentration of 0.1 g/dL (0.3 • Protection and promotion of breastfeeding: Exclusive
or 1 percent rise in hematocrit) from the fourth day breastfeeding till the age of 4 to 6 months and
onwards. promoting breastfeeding for as long as possible, even
• Approximately 2 months are required to achieve a up to 2 years. Human breast milk is low in iron about
normal Hb level. 0.5 mg/L. An infant taking 600 to 650 mL of breast
• Reticulocytes increase within 3 to 5 days and reach a milk daily ingests approximately 0.3 mg of iron/day.
maximum at 5 to 10 days, reticulocyte counts being 8 However, the bioavailability of this iron is quite high,
to 10 percent in severe anemia. (50%) as much as 0.15 mg of iron per day is absorbed
• The maximum rate of recovery from severe anemia in which is sufficient for an exclusively breastfed baby.
a child may be 0.25 to 0.4 g/dL per day increase in Hb Breast milk appears to be adequate to cover the dietary
Table 6: Iron content of food articles108-110 – Introduction of iron containing food after the age
of 4 to 6 months is the most important step in
Food Iron content Articles rich in iron (>10
prevention of anemia of infancy.
(mg/100 g) mg/100 g)
– Commercially prepared iron rich weaning foods
Cereals 2.5–14.0 Bajra, wild barley, kangri
though available in developed countries, are very
ragi, rice flakes, whole wheat
expensive and beyond the reach of the majority of
flour, kodra (Harik)
the families and so not recommended.
Pulses and 2.7–11.0 Bengal gram, cowgram, – Encourage natural homemade food items, initially
legumes soyabean
semisolids and then solid items so that child should
Leafy vegetables 0.9–40.0 Amaranth, beet greens, consume normal family diet by 9 to 12 months of
Bengal gram leaves, age.
coriander, alu leaves,
– Home made weaning foods rich in iron and
pudina, neem, radish top
rajgira leaves, turnip greens, vitamin C are cooked vegetables, raw fruits,
all types of green bhajis parents should be taught how to prepare mashed
(spinach, methi, lettuce, etc.) vegetables? Thick palak soup with boiled dal and
Roots and tubers 0.4–13.9 vegetable, citrous fruit juices, egg preparation,
minced mutton, etc. and motivated to introduce
Other vegetables 0.2–22.2 Amaranth seeds, dhaincha
these to the infants in early life after 4 to 6 months
seeds
of age.
Nuts and oil 2.5–10.0 Garden cress, gingelly, – Foods rich in vitamin C like orange, citrous food,
seeds mustard,
guava, tamarind are seasonably available and
Fruits 0.1–10.0 Dates, karvands, raisins are however expensive particularly in developing
Seafood 1.0–11.5 Most Indian fish, crab countries. In developing countries where meat
Meat 2.0–18.8 Beef intake is low, vitamin C (ascorbic acid) is the single
most important enhancer of iron absorption.
Milk 0.2–0.8
Adding as little as 50 mg of ascorbic acid to a
Miscellaneous Jaggery, yeast meal will double the iron absorption (an orange
or lemon, or cabbage 100 g or 200 g of amaranth
iron requirements of normal birth weight full term and will sufficient amount of vitamin C). The presence
infants up to the age of 6 months. From 6 months of age of vitamin C 25, 50, 100, 250, 500 mg in given meal
the iron requirement increases markedly and hence is associated with 2, 3, 4, 5, 6, fold enhancement
the iron from breast milk alone is no longer sufficient. of iron absorption respectively.5 Approximately
• Encouraging the timely introduction of iron containing 50 to 80 percent of vitamin C originally present in
weaning food is an important step in prevention of food can be lost during cooking. And hence should
anemia in early infancy and childhood. As maternal consumed a raw form. Inhibitors of iron absorption
transplacental transfer of iron from mother to child such as phytate and tannin. (Phytates are present
takes place during the last trimester of pregnancy, in wheat and other cereals). Tannin present in tea
iron storage in premature and low birth weight infant and to a lesser extent in coffee, nuts and legumens).
is affected and low. Hence, low birth weight infant hence should not be consumed with meals or
require iron supplementation from the age of two shortly after meals.
months. Approximately 50 to 80 percent of vitamin C originally
• Nutrition education and dietary modification: Food present in food can be lost during cooking. Vitamin C
based intervention: Anemia (IDA) is hardly ever content of food cooked and left standing decreases,
observed in exclusive breastfed infants till the age of reheating reduce still further. In poor households food
4 to 6 months even though mother is severely anemic. is cooked once and reheated 12 hours apart, in which
Breast milk has high bioavailability for iron absorption. case difficult to ensure that enough vitamin C is retained.
Breastfeeding along with appropriate complimentary Best would be to consume raw fruits but caution for
feed, including iron rich or iron fortified foods where hygienic care for GI infections. Common household
possible through the second year of life is important strategy, processing methods, germination, malting and
modality to prevent IDA. fermentation, increases vitamin C content and lower
tannin and phytic acid content or both, e.g. germination of • Dietary modification process like germination
some cereals and legumes for 24 to 48 hours is associated (sprouting of green grams) and intake of green leafy
with appearance of 10 to 70 mg ascorbic acid/100 gm and vegetables are also helpful in prevention of IDA.
reduction of 8 to 25 percent in tannin, 25 to 35 percent in Promotion of low viscocity, nutrient dense food for
phytic acid and bioavailability increases 2 folds. infants is useful. Germinating, malting, fermenting also
• Heme containing food like red-meat, fish and other increases iron absorption by increasing the vitamin C
sea-food have highly absorbable iron and promotes content and lowering tannin and phytic acid which
the absorption of the iron more than other less inhibits iron absorption from the gut. Germination
bioavailable food sources. However, it is a very increases the bioavailability by almost 2 folds. Just
small fraction of the total iron intake particularly inclusion of guava fruit/citrous food with lunch and
in persons from developing poor countries. Heme dinner have shown to raise Hb level by 2.2 g/dL.110
iron is directly absorbed (20–30% absorption). Its • For older children and adult, iron supplementation
bioavailability is little affected by the nature and can be done by cooking of the food in iron pots may
composition of the meal, like other associated foods increase the iron content of a meal several fold. This is
such as phytate, etc. especially true for soups containing vegetables. Frying
in iron pans does not increase the food’s iron content.
Approximate ascorbic acid content: Fruits and vegetables • Health program and nutrition education on dietary
vitamin C mg/100 g of food109,110 diversification are useful strategies to improve iron
Fruits Guava 326 intake in intervention area.
Lemon fresh (juice) 37–50 Strategies for iron fortification: Basic approach to
Orange fresh 46 prevention of IDA:
Pineapple fresh 37 • Food based strategies: The problem of IDA in children
Mango fresh 42 largely disappeared in North America when foods
Vegetables fortified with iron and other micronutrients became
Cabbage raw 54–60 available for children. In this group, the prevalence of
Cabbage boiled 15 IDA has fallen from 21 percent in 1974 to 13 percent in
Cauliflower raw 60 –96 1994.
Cauliflower boiled 20 • Nonfood based strategies: Food based strategies are
Potato raw 21 important for raising the iron status of populations. They
Potato boiled 12–18 will not be enough to improve the iron status rapidly.
Sweet potato raw 25–37
Sweet potato boiled 15 Current Approaches to Food Based
Spinach boiled 7–25 Fortification with Iron
Tomato raw 20–26 • Exclusive breastfeeding
Turnip boiled 17 • Avoid bottle-feeding
• Introducing of iron containing weaning foods from
• Nonvegetarian foods not only have a rich amount of 6 months of age
heme iron, but they enhance the absorption of the non- • Older children: Encourage diet containing iron—
heme iron contained in the rest of the meal. However, cereals wheat, ragi, jowar sprouted cereals, green
enhancing meat consumption is not practical by and leafy vegetables, jaggery, nonvegetarian food like
the poor rural people from developing and religious mutton, chicken, fish, egg and liver preparations.
objections to the consumption of meat also pose a • Commercially prepared iron rich weaning foods is not
problem. affordable to our poor patients
• Some breast milk substitutes particularly cow’s milk • Increasing utilization of iron in poor communities
is prone to cause gastrointestinal bleeding in infants • Increasing total consumption of habitual food so that
leading to IDA. their energy needs are fully met (Increased by 25–30
• Increasing total consumption of habitual food in percent when the energy shortage was corrected)
young children (Mothers should include to feed 4–5 • Enhancing the bioavailability of iron by germinating,
meals per day for young children.) so that their energy malting, fermenting by almost 2 folds
needs are fully met and ensure that total iron intake • Enhancer heme iron, vitamin C, meat, fish
is high even though the percentage of iron absorbed • Avoiding inhibitors of iron absorption like phytic acid,
from each meal remains low. phytates in plant based diets, tannin in tea and coffee.
Supplementation of Medicinal Iron meat, by the small amounts of iron from cereal diet. This
explains the high prevalence of iron deficiency.
Supplementation with oral iron tablet (syrup) is the most
Despite the fact that iron is one of the most abundant
widely used approach to control global problem of iron
metals on the earth crust, iron deficiency is the most
deficiency anemia. For the past 150 years or more, oral
prevalent deficiency in the world. PARADOX IS
ferrous sulphate syrups have been the primary strategy to
“POVERTY IN THE MIDST OF PLENTY”.
control IDA in infants and young children.3
It is useful for rapid treatment as well as for prophylaxis
1. Although pockets of infants and children remain at and prevention of anemia can be targeted at high-risk
risk, generally, the eradication of iron deficiency in group of becoming iron deficient, such as pregnant women
developed countries is recognized as a successful adolescent girls. Preschool and school and children,
public health accomplishment. This solution has not infants and ‘captive audience’ such as school children or
worked in developing countries where commercially plantation workers who can receive the supplements at
purchased fortified foods are not affordable or are not school or work. Problems encountered include side effect
used. of the drug and lack of motivation to continue the drug
2. However, adherence to the syrups is often limited for a minimum 3 to 4 months in patients who perceive
owing to a combination of their unpleasant metallic themselves not to be ill.
after taste, the dark stain they leave on the child’s teeth,
and abdominal discomfort.
In the developing world, there are three major Pregnant Women
approaches available to address iron deficiency: Pregnancy creates a larger demand for iron which is
1. Dietary diversification so as to include foods rich in needed for the development of the fetus and placenta and
absorbable iron. to expand the woman’s blood volume. Iron also is lost
2. Fortification of staple food items (such as wheat flour), with blood lost during delivery. About 100 mg of iron are
3. The provision of iron supplements. needed to cover the iron requirement of the mother and
Dietary or fortification strategies are not logistically the fetus during pregnancy. Dietary absorption of iron is
or economically feasible, supplementation of individuals reduced during the first trimester and morning sickness,
and groups at risk is an alternative strategy. nausea, vomiting adds to the problem. In most of the
For the past 150 years or more, oral ferrous sulphate developing countries 25 to 30 percent of women have little
syrups have been the primary strategy to control IDA in or no iron stores even before conception. Particularly in
infants and young children. pregnant teen age mothers the situation is farther critical.
The earth’s crust contains 4.5% iron whereas the Supplementation should be done primarily during the
human body contains a mere 0.005% of iron. Accordingly second half of pregnancy. Pregnant women are a priority
iron overload, should be more of a problem rather than group for iron supplementation. A number of programs—
iron deficiency. However, this is not so, because the form National Nutritional Anemia Program 1970, National
of iron in the environment as well as food is insoluble Nutritional Anemia Control Program (NNACP)111 1989
and difficult to assimilate. In early times the earth had have been implemented and dosage was revised from
a reducing atmosphere and an abundance of ferrous 60 mg elemental iron to 100 mg and 500 mg folic acid per
iron was available for incorporation into biological tablet. Poor compliance due to side effects like nausea,
molecules. Later with an increase in the atmospheric vomiting, pain in abdomen, constipation, loose motions,
oxygen, iron existed largely in its less available ferric etc. and lack of awareness regarding the real need for iron
form and special devices had to be developed by life- during pregnancy and the importance of iron for their
forms, for the acquisition of iron. Bacteria for example health, for the unborn fetus and the newborn.
synthesize and excrete high affinity chelating agents
that extract otherwise unavailable iron from the
surroundings environments. Roots of the plants also Adolescent Girls31-40
secrete substances that augment iron absorption. In
Why Concentrate on Adolescents Girls?
the mammalian species, mucosal transferrin appears to
perform this function. The greatly elevated iron requirement of pregnant
The body had been genetically designed to absorb woman indicates need for prepregnancy reserve. Daily
hemoglobin iron, as then human beings were hunters requirement of iron of pregnant women are three times
and carnivorous. The progressive change in diet that as compared to the need of nonpregnant women and total
began 10,000 years ago, with the cultivation of grain and requirement of iron during pregnancy is about 1000 mg.
vegetables led to replacement of highly available iron in Though food based strategies are important for raising the
iron status of population they will not be enough for many sulfate, orthophosphoric acid have been extensively
iron deficient women who become pregnant. Hence there used for the fortification of wheat flour, bread and other
is a higher requirement of iron during adolescent period bakery products, corn-soya-milk preparation (CSM)
and reproductive age and during lactation. In developing salt, sugar, fish sauce, rice, etc. Iron salt EDTA (ethylene
countries 24 to 30 percent women have no iron reserve. diamine tetra-acetate)112-117 has been successfully used
As poor iron store before conception is one of the cause of to fortify sugar/wheat flour in Guatemala115 (13 mg of
IDA during pregnancy, there is need to raise iron stores of iron/100 mg sugar). A formula for double fortified salt,
women (adolescents) before they become pregnant. Iron salt fortified with iodine and iron has been developed
loss during menstruation is estimated to be 16 mg/kg/day and has been found effective.112
and basal requirement during this period in both male
and females estimated to be 44 mg/kg/day or total of 3 mg/ Home Fortification with Sprinkles112-123
kg/day in female97 and hence need for supplementation of
iron in adolescent girls (10–18 years) 100 mg of elemental The idea of sprinkles was formulated in 1996, when a
iron daily to all adolescent girls every year for 100 days. group of consultants determined that the prevention of
Addition of 25 mg of vitamin C to iron folate tablet has childhood. IDA was a United Nations Children’s Fund
shown a higher Hb response as compared to iron folate priority, yet available interventions (syrups and drops)
tablet alone in adolescent girls.34 The most common form were not effective—single-dose sachets containing
of iron in iron tablet used are ferrous sulfate (20% iron) micronutrients in a powdered form, which are easily
fumerate (33% iron) and gluconate (12% iron). sprinkled onto any foods prepared in the household. This
would be a successful method to deliver iron and other
micronutrients to children at risk .
Iron Supplementation In sprinkles, the iron (ferrous fumarate) is encapsulated
• In breastfed term infants 1 mg/kg/ day of oral iron in within a thin lipid layer (soya-based hydrogenated lipid
single dose starting from 5th month of life will prevent layer) to prevent the iron from interacting with food.
IDA at later age. There are minimal changes to the taste, color, or texture
• In preterm and LBW babies one may give 2 to 3 mg/ of the food upon adding sprinkles. Other micronutrients
kg/day of oral iron and start it early, i.e. by 2nd and 3rd including zinc, iodine, vitamins C, D, and A, and folic
months of age. acid may be added to sprinkles sachets. Any homemade
• Iron ‘tonic’ to infants is more important than multi food can be fortified with the single-dose sachets, hence
vitamin drops! the term “home fortification”. Two formulations have
• Iron supplementation can also be given to pregnant been developed, a nutritional anemia formulation and a
women, school children, and other high-risk groups. complete micronutrient formulation.
• Community level: Fortification of staple foods like Encapsulation prevents the micronutrients from
cereals, grains, sugar or salt will be effective.Vitamin C oxidizing the food. No change in the color and taste, fixed
or meat increase the iron absorption. Salt fortification dose sachet (15–45 mg).
gives an iron content of 1 mg/g of salt in preparation. Home fortification can be done by, parents, health
Common112 salt fortified with iron orthophosphate and care giver and can be given as Intermittent Fe therapy
sodium hydrogen sulphate with ascorbic acid has been under supervision or can be integrated into existing health
found stable and effective in field trials in India. program. Sprinkles is a novel approach and this can be
• Similarly for infants fortification of formula feeds and sprinkled on any complementary food at containing
cereals have been successful in developed country. micronutrients in a powder form, which are easily sprinkled
• Fortification of foods with iron constitute the most onto any foods prepared in the household. Sprinkles have
desirable, cost effective and sustainable methods of been shown to be efficacious in the treatment of anemia in
preventing iron deficiency and is a long-term measure many developing countries.
for improving the iron status of the entire population. • Periodic de worming should be considered in endemic
• Fortify a staple food that is consumed in significant areas.
quantity regularly by most people. Widely consumed • Administration of one pediatric (small) tablet contain
condiment—salt, sugar, fish sauce, curry powder113 and ing 20 mg of iron and 100 ug of folic acid daily for 100
have all been successfully fortified with iron. In South days every year has been recommended by National
America both dried and liquid milk and milk products Nutritional Anemia Control Program.Various contact
like yogurt, fortified infant food have been fortified with points like measles (9 months) and DPT booster (16–
iron. Ferrous fumerate, ferrous gluconate, lactate and 18 months) in ICDS scheme should be utilized for the
ferrous sulfate, ferric orthophosphate, sodium acid distribution of iron folate.
Control of Viral, Bacterial and another alternative. All it needs realization of magnitude
Parasitic Infections of problem of IDA will and open eyes to prevent, detect
and treat IDA in time!
Effective, timely curative care could diminish the adverse
nutritional consequences of viral and bacterial disease. Did you Know!
Although the number of infective episodes is unlikely to
be reduced, the curative services reduce the duration • Thirty percent of world population suffers from IDA.
Out of which > 80 to 90 percent in developing countries.
and severity of infections, thereby improving iron status
• < 70 to 90 percent Indian children at 1 to 2 years of
even if there is no increase in dietary iron consumption.
age, adolescents, pregnant women, lactating mothers
Preschool children who have such would benefit from such
suffer from IDA.
improvements in health care. It is vital to educate families • Pica is a common symptom of IDA but can also be seen
frequent infections about proper feeding practices during in lead poisoning.
and after periods of infective illness, as the young children • Always suspect IDA in patient with breath holding
are often semistarved when ill , either because of their spasm.
poor appetite because of illness and myth of parents about • Exclusive breastfeeding protects a full term infant from
restrictions of diet during illness. Breastfeeding must be IDA till 6 months of age.
confined as it helps preventing infections apart from its • Preterms can develop IDA as early as 2 to 3 months.
direct effect on iron status. Along with encouragement • IDA early in life can reduce the intellectual potential
for taking timely immunizations, primary health care and cognitive functions of the child permanently.
measures like improvement in personal hygiene, environ • Adding vitamin C (fruit juices) or meat can increase
mental sanitation, provision of safe water go a long way in iron absorption by 3 to 4 folds.
improving the nutritional status and indirectly iron status • b-thalassemia is a common differential diagnosis of
of the children. The main culprits in causing anemia due to IDA. RDW helps in differentiation as it is normal in
chronic blood loss are parasites hookworm ankylostoma thalassemia minor and increased in IDA.
and necator and schitosoma. Heavy giardial infestation • Simple way to prove IDA is by iron therapeutic test.
can reduce iron absorption. Although routine deworming • HbA2 levels can be low in patients with thalassemia
is recommended in actual practice, it becomes costly, minor if they have coexisting IDA. Repeat HbA2 after
when advised to entire community and reinfections iron therapy and will show increased values.
are common. As it does not improve hemoglobin [Hb] • Cheapest and best form of iron is ferrous sulfate tablets.
value significantly, addition of supplemental iron or food
fortification with iron has better results for rise in (Hb)
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C H A P T E R 15
Megaloblastic Anemia
INTRODUCTION While Dr George Whipple (1), a pathologist along with his

colleagues at the Rochester university firmly established
Nutritional anemia is one problem that has haunted the the fact that properties of food affected blood formation,
developing world for ages now and still continues to do a concepted not previously accepted, George Minot and
so. Nevertheless the modern “zero figure trends” and the William Murphy (2) at Harvard Medical School developed
resultant increase in the followers of vegan diets has lead to a special diet that could reverse the pathology of pernicious
a surge in the number of anemia cases in the western world anemia and cure patients. These milestone discoveries
also. As science progressed, a better insight was gained lead another prominent investigator of the era WB Castle
into the causes, pathogenesis and molecular details of the (3) to put in decades of investigation into uncovering the
biology of the various kinds of anemias consequent to which complex pathophysiological mechanisms underlying
tremendous advances in the diagnostics and treatment of pernicious anemia.
the same have been made. Much has been discussed and Pernicious anemia is another example where an
spoken about the iron deficiency anemia which was so far effective treatment was found before an understanding
considered to be synonymous to nutritional anemia but of the underlying pathogenic processes was found. Even
as has been realized over the last few decades cobalamin before the therapy for megaloblastic anemia was described
and folic acid deficiency is an equally big problem in 1926, it was known that marrow morphology could
challenging the practicing pediatricians and hematologist, return to normal with reticulocytosis and correction of
if not more. This chapter intends to provide an overview anemia even in a previously documented megaloblastoid
of megaloblastic anemia in the pediatric population, both marrow. The observation that despite feeding the same
of the nutritional and other varieties with emphasis on liver extracts to various subjects the response to the
clinical approach, laboratory diagnosis as well as treatment pernicious anemia was variable lead Castle to think that
and preventive aspects of the disorder. some “intrinsic factor” must also influence the absorption
of the yet not discovered deficient nutrient and that it
HISTORY1-3 could be corrected by feeding beef stimulated gastric juice
In 1855, Thomas Addison at Guy’s Hospital described a from healthy patients along with the liver extracts. Later
lethal, idiopathic anemia that in 1872 was given the name on the fact that pernicious anemia was caused by inability
pernicious anemia by Biemer. For years it was believed of the stomach to absorb vitamin B12 because of atrophy of
that pernicious anemia was a result of the positive acting its mucosal lining was realized. A major clinical leap was
deleterious influence of an unknown infectious agent or when it was acknowledged that cobalamin and folic acid
biological product which caused increased destruction deficiencies could manifest as neurological symptoms
of the red blood cells. Ehrlich was amongst the first to like peripheral neuropathies, SCDSC and NTDs in the
describe megaloblastic bone marrow in 1891. In the 1920s unborn child of a folic acid mother. With advances in the
two milestones discoveries were made that changed the science, details of the absorption and transport of these
way in which the medical fraternity treats anemia today. compounds in the body, their role in the DNA synthesis
Chapter-15 Megaloblastic Anemia 127
and other single carbon transfer reactions in the body protein synthesis leading to a state of unbalanced cell
have been elucidated and this has also allowed clinicians growth and impaired cell division. These cells in a vain
to arrive at diagnoses in conditions with megaloblastosis attempt to divide increase their DNA contents by two to
where the nutrition seems to be adequate. Elevated levels four times and often get arrested in the S phase of the cell
of methyl malonic acid and homocysteine levels could be cycle. A larger than normal immature nuclei with finely
used as a means of diagnosing subclinical deficiencies of particulate chromatin afloat in the mature cytoplasm of a
vitamin B12 and folic acid, that these two correlated with large cells having unimpaired RNA and protein synthesis
the incidence arteriosclerotic diseases, bowel and other is the hallmark of megaloblastosis. Thus in a nutshell,
cancers and that correcting these levels could decrease the megaloblastosis results in a cell whose nuclear maturation
incidence of such events was also an outcome of elaborate is arrested while its cytoplasmic maturation proceeds
insights into the metabolism and physiological role of the normally independent of the nuclear events. Although
concerned nutrients. megaloblastic hematopoiesis commonly manifests as
anemia, it reflects a global defect in DNA synthesis which
DEFINITIONS affects all the proliferating cells of the body.
Megaloblastic anemia is used to describe a group of

disorders characterized by a distinct morphological pattern CAUSES OF MEGALOBLASTIC
in the hematopoietic cells most often macrocytosis of the
ANEMIA (TABLE 1)
red blood cells often accompanied by leukopenia and
thrombocytopenia. The prominent feature is a defective Although cobalamin and folate deficiency is amongst the
DNA synthesis with minimally altered RNA synthesis and major causes of megaloblastic anemia, it is not the sole
Table 1 Causes of megaloblastic anemia*4

Cobalamin deficiency Folate inhibitors: Antifolates (methotrexate, pyrimethamine,
sulfones, trimethoprime)
Nutritional cobalamin deficiency: Vegetarians, breastfed infants of Hereditary folate malabsorption (PCFT mutations)
mothers with pernicious anemia.
Abnormal intragastric events: Atrophic gastritis, hypochlorhydria, Damage to the ileal mucosa: Tropical and nontropical sprue,
PPI, H2 blockers regional enteritis, infiltrative disorders of the small bowel
(lymphoma)
Loss/atrophy of gastric oxyntic cells: Total or partial gastrectomy, Defective CSF transport
pernicious anemia, caustic damage
Insufficient pancreatic secretions: ZE syndrome Inherited disorders of folate utilization: Methylenetetrahydrofolate
reductase deficiency, methionine synthase deficiency18,19
Usurping of luminal cobalamin: By bacterial overgrowth in cases of Other causes
blind loop syndrome, diverticulosis; infection by D latum
Disorders of ileal mucosa/Cobalamin-IF receptors: Ileal bypass, Defects in purine and pyrimidine synthesis
nontropical sprue, Crohn’s disease Orotic aciduria
CUBAM receptor defects: Imerslund-Gräsbeck syndrome Myelodysplasia and leukemia, HIV
Drug effects: Metformin, neomycin Drug-induced
Congenital TCII deficiency Thiamine responsive megaloblast anemia
Metabolic disorders (cells not able to use vit B12 ): CbIA to CbIG Scurvy
disorders, nitrous oxide intoxication
Pyridoxine responsive anemia
Folate deficiency
Decreased dietary intake: Poverty, psychiatric illnesses, maternal
deficiency affecting fetus or infant, prolonged feeding of goat’s
milk
Increased requirements: Pregnancy, lactation, hemolysis,
hyperthyroidism, anticonvulsant therapy, Lesch-Nyhan
syndrome, prematurity, homocystinuria, psoriasis
*modified from ref no. 4
reason for a megaloblastic picture. Other conditions are withdrawn. However a deficiency would take longer to
also enumerated in the Table 1. set in due to an efficient enterohepatic circulation which
accounts for a turn over of 5–10 mcg/day of cobalamin.
COBALAMIN (VITAMIN B12)
Chemistry:5 Cobalamin, a member of the corrin family ABSORPTION AND TRANSPORT
has a structure described below in the Figure 1. A planar OF VITAMIN B12
corrin ring similar to heme, coordinates with a central
Cobalamin in the food is in the coenzyme form and
cobalt atom, with a 5, 6 dimethylbenzimidazole and
nonspecifically bound to protein. On reaching the
an upper axial ligand which varies in different biologic
stomach, the low pH causes proteolysis and releases
and pharmacological cobalamins like cyano-, methyl-,
the cobalamin which now preferentially binds to a high
hydroxyl-, 5’deoxyadenosylcobalamines. These axial
affinity (>intrinsic factor) 150 kda cobalamin-binding
ligands are attached when the central Co is in its most
protein, R protein (a haptocorrin) from gastric juice and
oxidized state, the cob(III) state. It can also exist in the
saliva. The cobalamin-R protein (holo R protein) complex
cob(II) and cob(I) states. These various forms confer a along with IF.
distinct identity to the cobalamin while it participates in Passes into the duodenum where the pancreatic
the various one carbon metabolism reactions. juices cleave the cobalamin from the complex. However
Nutrition:6-8 Cobalamin in the nature is produced only the IF does not undergo proteolysis and the cobalamin
by microorganisms and humans receive it solely from is now transferred to this 45kDa glycoprotein secreted by
diet. Certain bacteria and fungi produce this vitamin the oxyntic cells in the fundus and cardia of the stomach
in excess and form the major resources of cobalamin in response to food ingestion by membrane associated
for commercial purposes and therapy. Herbivores vesicular transport stimulated by vagal and hormonal
(and humans) obtain their cobalamin from plants signals. IF is produced in an amount far access of that
contaminated with cobalamin-producing soil bacteria required for absorption and only 2–4 mL of normal gastric
found in the roots of legumes. For all practical purposes, juice can correct cobalamin deficiency in adults lacking IF.
there is no uncontaminated plant source that could be While R binder binds both active cobalamin and its inactive
a source of vitamin B12. Nevertheless the colon of some analogs IF binds only the active forms. This property is
individuals contains cobalamin producing bacteria like used in order to excrete the inactive analogs secreted in the
Klebsiella pneumoniae, which can be absorbed. biliary secretions which are excreted along with R protein
Animal protein especially parenchymal meat is a while the active form attaches to IF and is reabsorbed thus
major dietary source of vitamin B12 for non-vegetarians providing an efficient system of enterohepatic circulation
(mcg/100 gm dry wt). Milk and milk products and eggs of cobalamin.
contain 1 to 10 mcg/100 gm dry weight. While an average This stable cobalamin-IF complex now passes into
non-vegetarian diet contains 5–7 mcg/day of cobalamin, the jejunum and into the ileum where the IF through its
the average vegetarian consumes only 0.25–0.5 mcg/day receptor binding site attaches to the receptors present on
(Table 2). the microvilli of the ileum. The functional IL cobalamin
Although heat does not influence the stability of receptors are composed of two proteins collectively
cobalamin, ascorbic acid readily changes the active forms known as CUBAM-cubulin and amnionless.8 The cubulin
of cobalamin into inactive analogs. With the liver storing is the larger extracellular portion of this complex which is
1 mg of the total 4–5 mg of the adult stores and an obligatory anchored to the membrane by the smaller amnionless.10
loss of 0.1 percent/day, it takes about 3–4 years to deplete These are very specific for the IF-cobalamin complex
the stores even if the dietary cobalamin is abruptly and do not bind any of the components when presented
singly or in combination with the R protein. The human
ileum contains cubam receptors to bind 1 mg of IF bound
cobalamin. Once internalized into the enterocyte the
cubam is recycled back to the surface. The cobalamin is
Table 2 Recommended daily allowance of vit B12
now released from the IF in the lysosome and is attached to
Recommended daily allowance (mcg) the transcobalamin(II) either within the eneterocyte itself
Men 2.4 or at the basal surface of the ileal enterocyte, while the IF
Nonpregnant women 2.4 is degraded. Holo-TC appears in the portal circulation in
about 3–5 hours and reaches peak levels in about 8 hours.
Pregnant and 2.6
lactating women Cobalamin when given in large doses can diffuse
passively through buccal, gastric and jejunal mucosa
Children 9–18 years 1.5–2
and less than 1 percent of such orally administered drug
Fig. 1 Absorption and transport of cobalamin (Image9 Adopted from: Dr Joseph Mercola, Alexa Natural Health Website)
then appears in the circulation within few minutes. This CELLULAR PROCESSING
property has been utilized to treat patients with impaired
absorption with oral supplements instead of parenteral The TCII-cobalamain complex is internalized13,14 via
therapy (Fig. 1). conventional receptor mediated endocytosis and within
In the blood the cobalamin is bound to three types of the lysosome, at the low pH the TC is cleaved off the
proteins. Transcobalamin (TC) I, II and III.11,12 >90 percent cobalamin and the cobalamin is transported into the
of recently absorbed cobalamin is bound to TCII which is cytosol. Here it can have two fates. It either goes to the
the specific transport proteins that delivers this important mitochondria to participate in reactions involving methyl
nutrient to the tissues. The TCII-cobalamin complex is malonyl-CoA or stays in the cytosol to be a part of the
rapidly cleared from the circulation in less than an hour as methionine synthase complex (Fig. 2).
it binds to the various cells with receptors for this complex In the mitochondria, cob(I) alamin is converted to its
and is internalized. Major circulating form which is the coenzyme form adenosyl cobalamin which along with
methyl form is never found free to in the plasma. The methylmalonyl-CoA mutase mediates transfer of a -CH
major (70%) circulating form found in the plasma is that moiety to convert methylmalonyl CoA to succinyl-CoA
bound to TCI which binds both active and inactive forms which can now take part in the Kreb’s TCA cycle and help
and is largely considered to be a plasma-storage protein generate ATP.14-17
for cobalamin. TCIII which is a asialoglycoprotein binds In the cytosol, cobalamin in its methylcobalamin form
all forms of cobalamin analogs with high affinity and acts as a coenzyme along with methionine synthase, a
within minutes delivers them to the liver through the complex enzyme requiring both folates and cobalamin
asialoglycoprotein receptors present on the surface of for carrying out one carbon metabolism reactions. First
hepatic cells and from there into the bile for fecal excretion. a methyl group is transferred from 5-methyl-tetra
Fig. 2 Intracellular metabolism of cobalamin
hydrofolate to methionine synthase–bound cob(I) alamin group to cob(I) alamin in a reaction involving methionine
to form methylcobalamin, followed by transfer of this synthase to form methylcobalamin. The approximate loci
methyl group to homocysteine to form methionine and for defects in cobalamin mutants, cblA to cblG, are shown.
regeneration of cob(I) alamin.14-17 MMCoA mutase, methylmalonyl-CoA mutase; SAH,
During these reactions sometimes the cob(I) alamin S-adenosylhomocysteine; SAM, S- adenosylmethionine
is spontaneously converted to the inactive form cob(II) (Fig. 2).15
alamine which needs to be reduced back to the active form
cob(I) alamin before it can accept and transfer a methyl DEVELOPMENT OF COBALAMIN DEFICIENCY
group. This is carried out by the enzyme methionine Nutritional deficiency: People following vegetarian
synthase reductase with the help of NADPH and SAM. diets can be either pure vegans who exclude all animal
This enzyme is defective in people with cbIE mutations. products from their diets and need to be routinely
Intracellular reactions involving cobalamin: In vivo supplemented with cobalamin or they could be those
substitutions include the replacement of hydroxocobalamin following lactovegetarian diets or lacto-ovo vegetarian
or cyanocobalamin by a 5′-deoxyadenosyl group attached by diets which incorporates milk and egg products into their
a covalent bond, giving rise to adenosylcobalamin (AdoCbl). food also need external supplementation, even if they
Methylcobalamin (MeCbl) is the main form in plasma. In are asymptomatic as they are too likely to suffer from
vivo, 5-methyl-tetrahydrofolate readily donates its methyl subclinical deficiencies.
Although severe cobalamin deficiency can often lead to of consuming monotonous diets low in animal-source
sterility, adverse pregnancy outcomes like preterm labor, foods prepared by unaware parents or self-imposed by the
intrauterine growth retardation, neural tube defects and adolescent to keep up with the so called “healthy vegan
recurrent miscarriages are also frequent manifestations of diet” trends and food fads. Low serum cobalamin levels
undetected cobalamin deficiency in the potential young have also been found in adolescents infected with HIV
mother. without having the frank immunodeficiency syndrome.
There is a critical period in prenatal and early postnatal Treatment with HAART resulted in improved status in
neurodevelopment when sufficient folate and cobalamin these candidates.
is required for the proper formation of neurologic circuits. Pernicious anemia:20-23 This lymphocyte mediate
Any perturbation of neurodevelopment during this destruction of the oxyntic cells of the stomach gives rise
period can give rise to subtle changes that can manifest to deficiency of the IF and constitutes one of the most
in behavioral abnormalities long after the folate and important causes of cobalamin deficiency in the adult
cobalamin deficiency is reversed. population. Autoantibodies are directed against the H+,
Cobalamin deficient mothers with low serum coba K+ ATPase of the parietal cells. Patients who lack IF after
lamin levels are not able to provide enough vit B12 stores to gastric resection and those with genetic mutations yielding
their fetuses at birth neither are they able to compensate defective or undetectable IF are not considered to have
for it when they breastfeed their child exclusively since pernicious anemia.
their breast milk cobalamin levels are also found to be The annual incidence of pernicious anemia is
equally low, often below 362 pmol/L. In India the duration approximately 25 new cases per 100,000 persons older
of exclusive breastfeeding is longer than the western world than 40 years. Although the average age of onset is about
and hence the relevance of supplementing the mother 60 years, pernicious anemia dose not avidly comply with
with B12 and folic acid supplements in order to prevent the boundaries of age, race, or ethnic origin. Although a
deficiency in the infant cannot be emphasized less. The genetic basis that predisposes one to develop pernicious
maternal stores can exert a strong effect on the vit B12 anemia has long been suspected, but neither the mode of
stores of the infant for around 12 months. inheritance nor the initiating events or primary mechanism
Children of mothers who have been on macrobiotic is precisely understood. There is a positive family history
diets (nearly vegan with occasional serving of animal for about 30 percent of patients, among whom the risk for
protein in form of fish) are also at risk not only because familial pernicious anemia is 20 times as high as in the
the cobalamin deficient mother is not able to provide the general population.
child with enough cobalamin stores during her pregnancy Other autoimmune diseases, including Grave’s disease
and lactation but she also tends to feed her child according (30%), Hashimoto thyroiditis (11%), vitiligo (8%), Addison
to the principles of macrobiotic diet. Elevated urinary disease, idiopathic hypoparathyroidism, primary ovarian
methylmalonic levels were found in 15 to 16 percent of failure, myasthenia gravis, type 1 diabetes mellitus, and
breastfed infants of vegetarian mothers who consumed adult hypogammaglobulinemia have been found to have
macrobiotic diets.18,19 significant associations with pernicious anemia.
Cobalamin deficient infants have also been found Histologic features of stomach in pernicious anemia
to be born to mothers who are on apparently balanced compared to normal: The normal gastric mucosa (A) is
nonvegetarian diets highlighting the fact that pregnancy contrasted to that seen in pernicious anemia (B), in which
places an additional stress on the mothers cobalamin there is atrophy of gastric glands, intestinal metaplasia
stores which needs to be corrected. This can negatively with goblet cells, and loss of parietal cells (not visible at
affect their breastfed infants’ cobalamin status at 6 weeks; this magnification) (Figs 3A and B).
indeed, over two-thirds of Norwegian infants of otherwise Abnormal events in the small bowel: Insufficient or
healthy mothers had a metabolic profile consistent with inactivated pancreatic proteases as occurs in the Zollinger-
cobalamin deficiency, which reverted to normal after Ellison syndrome, fail to cleave the cobalamin from the R
cobalamin replenishment. This emphasizes that many binder to allow it to bind to IF in order to get absorbed
more breastfed infants may need cobalamin supplements through the ileal receptors which are highly specific for the
early in life than previously realized. Such studies raise new cobalamin-IF complex.
questions as to whether the optimal intake of cobalamin in Bacterial overgrowth in the intestine as occurs during
women should be much higher than 2.4 mcg/day, and be bowel stasis in conditions like blind loop syndrome,
raised to 4–7 mcg/day. leads to usurpation of the cobalamin from the intestinal
The prevalence of cobalamin deficiency among older lumen making less it available for absorption. This type of
children and adolescents is also high, ranging from deficiency has been corrected with a 7–10 days antibiotic
40 percent to 80 percent in various communities, because course which sterilizes the gut.
mutations (in 80% of cases) involving either the cubulin

(CUBN) or amnionless (AMN) genes that constitute the
functional IF-cobalamin receptor (i.e., cubam) resulting
in selective cobalamin malabsorption. Presentation is
commonly between ages of 3–10 years with hematological
and neurological manifestations with low serum cobala
min. Consequent to the role of cubam in the renal tubular
absorption of several other proteins, a persistent but
benign proteinuria is also found in Imerslund-Gräsbeck
syndrome. Mutational analysis of gastric IF, CUBN and
AMN genes can give the diagnoses.
Nitrous oxide exposure: Nitrous oxide (N2O) by inacti
vating coenzyme forms of cobalamin by oxidizing the fully
reduced cob(I) alamin to cob(III) alamin causes a state
A
of functional intracellular cobalamin deficiency. First
identified in patients with tetanus who were given nitrous
oxide for up to 6 days, similar manifestations were also
found in persons exposed to nitrous oxide for open heart
surgery and through chronic exposure either accidental,
or occupational. These groups are considered to be at
high risk for developing megaloblastosis and cobalamin-
deficient neuromyelopathy. Megaloblastosis develops
within 24 hours and lasts less than 1 week after a single
exposure.
Inborn Errors of Cobalamin Metabolism20,21

Inborn errors of cellular cobalamin metabolism can affect
B synthesis of AdoCbl, synthesis of MeCbl, or synthesis of
Figs 3A and B (A) Histologic features of stomach in both cobalamin coenzymes, depending on which step
normal mucosa; (B) Pernicious anemia24 in metabolism is affected. Essentially, defects that affect
AdoCbl synthesis or directly affect methylmalonyl-CoA
Infestation by the fish tapeworm, Diphyllobothrium mutase result in isolated methylmalonic acidemia and
latum, which avidly usurps cobalamin for growth, affects aciduria; defects affecting synthesis of MeCbl result in hy
around 3 percent of the population and such people can perhomocysteinemia and homocystinuria; and defects re
develop frank cobalamin deficiency. Plerocercoids of this sulting in deficiency of both cobalamin coenzymes results
parasite reach the human intestine when they consume in combined methylmalonic aciduria and homocysteine.
partially cooked or raw fish containing, where they cblA: The cblA disorder is caused by mutations in the
develop into adult worms in the jejunum in about 6 weeks, MMAA gene on chromosome 4q31.1–q31.2. It plays a role
growing to a length of 10 m, with up to 4000 proglottids; in transfer of AdoCbl from cobalamin adenosyltransferase
when these worms lay eggs, the life cycle is repeated. Stool to methylmalonylCoA mutase and in stabilization of
examination showing the ova can give a diagnosis. This mutase-bound AdoCbl.
followed by praziquantel (10–20 mg/kg as a single dose cblB: Caused by mutations in the MMAB gene on chro
taken orally) which leads to expulsion of the worms and mosome 12q24 encoding cobalamin adenosyltransferase,
cobalamin replenishment is curative. it result in decreased synthesis of AdoCbl and therefore
Disorders of ileal IF-cobalamin receptors or mucosa: decreased activity of the AdoCbl dependent enzyme
Resection of only 1–2 feet of the distal ileum which has the methylmalonyl CoA mutase.
maximum density of the concerned receptors can cause Patients with the MUT disorder, caused by mutations
clinically significant cobalamin deficiency by reducing in the gene encoding methylmalonyl-CoA mutase itself
the number of interactions between the IF-cobalamin also have a similar clinical presentation although the
complex and its respective receptor. synthesis of AdoCbl is normal.
Imerslund-Gräsbeck syndrome25-28 is an autosomal When compared to serum and urine MMA levels in
recessive disorder in children arising from biallelic dietary cobalamin deficiency the levels here are very high.
Patients usually present in the first year of life, but late and Treatment of these disorders involves protein restric
benign presentations along with silent carrier states of tion, or feeding with formula deficient in valine, isoleucine,
these mutations are also known. Lethargy, failure to thrive, methionine and threonine, to limit the levels of the amino
hypotonia, recurrent vomiting and dehydration constitute acids that are the major source of methylmalonylCoA
common presenting symptoms. Any stress including within cells. Supplementation with OHCbl or CNCbl may
infections and even dietary changes can precipitate fatal be useful in cblA patients as well as some MUT and cblB
metabolic acidotic crises. patients. Therapy with carnitine has been advocated,
cblC: This disorder with more than 500 documented as has treatment with lincomycin and metronidazole
cases is the most common inborn error of cobalamin to reduce generation of propionate (the precursor to
metabolism is the most common inborn error of cobalamin methylmalonylCoA) by gut bacteria. Even with treatment,
metabolism. Around 70 different types of mutations outcomes may be poor.
affecting the MMACHC gene, which encodes a protein
that apparently plays a role as a cobalamin chaperone FOLATES
and in removal of the upper axial ligand of exogenous
cobalamins are likely to be the cause of this disorder. It is Chemistry:30,31 More than 100 compounds are known
found that there is a problem not with the uptake but with which are together known as folates. Folic acid (pteroy
the retention of cobalamin in the fibroblasts from cblC lmonoglutamate [PteGlu]) is the commercially available
patients, perhaps because it does not become associated parent compound. PteGlu consists of three basic compo
with cobalamin-binding enzymes. nents: a pteridine derivative, a p-aminobenzoic acid
There is decreased synthesis of both AdoCbl and residue, and an L-glutamic acid residue. This must be
MeCbl, and decreased activity of both cobalamin- first reduced at positions 7 and 8 to dihydrofolic acid
dependent enzymes. (H2PteGlu) and then to 5, 6, 7, 8-tetrahydrofolic acid
cblD: This disorder is caused by mutations in the (THF; H4PteGlu), and one to six additional glutamic
MMADHC gene, which encodes a gene of unknown acid residues must then be added by means of γ-peptide
function. Affection of the N-terminal domain of the bonds to the l-glutamate moiety (subscripted n in PteGlun
MMADHC protein resulted in variant 2, while mutations denotes polyglutamation) before it can play its part as a
affecting its C terminal domain caused variant 1. Although coenzyme (Fig. 4). The major role of folate coenzymes is in
the exact defect is still to be found, it is suggested that the donation or acceptance of one-carbon units in numerous
MMADHC protein plays a role in partitioning of cobalamin reactions in amino acid and nucleotide metabolism.
between the mitochondrial (methylmalonylCoA mutase)
and cytoplasmic (methionine synthase) compartments.
cblE: Mutations in the MTRR gene have been identified
in cblE patient’s locus on chromosome 5p15.3–p15.2,
which encodes methionine synthase. Both the cblE and
cblG disorders show rapid improvement in biochemical
and neurological parameters when treated with intra
muscular OHCbl while such is not the case with most cblB
patients which respond poorly.
cblF: This disorder is the result of mutations in the
LMBRD1 gene on chromosome 6q13, which encodes a
lysosomal membrane protein containing 9 transmemb
rane domains which leads to inability to transfer cobala
min freed from transcobalamin in the lysosome across
the lysosomal membrane into the cytoplasm. Cells from
cblF patients accumulate large amounts of free cobalamin
within lysosomes, but there is a deficiency of both
cobalamin coenzyme derivatives and decreased activity of
methylmalonylCoA mutase and methionine synthase.
cblG: Mutations at the MTR locus have been identified
in cblG patients.
All the above disorders can present in various combi
nations as well. Fig. 4 Folate structure29
Various substitutions in H4PteGlun occur at positions 5 or ABSORPTION AND TRANSPORT

10, or both; position 5 can be substituted by methyl (CH3),
formyl (CHO), or formimino (CHNH), and position 10 First the dietary folate which is mainly in the polyglutamate
can be substituted by formyl or hydroxymethyl (CH2OH). form is converted to folate monoglutamate form by γ-
Positions 5 and 10 can be bridged by methylene (–CH2–) or glutamyl hydrolase at the enterocyte brush border.31,32
methenyl (–CH=). This is followed by their transport through the duodenal
Nutrition: Vitamin B9 or folates are synthesized by and jejunal brush border by high-affinity membrane-
microorganisms and plants, including leafy vegetables associated, luminal surface–facing proton-coupled folate
(spinach, lettuce, broccoli), beans, fruits (bananas, transporters (PCFT). At pH 5.5, they act most efficiently and
melons, lemons), yeast, and mushrooms, and are also have equivalent affinity for transport of both physiologic
found in animal meats. reduced folates and folic acid, but at pH 6.5, reduced
Bioavailability of folates from various sources varies 5-methyl-tetrahydrofolate is transported more efficiently.
greatly mostly as result of the following factors. PCFT being a folate-hydrogen symporter results in a net
1. Food stability: The labile and susceptible reduced translocation of positive charge along with every folate
natural folate is damaged by oxidative cleavage by molecule transported.
nitrates or light exposure and prolonged cooking/ Within the enterocyte, after reduction to tetrahydro
boiling for over 30 minutes which can reduce the folate and methylated before release into plasma as
bioavailabilty by 50–80 percent. However folic acid 5-methyl-tetrahydrofolate the efflux from the basolateral
is much more stable. Similarly refrigeration of leafy membarane into the portal blood being aided by the
foods exposed to artificial fluorescent light in super multidrug resistance–associated protein 3 (MRP3). These
markets doubles the folate content and ascorbate proteins with a low affinity but high capacity can be best
when consumed along with folates increases the described to function as cellular “sump pumps” that eject
bioavailability. excess folates as well as antifolates out of cells. With the
2. Pureed foods allow easier access to the glutamate car help of MRP2, which mediates folate transport into the
boxypeptidase II (also known as folate-polyglutamate bile, an efficient enterohepatic circulation is maintained
hydrolase), which converts folate polyglutamates to thereby allowing the body to retain folates.
simpler folate monoglutamates before absorption; Less than 5 percent of average folate requirement can
any perturbation of this enzyme by organic acids be derived from that produced by the intestinal bacteria
(orange juice), sulfasalazine, or ethanol can preclude by absorption across the large intestine. However, this
absorption; conversely, folate-binding proteins in fraction is largely used up by the colonocytes themselves
human or cow’s milk can increase folate absorption for for purpose of nutrition.
infants and women. At high pharmacological concentrations passive dif
3. Interference with jejunal absorption of folaes due to fusion of folic acid is probably the primary mechanism of
intestinal disease. intestinal mucosal folate. Peak folate levels in plasma are
4. Drugs that interfere with the proton-coupled folate achieved 1 to 2 hours after oral administration.
transporter (PCFT) can also compromise folate Unlike cobalamin, folates are not privileged with a
absorption. specific serum transport protein which enhances their
The recommended daily allowances of folate are as cellular uptake. In the plasma, one-third of the folate is free,
follows (Table 3): two-thirds is nonspecifically bound to serum proteins, and
a very small fraction binds high-affinity, hydrophilic 40-
kDa folate-binding proteins, which are structurally related
Table 3 Recommended daily allowance of folate to hydrophobic (native) folate receptors. Specialized, high-
affinity, glycosyl-phosphatidylinositol-anchored (mem
Recommended daily allowance
brane) folate receptor-α, which takes up these folates at
Adult men and nonpregnant 400 mcg physiologic concentrations found in serum and transfer
women
into the intracellular compartment of proliferating cells.
Pregnant women 600 mcg The folate–folate receptor complex is then endocytosed.
Lactating women 500 mcg Acidification of the perinuclear endosomal compartment
Children 9-18 years 400 mcg to pH 6 cleaves the folate from folate receptor, thereby
releasing the folate to pass across the acidified endosome
1-6 years 3.3 mcg/kg/day
into the cytoplasm by a trans-endosomal pH gradient,
Infants 3.6 mcg/kg/day mediated most often by the PCFT.33,34
Folate receptor-α mediates the cellular uptake of blood-folate ratio in healthy humans of 3:1 the folate first
folates in proliferating normal and malignant cells binds to the folate receptor-α in the choroid plexus, and is
besides their transport across the placenta to the fetus, transported into the CSF with the assistance of the PCFT.
into the brain, and in renal conservation of folates. While A syndrome of severe developmental regression in
β receptors are expressed mostly in monocytes and early childhood associated with movement disturbances,
macrophages, α receptors are found in abundance in epilepsy, and leukodystrophy that is reversed by folinic
many kinds of malignant cells and these are now being acid occurs due to cerebral folate deficiency consequent
explored as potential diagnostic and therapeutic targets to a mutation in folate receptor-α, which perturbs folate
for certain malignancies. transport into the cerebrospinal fluid.
Similarly antifolate receptor-α antibodies that can
Placental Transport35 prevent uptake of folate into the cerebrospinal fluid,
lead to either infantile acute cerebral folate deficiency
Fetal and newborn blood folate is invariably more or one of two autism spectrum disorders (Rett syndrome
elevated than maternal blood folate which is proof and infantile low-functioning autism with neurologic
enough for existence of a placental mechanism for abnormalities). High doses of oral folinic acid can lead
preferential maternal-to-fetal folate transport. Transfer to partial or complete clinical recovery in 12 months by
receptors are abundant and polarized to the maternal- normalizing CSF folate levels.
facing microvillous membrane of the syncytiotrophoblast The role of PCFT in transport of folates across the
wherein they become the first to bind maternal folate choroid plexus is supported by the fact that mutations in
at physiologic concentrations and pH. For physiologic PCFT result in hereditary folate malabsorption with low to
transplacental folate transport a continued provision of undetectable cerebrospinal fluid folate values.
adequate dietary folate intake by the mother followed by
capture of maternal folate by placental folate receptors is
essential. This results in an intervillous blood concentration
Renal Retention of Folates (Cobalamin)
that is three times that of maternal blood and subsequent Once the folate reaches proximal tubule after filtration it is
concentration gradient based transfer of the folate into the bound to the folate receptor-α in the brush border memb
fetal circulation. Inadequate intake of folate by the mother ranes of these absorptive cells and is internalized rapidly
thereby leads to reduction in maternal-to-fetal folate by folate receptor-α–mediated endocytosis followed by its
transfer which in turn predisposes the embryo/fetus to dissociation in the acidic environment of the lysosome. It
very serious developmental defects. is then transported across basolateral membranes into the
blood, with recycling of apofolate receptor-α back to the
Folate Receptors in Embryonic and luminal brush border membrane. Megalin, a large 550-
Fetal Development36 kDa membrane protein interacts found in renal proximal
epithelial cells interacts with cubulin and functions as
Folate receptors-α are among the earliest genes activated in a multiligand receptor for a variety of macromolecules.
embryonic stem cells coinciding with the period when there Beside it also specifically binds to and mediates endo
is the need for increased folate requirements to support DNA cytosis of TCII-cobalamin complexes as well as filtered
synthesis during bursts of intense cell proliferation of initial folate bound to soluble folate-binding proteins in kidney
phases of organogenesis. They are abundantly expressed proximal tubules.
in early stage neural tube cells and neural crest cells and
their experimental perturbation can lead to profound Regulation of Folate Homeostasis37-39
abnormalities in neural tube closure and in heart, facial,
and eye development. A striking human correlate of such Upregulation of cell surface folate receptor-α in response
experimental studies is the significant increase in blocking to low extracellular and intracellular folate concentrations
autoantibodies against placental folate receptor-α seen in through transcriptional, translational, and post-transla
women with pregnancy complicated by neural tube defects. tional mechanisms allows it to bind all available folate and
thereby restore cellular folate homeostasis.
Cerebral Folate Transport Across The basic molecular mechanism has now been deci-
phered. Intracellular deficiency of folates leads to accumu
the Choroid Plexus lation of homocysteine which covalently binds to a protein
As has already been mentioned folate receptor-α and PCFT known as heterogeneous nuclear ribonucleoprotein-E1
are found in the basolateral membranes of the choroid (hnRNP-E1), which is already known to mediate the
plexus. To maintain the normal cerebrospinal fluid–to translational upregulation of folate receptor-α. Homo-
cysteinylation of hnRNP-E1 at specific cysteine–cysteine such that approximately 40 percent are in the mitochond
disulfide bonds leads to the unmasking of an underlying rial matrix, 50 percent in the cytoplasm, and 10 percent in
messenger RNA (mRNA)-binding pocket for which folate the nucleus.
receptor-α mRNA has a high affinity thereby triggering the After cellular uptake, 5-methyl-THF (which is the
biosynthesis of folate receptors which ultimately results major form that is transported intracellularly) it must
in a net increase of cell surface folate receptors to bind first be converted to THF via methionine synthase (in the
more available folate and thereby normalize cellular folate methylation cycle). This is because THF is the preferred
levels. physiologic substrate for polylpolyglutamate synthase,
which adds multiple glutamate moieties to THF. Only
Intracellular One-Carbon Metabolism once this is accomplished the polyglutamylated form of
THF participate in one-carbon metabolism where it can
Polyglutamylation is important not only because it is the
be converted to either 10-formyl-THF—used in de novo
folate form that can be retained within the cell but also
biosynthesis of purines, or to 5,10-methylene-THF—used
because, polyglutamylated folates are more efficient sub
for synthesis of thymidylate. Also 5, 10 -methylene-THF and
strates for folate-dependent enzymes. In human eryth
10-formyl-THF can be interconverted by intermediates.
rocytes, folate is accumulated at earlier stages within
The mitochondrial compartment contains its comple
the marrow by folate receptors;8 on maturation, more
ment of folate cofactors, and homologues of the major
than 90 percent of H4PteGlu(n) molecules interact with
cytosolic enzymes. Other one-carbon donors like serine,
hemoglobin, which, because of its high capacity, assists in
intracellular folate retention (Fig. 5). glycine, dimethylglycine, and sarcosine also enter mito
chondria and ultimately generate formate that crosses
Compartmentalization and Channeling back into the cytoplasm. In the cytoplasm, C1-THF
synthase uses this mitochondria-derived formate with
of Folate Metabolism THF to form 10-formyl-THF, which is required for the de
Folate metabolism and folate-dependent enzymes are novo synthesis of purines; this enzyme can also catalyze the
very strategically compartmentalized with a distribution interconversion of THF, 10-formyl-THF, 5, 10-methenyl-
Fig. 5 One carbon metabolism40 (Abbreviations: B2: Riboflavin; B6: Pyridoxal phosphate; B12: Cobalamin; DHF: Dihydrofolate; DHFR:
dihydrofolate reductase; DMG: Dimethylglycine; dTMP: Deoxythymidine 5’-phosphate; dUMP: 2’-deoxyuridin-5’-phosphate; MS:
Methionine synthase; 5-MTHF: 5-methyltetrahydrofolate; MTHFR: Methylene tetrahydrofolate reductase; SAM: S-adenosylmethionine;
SAH: S-adenosylhomocysteine; SHMT: Serine hydroxymethyltransferase; THF: Tetrahydrofolate; TS: Thymidylate synthetase; UMFA:
Unmetabolized folic acid. Adopted from ref no. 40)
THF, and 5, 10-methylene-THF. Thus a continued delivery plication of hematopoietic precursors and causing lethal
of mitochondrial formate helps perpetuate cytoplasmic anemia. In these circumstances the methylation reactions
one-carbon metabolism. Formation of 5, 10-methylene- are also partly protected by the reduced rate of cell
THF from serine (which is derived from glycolytic division. Hence when only folic acid is administered in
intermediates) forms another major entry point of one- cobalamin deficiency without simultaneous supplemen
carbon units into cytoplasmic folate metabolism. After 5, tation of B12, cell division is induced and the subsequent
10-methylene-THF is converted to 5-methyl-THF by the consumption of methionine in protein synthesis, impairs
enzyme methylenetetrahydrofolate reductase, it can be methylation of myelin and precipitates or exacerbates
used in the methylation cycle that involves methylation of subacute combined degeneration (SCD).
homocysteine via methionine synthase to form methion The selective use of available folate to conserve
ine and tetrahydrofolate. methionine, together with the ability of nerve tissue to
The methionine that is generated can be converted to concentrate folate from the plasma, explains the absence
a methyl donor through its adenosylation to SAM which of SCD in folate deficiency.
in turn is a universal donor of methyl groups for critically
important biologic methylation reactions involving over Development of Folate Deficiency
80 proteins, membrane phospholipids, the synthesis of
neurotransmitters. Nutritional: The body stores of folate are adequate for
5, 10-methylene-THF can be converted to 10-formyl- only about 4 months any additional stress that increase
THF, it can be used for de novo synthesis of purine folate requirements like illness, hemolysis, anorexia will
nucleotides for DNA and RNA or can also be used in the tip an individual who was chronically in a negative folate
thymidylate cycle via the enzyme thymidylate synthase to balance to develop frank folate deficiency.
generate thymidylate for DNA synthesis. Socioeconomic status has a major impact as folate
deficiency often coexists with poverty, malnutrition, and
chronic bacterial, viral and parasitic infections. ignorance
Methylfolate Trap41-45 about cooking practices like overheating food also contri
The methylfolate trap is a normal physiological response butes to the nutritional losses of folates.
to impending methyl group deficiency resulting from a In the economically well off countries food faddism,
very low supply of methionine which decreases cellular alcoholism, or unbalanced slimming diets usually lead to
S-adenosyl-methionine (SAM) thereby endangering im decreased folate intake in adolescents.
portant methylation reactions, including those required to Pregnancy and infancy: Folate requirements (over 400
maintain myelin. To protect against such catastrophy and mcg/day) during pregnancy and lactation are increased
considering availability of SAM as its utmost priority the tremendously for growth of the fetus, placenta, breast,
cell behaves as explained as: and other maternal tissues. There is also increased urinary
• Decreased SAM causes the folate co-factors to be di loss of folate in pregnancy (about 14 mcg/day versus
rected through the cycle involving 5-methyl-tetra approximately 4.2 mcg/day in nonpregnant women)
hydrofolate (5-methyl-THF) and methionine synthe because of a lower renal threshold. Additional folate
tase and away from the cycles that produce purines and during pregnancy is required to prevent both pregnancy
pyrimidines for DNA synthesis. This not only enhances complications (pre-eclampsia, placental abruption or
the remethylation of homocysteine to methionine and infarctions, recurrent miscarriage) and poor pregnancy
SAM but by restricting DNA biosynthesis decrease outcomes (preterm delivery, NTDs, congenital heart
the requirement of methionine for protein synthesis defects, and intrauterine growth retardation).
and with it cell, division thereby allowing the limited The rapidly proliferating tissues in children also have
methionine to be conserved for the vital methylation an absolute requirement for exogenously supplied folate.
reactions in the nerves, brain, and elsewhere. Although human milk can maintain folate balance in
• Since in the absence of methionine homocysteine breastfed infants, the breast milk content of folate is low
cannot be formed which as discussed earlier is when the mother’s folate status is poor.
essential to allow folate to be retained intracellularly, Folates and neurodevelopment: All inborn errors
there sets in a state of intracellular folate deficiency of folate metabolism, which result in reduced folate
which restricts the rate of mitosis in the cells and hence availability to the developing brain, give rise to mental
decreases requirements of methionine further. retardation and related mental health problems. Since the
Vitamin B12 deficiency is mistakenly perceived as fetal brain is dependent on sufficient provision of maternal
methione deficiency by the cells, thus resulting in an folate during embryogenesis maternal folate deficiency
inappropriate response of downregulating the multi can compromise the delivery of folate to the developing
fetal brain, and depending on the degree of deficiency, Table 4 Morphology49

there could be a spectrum of neurologic abnormalities
Morphology in megaloblastosis from cobalamin and
ranging from full-blown NTDs to more subtle changes
folate deficiency
that manifest in childhood as behavioral abnormalities.
Many studies have confirmed this theory and marked Peripheral smear
• Increased mean corpuscular volume (MCV) with macro-
improvement in pregnancy outcomes in form of reduced
ovalocytes (up to 14 mm), which is variously associated with
incidences of NTDs as well as better neurocognitive anisocytosis and poikilocytosis
status in the offsprings of mothers who were adequately
supplemented with folate perinatally. • Nuclear hypersegmentation of polymorphonuclear
neutrophils (PMNs) (one PMN with six lobes or 5% with five
Folates and intrinsic hematologic disease: Because
lobes)
folate is necessary for hematopoiesis, folate requirements
are increased when there is significant compensatory • Thrombocytopenia (mild to moderate)
erythropoiesis in response in hemolytic disorders, abnor • Leukoerythroblastic morphology (from extramedullary
mal hematopoiesis, or infiltration by abnormal cells in hematopoiesis)
marrow. In fact folate deficiency developing in hemolytic Bone marrow aspirate
disorders can lead to an acute aplastic crisis and hence the • General increase in cellutarity of all three major hemato
recommendation of routine prophylactic administration poietic elements
of folate in all follow up cases of hemolytic anemias has • Abnormal erythropoiesis—orthochromatic megaloblasts
been laid down. An unexpected increase in transfusional • Abnormal leukopoiesis—giant metamyelocytes and “band”
requirement or a fall in platelets can also suggest folate forms (pathognomonic), hypersegmented PMNs
deficiency.
• Abnormal megakaryocytopoiesis—pseudohyperdiploidy
Drugs and folates: Although trimethoprim and py
rimethamine bind to bacterial and parasitic dihydrofolate
reductase with much greater affinity than to human are arrested (as megaloblastic cells) at various stages of
dihydrofolate reductase, but patients with underlying the cell cycle is the hall mark. Although megaloblastosis
folate deficiency appear to be more susceptible to the affects all proliferating cells including those of the
effects of these drugs. The megaloblastosis can be reversed intestinal lumen, cervix, uterus, changes are most striking
by folinic acid (5-formyl-tetrahydrofolate [5-formyl-THF]; in the blood and the bone marrow.
leukovorin). Methotrexate binds with high affinity to human As the megaloblastic erythroid cells are prone to
dihydrofolate reductase and leads to trapping of folate as programmed cell death, ineffective hematopoiesis extends
a metabolically inert form (dihydrofolate). This leads to into long bones, and the bone marrow aspirate exhibits
a true depletion of THF within hours and consequently trilineal hypercellularity, especially of the erythroid series.
to functional deficiency of 5, 10-methylene-THF and This apparent exuberant cell proliferation seen within
reduced thymidylate synthesis. Although megaloblastosis the marrow with numerous mitotic figures is misleading
can develop rapidly, the toxic effects of methotrexate can because these cells are actually proliferating very slowly.
be avoided by rescue with 5-formyl-THF (leukovorin). Elevated LDH, increased bile pigments and iron are
Sulfasalazine produces megaloblastosis in up to two-thirds outcomes of this ineffective erythropoiesis.
of patients taking full doses (over 2 g/day) by decreasing The mature erythrocytes are of various sizes with
absorption of folates and induction hemolytic anemia higher mean corpuscular volumes (MCV). In fact increase
(i.e. increased requirements). Anticonvulsants can induce in (MCV) with macro-ovalocytes (up to 14 μm) is one of
NTD, and guidelines have stressed the importance of the earliest manifestations of megaloblastosis. Because
ensuring that pregnant women and children with epilepsy these cells have adequate hemoglobin, the central pallor,
be prescribed folates together with anticonvulsants. The which normally occupies about one-third of the cell, is
only caveat is to correct B12 deficiency before prescribing decreased. Poikilocytosis and anisocytosis are seen when
long-term folates. severe anemia is present. Cells containing remnants of
DNA (Howell-Jolly bodies), arginine-rich histone, and
PATHOLOGY OF MEGALOBLASTIC ANEMIA nonhemoglobin iron (Cabot rings) may be observed.
Extramedullary megaloblastic hematopoiesis may give
Hematological Manifestations46-48
rise to a leukoerythroblastic picture.
Peripheral smear and bone marrow examination (Table 4): Nuclear hypersegmentation of DNA in PMNs is
Widening disparity in nuclear-cytoplasmic asynchrony as found to be a strong and pretty consistent indicator of
a cobalamin- or folate-deficient cell divides, until the more megaloblastosis when associated with macro-ovalocytes.
mature generations of daughter cells die in the marrow or In megaloblastosis greater than 5 percent PMNs with more
than five lobes or a single PMN with more than six lobes process is a patchy demyelination affecting both the
supports the diagnosis and a formal lobe count/PMN (i.e. brain and the spinal cord. It begins as a swelling of the
lobe index) above 3.5 may be obtained. myelin sheath followed by its breakdown and eventual
Thrombocytopenia and neutropenia often accompany axonal degeneration which is often followed by secondary
severe anemia although they may be seen even in the Wallerian degeneration of the long tracts. Lesions first
absence of anemia. involve the dorsal columns in the thoracic segments
Erythroid hyperplasia reduces the myeloid-to-erythroid spreading contiguously to engulf the corticospinal tracts
ratio from 3:1 to 1:1. Proerythroblasts often do not exhibit and ultimately affecting the spinothalamic and spino
much abnormality except for a larger size but the later cerebellar tracts as well. Degeneration of the dorsal root
precursors show many abnormalities. These megaloblasts ganglia, celiac ganglia, the Meissner plexus, and the
are not only larger but instead of having a densely packed Auerbach plexus also occurs. Demyelination may also
chromatin they have an open, finely stippled, reticular, extend to the white matter of the brain. Whether the
sieve-like pattern. The orthochromatic megaloblast, with peripheral neuropathy is caused by a distinct lesion or
its hemoglobinized cytoplasm, continues to retain its results from spinal cord disease is still to be found.
large sieve-like immature nucleus, in sharp contrast to the
clumped chromatin of orthochromatic normoblasts.80-90
percent of the potential progeny of proerythroblasts that
develop into later megaloblastic die in the bone marrow.
Effective scavenging dead or partially disintegrated
megaloblasts by the marrow macrophages forms the basis
for ineffective erythropoiesis (intramedullary hemolysis).
Leukopoiesis is hit as well. There is an absolute
increase in the myeloid precursors, which are large and
have similar sieve-like chromatin. Spectacular giant (20–
30 μm) metamyelocytes and “band” forms are often seen
and are pathognomonic for megaloblastosis. Their clinical
relevance lies in the fact that such giant metamyelocytes
and band forms are not seen in the megaloblastoid bone
marrow of leukemia and MDS. They may persist in the
marrow for 10–14 days after the initiation of treatment for
megaloblastosis (Figs 6A to E).
Megaloblastosis50,51 when affects the rapidly prolifera
ting cells of the gastrointestinal tract leads to their atrophy
which in turn cause further malabsorption of cobalamin
and folate thereby fueling a vicious cycle wherein mega
loblastosis begets more megaloblastosis which can be
adequately interrupted by specific therapy with folates A
and cobalamin.
Megaloblastosis versus Macrocytosis

The central pallor that normally occupies about one-third
of the normal red blood cell is decreased in macro-ovalo-
cytes. This contrasts with the finding of thin macrocytes,
in which the central pallor is increased. This is because the
hemoglobinization in cobalamin and folate deficiency is
only increased as the cell now takes more time to mature
and hence the decrease in central pallor (Table 5).
Neurological Manifestations B
Cobalamin deficiency can manifest as a myriad of Figs 6A and B The peripheral smear (A) Hypersegmented polys;
neurological defects. The basic underlying pathogenic (B) Macro-ovalocytes
CLINICAL FEATURES
History
History often reveals an exclusively breast fed infant born
to an apparently anemic mother who is either vegetarian
by choice or forced due to socioeconomic factors. The
infant would have gradually curtailed his activities and the
mother is unable to notice the gradually developing pallor
until it becomes severe enough that the child needs to be
taken to a doctor. Occasionally children may present with
shortness of breath and impending failure. Since this is a
chronically developing anemia such manifestations are
seen only when the hemoglobin falls below 5 gm/dL.
C Gastrointestinal symptoms may predominate in some
including loss of appetite, weight loss, diarrhea, nausea,
vomiting, and glossitis aggravated by spicy foods.
The neurological symptomatology may vary from vague
complaints like decreased memory, lethargy, irritability,
mild degree of cognitive impairment to severe peripheral
neuropathies a subacute combined degeneration of the
spinal cord which is the most feared complication of this
nutritional deficiency. SCDSC may manifest as loss of
D
vibration sense, parestherias and weakness, all affecting
the lower limbs much more than the upper.
Further enquiry regarding parity of the mother, birth
history and dietary details as well as past illnesses and
surgeries, drug ingestions (antiepileptics, pyrimethamine),
worm infections and affection of other family members
may aid in coming to an etiology of the present condition.
E
Figs 6C to E Microscopic picture of megaloblastosis49 (C). Giant Physical Examination
metamyelocyte (Band form cell) Details from the cells in the Physical examination reveals different features in well-
aspirate (D) compared with normal hematopoiesis at same
nourished patients and poorly nourished individuals. The
magnification (E)
latter show evidence of significant weight loss or other
stigmata of multiple deficiencies due to “broadspectrum”
malabsorption. Angular cheilosis, bleeding mucous mem
Table 5 Macrocytosis versus megaloblastosis51 branes, dermatitis, and chronic infections hint to associated
Macrocytosis without megaloblastosis vitamin A, D, E, K deficiency with PEM. Various degrees of
• Reticulocytosis pallor with lemon-tint icterus (i.e. a combination of pallor
• Liver disease and icterus best observed in fair-skinned individuals) are
• Aplastic anemia common features of megaloblastosis.
• Myelodysplastic syndromes (especially 5q-) The skin may be diffusely pigmented or have abnor
• Multiple myeloma
mal blotchy tanning. A macular hyperpigmentation41
• Hypoxemia
• Smokers
with follicular accentuation may be observed in the axilla
and groin; hyperpigmentation can also involve the dorsal
Spurious increases in MCV without macro-ovalocytosis51 acral distal interphalangeal joints with special emphasis
• Cold agglutinin disease on pigmentation of the nail beds and skin creases. Unlike
• Marked hyperglycemia Addison’s disease there is no staining of the mucous
• Leukocytosis membranes. Premature graying, observed in light-and
• Older individuals dark-haired individuals, is reversible within 6 months of
cobalamin therapy. These pigmentation changes often changes in the activity of AdoMet-dependent methyltrans-
resolve within 2 months of cobalamin replacement. ferase.
Glossitis with a smooth (depapillated), beefy red tongue
with occasional ulceration of the lateral surface or gingival Subclinical Cobalamin Deficiency57-59
hyperplasia are found on oral examination. Increased
jugular venous distention along with gallop, cardiomegaly The issue of subclinical cobalamin deficiency has been
(with or without pericardial effusions), pulmonary basal a semantic dilemma. Many persons present with subtle
crepitations, pleural effusion, tender hepatomegaly, and symptoms including fatigue, cognitive changes, lower
pedal edema should alert the clinician towards a cardiac quality-of-life measures, and subtle symptoms of neuro
failure due to severe anemia. Very rarely nontender pathy that cannot be directly attributed to cobalamin
hepatomegaly, but more often splenomegaly may hint deficiency, despite the fact that these very symptoms
towards extrameduallary hematopoiesis (Table 6). are often seen in symptomatic cobalamin deficiency;
Also look for signs of associated hypo-or hyperthyroid often this triggers testing with a serum cobalamin test,
ism like neck swellings and loss of eyebrows, etc. and a borderline result generates a new set of problems,
Anemia and neurological signs52-56 are found to be including the need to label this entity and thereby make
inversely related. SCDSC can have all or few of the features clinical decisions. The incidence of subclinical cobalamin
which include decreased vibration sense below the iliac deficiency is found to be 10 times higher in the US
crests (mobile phone sign), loss of position sense in feet population than the classical overt type of megaloblastic
and ataxia all due to affection of posterior spinal columns. anemia. Demonstration of an increase in metabolites (i.e.
Weakness and progressive spasticity with increased serum homocysteine and MMA test results), helps pick-up
muscle tone, exaggerated deep tendon reflexes with many individuals having subclinical cobalamin deficiency,
clonus, extensor plantar response, and in-coordinate provided they had no subtle cognitive abnormalities.
or scissor gait, which may progress to spastic paraplegia A cut-off value of 148 pmol/L (less than 200 pg/mL) is
indicate UMN type of lesion because of pyramidal tract consistent with 3 standard deviations (SDs), and will miss
involvement. The involvement of peripheral nerves may about 3 to 5 percent of patients with clinical cobalamin
markedly modify these signs to include flaccidity and deficiency.
the absence of deep tendon reflexes. A positive Romberg However, the literature does not provide the clinician
sign as well a positive Lhermitte sign may be elicited. with set guidelines about how to manage the entity of
Loss of sphincter and bowel control, altered cranial nerve subclinical cobalamin deficiency, which is defined as
dysfunction with altered taste, smell, and visual acuity biochemical evidence for cobalamin deficiency—reflected
or color perception, and optic neuritis are other rare but by a low cobalamin value (and increased MMA and
proven manifestations of cobalamin deficiency.56 homocysteine) but without overt clinical manifestations.
Data suggests that changes of SCDSC is a result of Although some experts do not feel obliged to treat,
decreased remethylation of homocystiene as a result of preferring to wait until there are overt symptoms, there
is another school that feels ethically bound to treat even
without overt clinical manifestations rather than allow
Table 6 Clinical features of B12 and folate deficiency57
them to develop and make the patient suffer.
System Manifestations
Hematologic Pancytopenia with megaloblastic marrow DIAGNOSTIC ISSUES AND INVESTIGATIONS
Cardiopulmonary Congestive heart failure
Peripheral Smear and Bone Marrow Aspirate
Gastrointestinal Beefy-red tongue and added stigmata of
broadspectrum malabsorption in folate Macro-ovalocytes, although not specific for megaloblastic
deficiency anemia are the hallmark of megaloblastosis. Similarly,
Dermatologic Melanin pigmentation and premature though the importance of MCV values in suspecting mega
graying loblastic anemia is immense only half of the patients with
Genital Cervical or uterine dysplasia MCVs greater than 105 fL may have vitamin deficiency.
Macrocytosis per se is not associated with megaloblastosis
Reproductive Infertility or sterility
in around 50 percent of the cases and a complete diagnosis
Psychiatric Depressed affect and cognitive dysfunction may be reached only after carrying out several other
Neuropsychiatric Unique to cobalamin deficiency with additional tests.
cerebral, myelopathic, or peripheral The frequency of hypersegmented neutrophils
neuropathic disturbances, including optic (5 percent with five lobes or 1 percent with six-lobed PMNs)
and autonomic nerve dysfunction in patients with megaloblastic hematopoiesis is 98 percent
with the specificity of this finding being approximately 95 Biochemical Evidence56,61

percent. Hypersegmented PMNs and macro-ovalocytosis,
together give results that are much more accurate with Serum levels of cobalamin: The sensitivity of cobalamin
a specificity of 96 percent to 98 percent, and the positive concentration less than 200 pg/mL (or less than 148
predictive value of folate or cobalamin deficiency of about pmol/L) exceeds 95 percent when the clinical spectrum
94 percent. suggests and smear examination reveals megaloblastosis.
Diagnostic features of a megaloblastoid marrow have A serum cobalamin level of more than 300 pg/mL
been mentioned in previous sections. A question now predicts folate deficiency or another hematologic or
being frequently raised is that whether a bone marrow neurologic disease while 99 percent of patients with occult
examination always necessary to make a diagnosis of deficiencies will have levels less than 300 pg/mL. In view of
folate and cobalamin deficient megaloblastic anemia. lack of transparency related to these tests, poor validation,
There are many schools of thought. With highly sensitive and poor tracking of assay performance, if the clinical
serum tests for the specific diagnosis of cobalamin and picture is consistent with cobalamin deficiency, and the
folate deficiency now available, it would be reasonable serum cobalamin level is normal or borderline low, it is
to say that the urgency of the diagnosis should dictate the entirely appropriate to treat as for a cobalamin deficiency.
need for a bone marrow. For example, an urgent bone Cobalamin deficiency can falsely raise serum folate by
marrow aspirate examination showing megaloblastosis 20 to 30 percent via methyl-folate trapping. In patients
for immediate diagnosis is indispensible in a case of florid with megaloblastic anemia, the finding of a normal to
hematologic disease with or without neurologic disease increased level of serum folate, along with a reduced
suggestive of cobalamin or folate deficiency. However, in ratio of RBC to serum folate provides strong although an
stable and non-urgent cases with characteristic peripheral indirect evidence of cobalamin deficiency (Table 7).
smear, or for a patient with a primary neuropsychiatric Serum folate levels: When negative folate balance
presentation, proceeding with measurement of serum continues, hepatic folate stores are depleted in about
levels of vitamins or metabolites without a bone marrow 4 months. This leads to tissue folate deficiency, which
aspiration is a reasonable option. clinically correlates with a decrease in RBC folate (less
than 150 ng/mL) by the microbiologic assay.
Masked Megaloblastosis60
Serum MMA and Homocysteine Level
Conditions wherein true cobalamin or folate deficiency
with anemia is not accompanied by classic findings of Basis: Perturbation of methionine synthase activity by
megaloblastosis in the peripheral blood and bone marrow cobalamin deficiency results in substrate (homocysteine)
constitute the phenomenon of masked megaloblastosis buildup and elevated serum levels of homocysteine,
any condition that compromises a cells capacity to carry which can be measured by a sensitive assay. Additionally
out hemoglobinization such as iron deficiency anemia cobalamin deficiency also affects the activity of methyl
malonyl-CoA mutase negatively, which leads to elevated
and thalassemia will simultaneously decrease the ten
dency to form megaloblastic cells. However certain
points can help unveil this occult megalblastosis. A Table 7 Cobalamin levels and B12 deficiency56
wide RBC distribution width (RDW) with a normal MCH Falsely low serum cobalamin in the absence of true56
and/or MCV on the Coulter counter readout may reflect cobalamin deficiency
megaloblastic anemia 9 or dimorphic anemia (macro- • Folate deficiency (one-third of patients)
ovalocytes plus microcytic hypochromic RBCs). Since • Multiple myeloma
hemoglobinization has got no business with the white • TCI deficiency
blood cells and their pecursors, these pathognomonic • Megadose vitamin C therapy
findings (giant myelocytes and metamyelocytes, and Falsely raised cobalamin levels in the presence
hypersegmented PMNs) remain unaltered and can be of of a true deficiency62
great help in suspecting an underlying folate or cobalamin
• Cobalamin binders (TCI and II) increased (e.g.
deficiency. The latter may persist for up to 2 weeks after myeloproliferative states, hepatomas, and fibrolamellar
replacement with cobalamin or folate. Once masked hepatic tumors)
megaloblastosis has been recognized investigations to • TCII-producing macrophages are activated (e.g.
rule out iron deficiency, anemia of chronic disease, or autoimmune diseases, monoblastic leukemias and
hemoglobinopathies is indicated. Without correction of lymphomas)
the iron deficiency, cobalamin or folate will not elicit a • Release of cobalamin from hepatocytes (e.g. active liver
maximal therapeutic benefit. Conversely, treating with disease)
iron alone would unmask the megaloblastosis. • High serum anti-IF antibody titer
serum MMA levels. Thus homocysteine and MMA are is more cost effective it is recommended to first use the
sensitive tests for cobalamin deficiency. Early manifes cheaper tests that can assist in the diagnosis of cobalamin
tations of negative cobalamin balance are increased serum and folate deficiency (Table 8).
methylmalonic acid (MMA) and total homocysteine
levels. This occurs when the total cobalamin in serum is Tests to Assess Absorption and Transport
still in the low-normal range. Normal levels of MMA and
homocysteine rule out clinically significant cobalamin Schilling test: This test is rarely used these days since less time
deficiency with virtually 100 percent certainty. consuming and less tedious ways of diagnosing pernicious
Values: The normal value for serum homocysteine is anemia are now available. However for historical purposes
5.1 to 13.9 μM and serum MMA is 73 to 271 nM, and in the principle of this test is mentioned in brief. It is important
general the higher the values, the more severe the clinical to remember that this test intends to define an etiology of
abnormalities.9 However age-, creatinine-, gender-, diet-, cobalamin deficiency already established by other tests and
and race-dependent can cause the values to fluctuate over not to diagnose cobalamin deficiency per se.
a wide range. In the first part of the test, the patient is given radio
Application: When cobalamin and/or folate defi labeled vitamin B12 orally followed by an intramuscular
ciency is suspected strongly and the cobalamin levels injection of unlabeled vitamin B12 given an hour later
are suggestive but not definitive, then the MMA and which is only enough to temporarily saturate B12 receptors
homocysteine tests are an excellent means to confirm in the liver with enough normal vitamin B12 to prevent
a clinical diagnosis. Patients with clinical cobalamin radioactive vitamin B12 binding in body tissues (especially
deficiency usually have MMA values over 1000 nM and in the liver). Normally, the ingested radiolabeled vitamin
homocysteine values that are over 25 μM. The MMA and B12 will be absorbed into the body. Since the body already
homocysteine test results exceed cobalamin levels by has liver receptors for vitamin B12 saturated by the
quite an amount when sensitivities are compared as they injection, much of the ingested vitamin B12 will be excreted
increase much earlier and more consistently than the in the urine.
drop in cobalamin levels. • A normal result shows at least 10 percent of the
Since running serum cobalamin and folate levels radiolabeled vitamin B12 in the urine over the first 24
compared with serum MMA and homocysteine levels hours.
Table 8 Interpretation of serum levels62

Cobalamin* Folate (ng/mL) Provisional diagnosis Proceed with metabolites?
(pg/mL)
>300 >4 Cobalamin or folate deficiency is unlikely No
<200 >4 Consistent with cobalamin deficiency No
200–300 >4 Rule out cobalamin deficiency Yes
>300 <2 Consistent with folate deficiency No
<200 <2 Consistent with (1) combined cobalamin plus folate Yes
deficiency or (2) isolated folate deficiency
>300 2–4 Consistent with (1) folate deficiency or (2) an anemia Yes
unrelated to vitamin deficiency
Test results on metabolites: serum methylmalonic acid and total homocysteine
Methylmalonic Total Diagnosis
acid (Normal, homocysteine
70–270 nM) (Normal, 5–14 mM)
Increased Increased Cobalamin deficiency confirmed; folate deficiency still possible (i.e. combined
cobalamin plus folate deficiency possible)
Normal Increased Folate deficiency is likely
Normal Normal Cobalamin and folate deficiency is excluded
*Table adopted from ref no. 62
• In patients with pernicious anemia or with deficiency

due to impaired absorption, less than 10 percent of the
radiolabeled vitamin B12 is detected.
If an abnormality is found, i.e. the B12 in the urine is
only present in low levels, then in the second part the
test is repeated, with additional oral intrinsic factor.
• A normal urine collection shows a lack of intrinsic
factor production, i.e. pernicious anemia.
• A low result on the second test also implies other
causes of malabsorption, including infestation with D
latum or giardia, celiac disease, Whipple’s disease, etc.
Single–sample stool excretion test: Labeled cobalamin
was fed with a nonabsorbable dye together with nonab
sorbable chromium chloride (Cr51). A sample of such
stained stool was counted for Cr51 and Co57 and the
Fig. 7 Therapeutic response
ratio of the counts compared with that of the sample that
was fed. Normally 36–88 percent of the dose should be
absorbed. This test is not readily available. • Decrease in the neutrophil lobe count to normal over a
4 weeks period.
Miscellaneous Accelerated turnover of normal DNA in erythroid
precursors also increases serum rate level, peaking by the
• Increase LDH, serum bilirubin and serum iron levels fourth day along with increased cellular phosphate uptake
reflect ineffective erythropoiesis-non-specific. for nucleotide synthesis. These together may precipitate
• Serum lipid, cholesterol and immunoglobins may be an attack of gout if the patient has a “gouty predisposition”.
decreased-non-specific. If by the end of the third week, the RBC count is not
• Increased serum gastrin and pepsinogen levels. above 3 × 106/mm3 additional causes of underlying
• Antibodies to IF in the serum is highly specific and iron deficiency, hemoglobinopathy, chronic disease, or
indicates either present or imminent cobalamin hypothyroidism should be considered.
deficiency.
• Serum transcobalamin II levels: As an early marker
of cobalamin homeostasis, as a surrogate for the
TREATMENT OF MEGALOBLASTOSIS67-74
Schilling test, or to diagnose cobalamin deficiency in Principles
lieu of serum cobalamin values is still in the process of
validation. • Routinely, treatment with full doses of parenteral
cobalamin (1 mg/day) and oral folate (1–5 mg)
before knowledge of the type of vitamin deficiency
Positive Therapeutic Response63-66
is established should be reserved for the severely ill
Clinical, hematological and biochemical response to patient.
therapy with cobalamin and folic acid is another way to • Patients with vitamin B12 deficiency despite a normal
diagnose the deficiency in retrospect. A single injection absorption, such as vegetarians and vegans, only
of cyanocobalamin is given and disappearance of need a daily supplement in the form of a vitamin pill
megaloblastoid changes in the bone marrow is looked for containing at least 6 μg of vitamin B12.
in the next 48 hours besides two of the following, in order • Patients with an irreversible cause of vitamin B12
to call the test positive (Fig. 7). deficiency are destined to lifelong treatment with a
• 50 percent decrease in serum iron or LDH within 48 pharmacological dose of vitamin B12.
hours. Severe deficiency: When a patient of suspected mega
• Increase in retic count 5 to 10 days after treatment. loblastic anemia presents in failure either due to anemia
• Correction of neutropenia and thrombocytopenia over itself, due to sodium retention or due to myocardial
a period of 2 weeks. hypoxia the treatment includes oxygen administration
• Once reticulocytosis subsides MCV is decreased by 5 fL with slow transfusion of packed cells under cover of
or more. diuretics to avoid disastrous conditions of fluid overload.
• Plasma MMA and homocysteine in 2 weeks. Giving high initial doses of vitamin B12 can cause severe
• Correction of anemia of 2 to 4 weeks. metabolic disturbances like hypokalemia by shifting
extracellular potassium intracellularly in response to hematologic recovery is documented although if the

the sudden increase in the cell replication. This is also underlying etiology for the folate deficiency is not corrected
exaggerated further by the delay in renal potassium a lifelong folic acid supplemented is also warranted for.
retention. A clinician should anticipate such complications Folinic acid (i.e. 5-formyl-THF [leukovorin]) should be
and keep a watch on the potassium levels besides starting reserved only for rescue protocols involving antifolates
prophylactic potassium supplements in such cases. Hence (methotrexate or trimethoprim-sulfamethoxazole), for
it is recommended to give small doses of 10 µg (0.2 µg/kg for 5-fluorouracil modulation protocols, after nitrous oxide
severely deficient children) CNbl subcutaneously for first toxicity, or in pediatric cases involving cerebral folate
2 days which should suffice to normalize serum LDH as deficiency or inborn errors of folate metabolism.
well as iron levels besides inducing reticulocytosis within
approximately a week. However, more is needed before Prophylaxis
serum MMA and homocysteine levels are normalized and
body stores are replenished. Prophylaxis with Cobalamin
Conventional therapy:75-78 In a stable child it is advisable • 5 to 10 mcg for nutritional causes and 1000 mcg/day for
to collect samples of bone marrow aspirate, those for problems of malabsorption
serum folate and cobalamin levels. Conventional therapy • Infants on specialized diets
includes once daily injection 1000 µg for a week which is • Premature infants
succeeded by 100 µg of cyanocobalamin injection for a • Infants of mothers with pernicious anemia
month and then monthly injections. • Infants and children of mothers with nutritional
Different forms of vitamin B12 can be used, including cobalamin deficiency
cyano-, hydroxy-, and methylcobalamin. • Vegetarianism and poverty-imposed near-vegetari
Hydroxycobalamin may have advantages due to a anism
slower metabolism however, the depot preparation of • Total gastrectomy.
cyanocobalamin (cyanocobalamin-tannin complex sus
pended in a sesame oil-aluminium monostearate gel) is
Prophylaxis with Folic Acid
metabolized even slower than hydroxycobalamin.
All inborn errors of cobalamin metabolism adequately • 4 g/day periconceptionally
justify use of parenteral therapy with cobalamin. There • All women contemplating pregnancy (at least 400
is no major advantage of other preparations over generic mcg/day)
cyanocobalamin. Oral 2 mg cobalamin tablets consumed • Pregnancy and lactation, premature infants
daily is found to be equivalent to traditional monthly • Mothers at risk for delivery of infants with neural tube
parenteral treatment with 1 mg of intramuscular/subcu defects
taneous cobalamin among those requiring long-term • Hemolytic anemias/hyperproliferative hematologic
cobalamin as at such high doses cobalamin is absorbed states
passively across the mucous membranes of oral cavity and • Patients with rheumatoid arthritis or psoriasis on
stomach. This option is especially helpful in patients in whom therapy with methotrexate
parenteral therapy becomes less feasible due to refusal for • Patients on antiepileptic drugs
daily injection, those with coagulation disorders and in them • Patients with ulcerative colitis.
cobalamin (1–2 mg/day as tablets) can be recommended
despite cobalamin malabsorption. Meals can decrease the Thiamine-responsive Megaloblastic
bioavailability of cobalamin by 40 percent and taking the
same doses empty stomach decreases the losses in stool.
Anemia Syndrome79
Certain points should be emphasized when initiating It is caused by mutations in SLC19A2, encoding a thiamine
treatment with vitamin B12. Once vitamin B12 has been transporter protein. It is usually associated with diabetes
administered, the increase in red cell production will mellitus, anemia and deafness. With an onset generally
increase the demand on iron stores and, therefore, it seen during infancy or at early childhood and most of
is important to monitor–and correct–any signs of iron the thiamine-responsive megaloblastic anemia (TRMA)
deficiency. A drop in plasma folate after initiation of patients are originated from consanguineous families
vitamin B12 treatment is a sign of unmasking of hitherto and is thus an autosomal recessive disease whereby
occult folate deficiency. active thiamine uptake into cells is disturbed. Thus, at
Even when malabsorption is a problem oral folate physiological concentrations (food as the only source),
(folic acid) at doses of 1 to 5 mg/day results in adequate thiamine is not transported normally and intracellular
absorption. Therapy should be continued until complete thiamine deficiency leads to decreased activity of enzymes
associated with thiamine pyrophosphate. The role of 12. Beedholm-Ebsen R, van de Wetering K, Hardlei T,
thiamine in DNA metabolism and heme synthesis explains et al. Identification of multidrug resistance protein 1
the megaloblastic anemia. Thrombocytopenia has been (MRP1/ABCC1) as a molecular gate for cellular export of
less commonly reported in TRMA patients. The rarity of cobalamin. Blood. 2010;115:1632.
13. Shah NP, Beech CM, Sturm AC, et al. Investigation of the
leukopenia is in these patients is probably accounted for
ABC transporter MRP1 in selected patients with presumed
by the different needs of the hematopoietic progenitor
defects in vitamin B12 absorption. Blood. 2011;117:4397.
cells to the intracellular thiamine. Diabetes mellitus in 14. Quadros EV, Nakayama Y, Sequeira JM. The protein and
TRMA patients is a consistent finding and is most likely the gene encoding the receptor for the cellular uptake of
secondary to impairment of islet cell function by the transcobalamin-bound cobalamin. Blood. 2009;113:186.
intracellular thiamine deficiency. 15. Hematology. Basic Principles and Practice (Embargo:)
While the costs for health care delivery move farther Your Access: (Embargo:), by Hoffman and Ronald, 6th
and farther out of the reach of the common man, and edn, Chapter 37 Megaloblastic Anemia, Asok, C. Antony,
the ongoing debate on ways to reduce these costs, few p.476.
instances in internal medicine and hematology yield 16. Quadros EV. Advances in the understanding of cobalamin
more satisfying dividends than diagnosing and treating assimilation and metabolism. Br J Haematol. 2010;148:195.
17. Kim J, Gherasim C, Banerjee R. Decyanation of vitamin
cobalamin and folate deficiency using generic vitamins
B12 by a trafficking chaperone. Proc Natl Acad Sci USA.
that are “dirt-cheap”—costing only one or two cents a
2008;105:145-51.
day. These conditions are devastating when undiagnosed 18. Koutmos M, Datta S, Pattridge KA, et al. Insights into
or misdiagnosed or when cobalamin deficiency is treated the reactivation of cobalamin-dependent methionine
with folate alone. Recognition of various populations at risk synthase. Proc Natl Acad Sci USA. 2009;106:18527.
and the clinical scenarios in which folate and cobalamin 19. Olteanu H, Banerjee R. Human methionine synthase
deficiency are likely to be present, and the availability of reductase, a soluble p. 450 reductase-like dual flavoprotein,
sensitive and specific tests, should reduce uncertainty in is sufficient for NADPH-dependent methionine synthase
diagnosis. activation. J Biol Chem. 2001;276:355-8.
20. Hansen MR, Nexo E, Svendsen I, et al. Human intrinsic
factor: its primary structure compared to the primary
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C H A P T E R16
Anemia of
Chronic Disease
Anemia of chronic disease (ACD), the second most prevalent anemia after anemia caused by iron deficiency, occurs in patients
with acute or chronic immune activation. The condition has thus been termed “anemia of inflammation.”1 ACD is an anemia of
underproduction that usually is normocytic, normochromic, and relatively mild, with a hemoglobin level greater than 10 g/L.
However, the anemia can be severe, and the mean corpuscular volume may be reduced. Hypochromia (mean corpuscular hemoglobin
concentration, 26 to 32 g/dL) is more common than microcytosis.
The most frequent conditions associated with anemia of Table 1 Causes of anemia of chronic disease
chronic disease are listed in Table 1.
Chronic infections
• Tuberculosis
ANEMIA • Pulmonary infections
• Microcytosis in ACD is usually not as striking as that • Subacute bacterial endocarditis
commonly associated with iron deficiency anemia; • Pelvic inflammatory disease
values for MCV <72 fL are rare. Another distinction • Osteomyelitis
from iron deficiency is that hypochromia typically • Chronic urinary tract infections
precedes microcytosis in ACD but typically follows • Chronic fungal disease
the development of microcytosis in iron deficiency.2 • Human immunodeficiency virus infection
The hematocrit usually is maintained between 0.25 Chronic noninfectious inflammations
and 0.40, but significantly lower values are observed in • Rheumatic fever
20 to 30 percent of patients.2,3 • Severe trauma
The percentage of reticulocytes is normal or • Thermal injury
reduced; although on rare occasions, it may be slightly Malignant diseases
increased.
• Lymphoma
• Red cell distribution width may be normal initially but
• Leukemia
is typically elevated to a moderate degree, and generally
• Multiple myeloma
does not help in distinguishing iron deficiency and ACD.
Autoimmune
• The degree of anemia is proportional to the severity of
• Rheumatoid arthritis
the underlying disease. The severity of the anemia and
the activity of rheumatoid arthritis are judged by fever, • Systemic lupus erythematosus
severity of joint swelling and inflammation, and the eryth- • Vasculitis
rocyte sedimentation rate (ESR). In patients with malig- Miscellaneous
nant disease, anemia is more severe when metastases are • Chronic renal disease
widespread than when the disease is localized. Serum • Chronic liver disease
iron concentration and total iron binding capacity (TIBC) • Endocrine disorders
is decreased. Transferrin saturation is subnormal.2 Graft rejection
The abbreviation sTfR/log ferritin denotes the ratio of ACD.6 However, FEP increases more slowly in anemia of
the concentration of soluble transferring receptor to the chronic disorders than it does in iron deficiency.
log of the serum ferritin level in conventional units.
In patients with ACD, however, the serum ferritin level PATHOGENESIS
indicative of adequate reticuloendothelial iron stores
requires upward adjustment. Serum ferritin values usually The pathogenesis of anemia of chronic disease has been
increase in patients with inflammatory diseases and attributed to following:
extreme elevations of serum ferritin may be a nonspecific • Shortened erythrocyte survival
indicator of significant underlying disease.4 When iron • Impaired marrow response
deficiency coexists, the serum ferritin level falls but do not • Disturbance in iron metabolism.
reach values as low as those found in uncomplicated iron The shortening of the erythrocyte survival creates an
deficiency. A patient with chronic inflammatory disease increased demand for red cell production on the marrow
and a serum ferritin <30 µg/L is certainly iron-deficient, and the marrow is unable to respond fully because of a
and a patient with a serum ferritin >200 µg/L is certainly combination of a blunted erythropoietin response, an
not iron-deficient. Examination of a Prussian blue–stained inadequate progenitor response to erythropoietin, and
marrow specimen can confirm the ferritin status. Flow limited iron availability.
chart 1 shows differences between anemia due to iron ACD is one manifestation of the systemic response to
deficiency from anemia of chronic disease. immunologic or inflammatory stress, which results in the
A determination of the levels of soluble transferrin production of various cytokines: the cytokines most often
receptors by means of commercially available assays implicated in the pathogenesis of ACD are TNF7, IL-18, IL-
can be helpful for differentiating between patients with 69, and the interferon,10,11 concentrations of which have
anemia of chronic disease alone (with either normal or been reported to be increased in the serum or plasma of
high ferritin levels and low levels of soluble transferring patients with disorders associated with ACD.11,12
receptors) and patients with anemia of chronic disease
with accompanying iron deficiency (with low ferritin levels Shortened Erythrocyte Survival
and high levels of soluble transferrin receptors).5 The rate of survival of cells from patients with arthritis,
The concentration of free protoporphyrin in the when transfused into normal subjects, is normal, and
erythrocytes (FEP) tends to be elevated in patients with the survival of red cells from normal individuals in the
circulation of patients with arthritis is less than the normal
Flow chart 1 Algorithm for the differential diagnosis among iron
rate. Therefore, shortened red cell survival in patients
deficiency anemia, anemia of chronic disease, and anemia of
with chronic inflammatory disorders is attributed to an
chronic disease with iron deficiency
extracorpuscular mechanism. IL-1 levels and shortened
red cell survival are correlated in anemia patients with
rheumatoid arthritis, and mice that become anemic after
exposure to TNF in vivo also exhibit a shortened red cell
survival.
Neocytolysis, a selective hemolysis of newly formed
erythrocytes associated with erythropoietin deficiency,
can also contribute to shortened red blood cell (RBC)
survival in ACD.
IMPAIRED MARROW RESPONSE

The bone marrow which normally should compensate for
decreased erythrocyte survival does not show increased
marrow response.
The three proposed possible mechanisms are:
1. Inappropriately low erythropoietin secretion
2. Diminished marrow response to erythropoietin
3. Iron-limited erythropoiesis.
The erythropoietin response to anemia is blunted. This
impaired erythropoietin response is cytokine-mediated.
IL-1, TNF-α, and transforming growth factor-β inhibit
Chapter-16 Anemia of Chronic Disease 151
production of erythropoietin by various hepatoma cell by TNF-α. Hepcidin appears to promote macrophage
lines. This reduces the tissue oxygen consumption so iron retention by causing internalization of the iron
that normal oxygenation is maintained despite reduced transport protein ferroportin.15 Also causes decreased
hemoglobin levels. There is also increased erythrocyte duodenal absorption of iron. Under certain conditions,
2, 3-diphosphoglycerate levels along with decreased hepcidin may be associated with impaired erythroid
hemoglobin oxygen affinity colony formation in vitro.16 The mechanisms leading
TNF, IL-1, and interferon have all been reported to to anemia in anemia of chronic disease are given in
inhibit erythropoiesis in vivo and in vitro. TNF induces IL-1 Figure 1.
production by macrophages, IL-1 induces interferon-γ • Apoferritin is normally synthesized in response to
production by T-lymphocytes, and interferon-γ can increased intracellular iron concentration.17 It has
exhibit positive or negative feedback on production of IL-1 been suggested that excess apoferritin is made in infla
and TNF. mmatory and malignant conditions, and the surplus
Treatment with recombinant human erythropoietin binds a larger-than-usual amount of iron entering the
can correct ACD in many cases. cell.18 In effect, such a mechanism would divert iron
from the rapid to the slow pathway of iron release.
Rodents injected with recombinant TNF developed a
ABNORMAL IRON METABOLISM hypoferremic anemia associated with impaired storage
A hallmark of anemia of chronic disease is the develop iron release and incorporation into erythrocytes.19
ment of disturbances of iron homeostasis, with inc IL-1 increases translation of ferritin messenger RNA
reased uptake and retention of iron within cells of the and this additional ferritin act as a trap for iron that might
reticuloendothelial system. This leads to a diversion of iron otherwise be available for erythropoiesis.20
from the circulation into storage sites of the reticuloendo
Nitric oxide, which is a common mediator of cytokine
thelial system, subsequent on-restricted erythropoiesis. effects, has similar effects on ferritin expression.21
It has been proposed that lack of iron for erythropoiesis
Lactoferrin is a transferring like protein in neutrophil-
contributes to the inadequate marrow response in ACD. specific granules,22 released from the neutrophil during
Evidence of a functional iron deficiency in this syndrome phagocytosis or stimulation by IL-1.23 Lactoferrin-bound
includes erythrocyte microcytosis, increased FEP, reduced iron is not immediately available for erythropoiesis as it
transferrin saturation, and decreased marrow sideroblasts. transfers iron from its transferrin-bound, circulating state
• The major contributor to hypoferremia in patients to a storage state.
with ACD is probably a shift of iron from a transferrin- In Panel A, the invasion of microorganisms, the
bound, available state to a ferritin-incorporated emergence of malignant cells, or autoimmune dysregu
storage state. Iron absorption appears to be normal, lation leads to activation of T cells (CD3+) and monocytes.
but iron tends to remain in the mucosal cell and in These cells induce immune effector mechanisms, thereby
hepatocytes and there is a limited availability of iron producing cytokines such as interferon-α (from T cells)
for erythroid progenitor cells. Macrophages, the major and tumor necrosis factor-a (TNF-α), interleukin-1,
site from which iron is obtained for erythropoiesis, also interleukin-6, and interleukin-10 (from monocytes or
exhibit increased iron storage. macrophages).
Macrophage iron becomes available for erythropoiesis In Panel B, interleukin-6 and lipopolysaccharide
through two mobilization pathways: stimulate the hepatic expression of the acute-phase
• Rapid pathway, associated with almost immediate protein hepcidin, which inhibits duodenal absorption of
return of the iron retrieved from senescent red cells; iron.
• Slower pathway, consisting of iron mobilized from In Panel C, interferon-α, lipopolysaccharide, or both
storage13 increase the expression of divalent metal transporter
• In ACD, the slower pathway predominates, and 1 on macrophages and stimulate the uptake of ferrous iron
iron tends to accumulate. The acquisition of iron by (Fe2+). The anti-inflammatory cytokine interleukin-10 up
macrophages most prominently takes place through regulates transferrin receptor expression and increases
erythrophagocytosis and the transmembrane import of transferrin receptor–mediated uptake of transferrin
ferrous iron by the protein divalent metal transporter 1 bound iron into monocytes. In addition, activated macro
(DMT1). Recent studies of the role of the liver-produced phages phagocytose and degrade senescent erythrocytes
antimicrobial peptide hepcidin strongly suggest that it for the recycling of iron, a process that is further induced
is the dominant factor in this process.14 by TNF-α through damaging of erythrocyte memb
• Hepcidin is an acute-phase-reacting peptide, which ranes and stimulation of phagocytosis. Interferon a
is induced by IL-6, lipopolysaccharides and inhibited and lipopolysaccharide down-regulate the expression
Fig. 1 Pathophysiological mechanisms underlying anemia of chronic disease
of the macrophage iron transporter ferroportin 1, thus In summary, these mechanisms lead to a decreased
inhibiting iron export from macrophages, a process that iron concentration in the circulation and thus to a limited
is also affected by hepcidin. At the same time, TNF-α, availability of iron for erythroid cells.
interleukin-1, interleukin-6, and interleukin-10 induce In Panel D, TNF-α and interferon-g inhibit the
production of erythropoietin in the kidney.
ferritin expression and stimulate the storage and retention
In Panel E, TNF-α, interferon-g and interleukin-1 directly
of iron within macrophages.
inhibit the differentiation and proliferation of erythroid
progenitor cells. In addition, the limited availability of iron Erythropoietic Agents

and the decreased biologic activity of erythropoietin lead
to inhibition of erythropoiesis and the development of The therapeutic effect involves counteracting the antipro
anemia. Plus signs represent stimulation, and minus signs liferative effects of cytokines,25 along with the stimulation of
inhibition. iron uptake and heme biosynthesis in erythroid progenitor
cells. Accordingly, a poor response to treatment with
erythropoietic agents is associated with increased levels
Treatment Options of proinflammatory cytokines, on the one hand, and poor
Moderate anemia warrants correction, especially in iron availability.24
patients with additional risk factors (such as coronary Three erythropoietic agents are currently available:
artery disease, pulmonary disease, or chronic kidney 1. Epoetin alfa
disease), or a combination of these factors. In patients 2. Epoetin beta
with renal failure who are receiving dialysis and in 3. Darbepoetin alfa.
patients with cancer who are undergoing chemotherapy, These differ in terms of their pharmacologic com
correction of anemia up to hemoglobin levels of 12 g per pounding modifications, receptor-binding affinity,
deciliters is associated with an improvement in the quality and serum half-life, thus allowing for alternative
of life. dosing and scheduling strategies.
In a retrospective review of nearly 100,000 patients The long-term administration of epoetin has been
undergoing hemodialysis, levels of hemoglobin of 8 g per reported to decrease levels of TNF-a in patients with
deciliters or less were associated with a doubling of the chronic kidney disease; reportedly, those who respon
odds of death, as compared with hemoglobin levels of ded well to epoetin therapy had a significantly higher
10 to 11 g per deciliter. Guidelines for the management of level of expression of CD28 on T cells and lower levels of
anemia in patients with cancer or chronic kidney disease interleukin-10, interleukin-12, interferon-γ, and TNF-a
recommend a target hemoglobin level of 11 to 12 g per than did those with a poor response. Such anti-infla
deciliter. mmatory effects might be of benefit in certain diseases
such as rheumatoid arthritis, a disease in which combined
Transfusion treatment with epoetin and iron not only increased
hemoglobin levels but also resulted in a reduction of
Transfusions are particularly helpful in the context of
disease activity.
either severe anemia (in which the hemoglobin is less than
The production of erythropoietin receptors by cancer
8.0 g per deciliter) or life-threatening anemia (in which the
cells appears to be regulated by hypoxia, and in clinical
hemoglobin is less than 6.5 g per deciliter), particularly
cancer specimens the highest levels of erythropoietin
when the condition is aggravated by complications that
receptors were associated with neoangiogenesis, tumor
involve bleeding.
hypoxia, and infiltrating tumors. Erythropoietin increases
Iron Therapy inflammation and ischemia-induced neovascularization
by enhancing the mobilization of endothelial progenitor
Oral iron is poorly absorbed because of the down cells.26, 27
regulation of absorption in the duodenum. Only a fraction
of the absorbed iron will reach the sites of erythropoiesis, Monitoring Therapy
owing to iron diversion mediated by cytokines, which
directs iron into the reticuloendothelial system. Before the initiation of therapy with an erythropoietic
In addition, iron therapy for patients with anemia agent, iron deficiency should be ruled out. Hemoglobin
of chronic disease is controversial. By inhibiting the levels should be determined after four weeks of therapy
formation of TNF-α, iron therapy may reduce disease and at intervals of two to four weeks thereafter. If
activity in rheumatoid arthritis or end-stage renal disease. the hemoglobin level increases by less than 1 g per
In addition to possible absolute iron deficiency deciliters, the iron status should be reevaluated and iron
accompanying the anemia of chronic disease, functional supplementation considered.28 If iron-restricted erythro
iron deficiency develops under conditions of intense poiesis is not present, a 50 percent escalation in the
erythropoiesis24 during therapy with erythropoietic agents, dose of the erythropoietic agent is indicated. The dose
with a decrease in transferrin saturation and ferritin to of the erythropoietic agent should be adjusted once the
levels 50 to 75 percent below baseline.24 hemoglobin concentration reaches 12 g per deciliter.29 If
Iron supplementation should also be considered for no response is achieved after eight weeks of optimal dosage
patients who are unresponsive to therapy with erythropoi- in the absence of iron deficiency, a patient is considered
etic agents because of functional iron deficiency. nonresponsive to erythropoietic agents.
ANEMIA OF CHRONIC RENAL INSUFFICIENCY Reticulocyte count: The reticulocyte count often is within
normal limits,35 but it may be moderately increased.36
The term anemia of chronic renal insufficiency refers to The highest values were observed with extreme azotemia
that anemia resulting directly from failure of the endocrine (BUN, 300 to 350 mg/dL).
and filtering functions of the kidney. The kidney is the
major source of erythropoietin and the ability to secrete Leukocyte count: The leukocyte count typically is normal,
this hormone is lost as the kidney fails. Renal failure is but slight neutrophilic leukocytosis may be observed.35
also associated with other pathologic processes that either Platelet count and function: The platelet count is either
inhibit erythropoiesis or shorten erythrocyte survival. Lack normal or slightly increased,35 but platelet function may
of sufficient erythropoietin is the most important factor be severely impaired resulting in defective hemostasis
in causing anemia; consequently, the hypoproliferative disease (×1500).
features of the anemia tend to predominate.In clinical
settings, in chronic renal failure, additional factors may Bone marrow examination: The bone marrow tends to
also contribute to the development of anemia such as : be hypercellular and slight erythroid hyperplasia may
• The presence of infection or inflammation be observed. The myeloid-to-erythroid ratio averaged
• Iron deficiency anemia due to blood loss from the 2.5:1.0.35 Erythroid maturation remains normal. In some
gastrointestinal tract or hematuria or from retention of instances, especially when renal failure is relatively acute,
blood in the hemodialysis apparatus tubing30 hypoplasia of erythroid elements is noted.
• The megaloblastic anemia because of folate deficiency Liver function test: The serum bilirubin level is usually
in patients on dialysis31 within normal limits but the hemolytic index (a measure
• Certain types of renal disease, including the hemolytic-
uremic syndrome or thrombotic thrombocytopenic
purpura, are associated with microangiopathic hemo
lytic anemia
• Aluminum intoxication can cause microcytic anemia
in dialysis patients.32
Clinical Description
The nature of the underlying disease has little relation to
the degree of anemia, although anemia may be less severe
in patients with hypertensive renal disease and with
polycystic disease.33
The severity of the anemia bears a rough relationship
to the degree of renal insufficiency.
Anemia is not routinely observed until the creatinine
clearance falls to <40 mL/minute, which corresponds A
roughly to a serum creatinine of 2.0 to 2.5 mg/mL in
an average-sized adult. At creatinine clearance rates
below that, a statistically significant correlation between
creatinine clearance and hematocrit has been reported.34
Laboratory Findings
Hemoglobin and PCV: Anemia tends to become more
severe as renal failure worsens, but in most patients the
hematocrit ultimately stabilizes between 0.15 and 0.30.2
The apparent degree of anemia may be exaggerated or
minimized by alterations in plasma volume.
Peripheral blood film (PBF): The erythrocytes usually are
normocytic and normochromic. The majority of red cells
appear normal on blood smears. Occasionally, “burr” cells B
(Figs 2A and B) are observed along with some triangular, Figs 2A and B (A) Crenated cells in renal; (B) Burr cells in renal
helmet-shaped, or fragmented cells. disease (X 3000)
of urobilinogen excretion in relation to total circulating – Impaired erythrocyte glycolysis has been found
hemoglobin) may be increased in ~40 percent of patients.37 in uremic patients. Hemoglobin oxygen affinity
Serum iron: It is normal in mild renal failure. Serum is reduced,46 because of increased erythrocyte
iron decreases in severe disease, or hyperferremia. The adenosine triphosphate and 2, 3-DPG levels.
gastrointestinal absorption of iron is also reduced in – Neocytolysis, the selective hemolysis of newly
patients with chronic renal failure.38 FEP may be normal formed red cells, has been reported after
or moderately increased, but the increased values seem to erythropoietic with-drawal in dialysis patients
occur only in patients with hypoferremia.39 The erythrocyte and may contribute to shortened red cell
lactate dehydrogenase level is within normal limits. survival in dialysis patients.
HbA1c: The glycated fraction (A1) of hemoglobin tends to
increase in chronic renal failure. Hemoglobin A1 value Management and Course
averaged 10.8 percent in patients with uremic patients
compared with 7.1 percent in nonuremic individuals.40 In • Recombinant erythropoietin: Recombinant human
uremic patients treated with dialysis, the value averaged 8.8 erythropoietin has been available for treatment of
percent. The increase is thought to result from carbamylation anemia of renal disease which has revolutionized
of the hemoglobin molecule by urea-derived cyanate; it the approach to this disorder. Erythropoietin can
can be detected by using column chromatography. The be administered intravenously or subcutaneously.
increase in the A1 fraction may continue after successful Although erythropoietin was originally given three
renal transplantation has brought the azotemia under times weekly (to coincide with dialysis schedules),
control because of disturbed carbohydrate metabolism.41 single weekly doses are similarly efficacious if the total
weekly dose is increased appropriately.48 A standard
Pathogenesis starting dose would be 100 to 150 U/kg/week, given
The three factors involved are: as a single or in divided doses. Higher doses generally
1. Erythropoietin deficiency result in faster correction of anemia; target hemoglobin
2. Suppression of marrow erythropoiesis is typically attained within 6 to 8 weeks.47 Iron
3. Shortened red cell survival. supplementation is necessary, particularly in patients
on hemodialysis. The target hemoglobin/hematocrit
• As renal function deteriorates, renal erythropoietin range is usually 12 g/dL/0.360.
secretion decreases.42 Measured erythropoietin values • Recombinant human erythropoietin (rHuEPO): Deter
may be lower than normal, higher than normal, or mine the baseline serum erythropoietin and ferritin
normal.42 levels prior to starting rHuEPO therapy. If ferritin is
• Suppression of bone marrow less than 100 ng/mL, give ferrous sulphate 6 mg/kg/
– Retained uremic toxins depress erythropoiesis day aimed at maintaining a serum ferritin level above
directly.36,43 100 ng/mL and a threshold transferrin saturation of
– Hyperparathyroidism may also contribute to 20 percent.
marrow suppression. It is effects by causing marrow • Start with rHuEPO treatment in a dose of 150 units/kg/
fibrosis. day subcutaneous three times a week.
– Cytokine-mediated anemia mechanisms typically • Monitor blood pressure closely (increased viscosity
associated with ACD may be active in renal failure. produces hypertension in 30 percent of cases) and
– 20 to 70 percent of uremic patients show shortened perform complete blood count (CBC) weekly.
red cell survival related to degree of azotemia.36 In
some patients, splenic sequestration of red cells Titrate the dose:
may be a contributory factor. If no response, increase rHuEPO to 300 units/kg/day SC
– In 20 percent patients, red cell pentose phosphate three times a week.
pathway is impaired.44 ↓
– Oxidant drugs, such as primaquine or sulfonamides, If hematocrit (Hct) reaches 40 percent, stop rHuEPO until
produce a Heinz body hemolytic anemia in patients Hct is 36 percent and then restart at 25 percent dose.
with the pentose phosphate pathway defect. ↓
Contamination of dialysate water by chloramines, If Hct increases very rapidly (>4% in 2 weeks), reduce dose
which inhibit phosphoglyceromutase and thus by 25 percent.
cause accumulation of glycolytic intermediates, Folic acid 1 mg/day is recommended because folate is
may worsen this defect.44,45 dialyzable.
Side Effects/Adverse Reactions Oral iron supplements, though cheap, are often
insufficient to maintain iron stores, especially in HD
• Erythropoietin is generally safe. When used for patients, due to excessive blood loss, poor absorption,
anemia in renal disease, hypertension is an important poor compliance with medications and gastrointestinal
complication which is transient, confined to the first side effects.
3 to 6 months of treatment.49 Rarely, the hypertension is The current NKF-KDOQI guidelines recommend oral
abrupt and severe with encephalopathy and seizures. iron therapy to be given in doses ranging from 2 to 3 mg/
The pathogenesis is multifactorial. An increase in kg up to 6 mg/kg of elemental iron per day in two to three
peripheral vascular resistance because of decrease in divided doses per day. Maintenance therapy aims to
cardiac output, heart rate and stroke volume due to provide 1–2 mg/kg of elemental iron per week to achieve a
correction of anemia may be responsible. TSAT between 20 and 50 percent and serum ferritin levels
• Anaphylaxis in response to erythropoietin has been of 100 to 800 ng/mL.
described but is extremely rare.
RENAL REPLACEMENT THERAPY
Erythropoietin Resistance
Renal replacement approaches (transplantation and
The failure of patients to respond optimally to erythro dialysis) aim to restore or substitute for lost renal function.
poietin therapy or a requirement for unusually high doses As such, they may have some effects on anemia associated
is referred to as an erythropoietin resistance. with renal failure.
Iron deficiency is the most common cause.50 Patients
with serum ferritin levels <100 to 200 µg/L or with transferrin RENAL TRANSPLANTATION
saturation values <20 to 25 percent50,51 require iron supple
Renal transplantation is the complete and satisfactory
mentation. The best early predictors of erythropoietin
treatment for renal insufficiency. Anemia is usually
response are serum TfR and serum fibrinogen. Response
corrected over an 8- to 10-week period due to erythropoietin
rates approaches 100 percent when both are low and
secretion by the grafted kidney.57
29 percent when both are high, reflecting both the patients
Two peaks of erythropoietin secretion have been
iron status and the presence or absence of inflammation. documented. An early peak at 7 days and a second
Inadequate hemodialysis is associated with erythro more sustained increase in erythropoietin levels, on
poietin resistance.52 As discussed earlier, secondary hyper approximately day 8, accompanied by reticulocytosis and
parathyroidism often accompanies renal failure and the a gradual increase in hemoglobin levels. Erythropoietin
associated marrow fibrosis may contribute to the anemia values return to normal when the hematocrit reaches 0.32.
and to erythropoietin resistance. Approximately, 80 percent of patients experience an
Treatment with vitamin D3 can decrease recombinant increase in blood hemoglobin concentration after renal
erythropoietin requirements and improve hemoglobin allograft.57 Improvement in erythropoiesis occurs earlier
values.53 If erythropoietin resistance is associated with an when cyclosporin is used for immunosuppression rather
increased MCV, folate supplementation is warranted. than with antilymphocyte globulin (ALG).
The use of angiotensin-converting enzyme inhibitors
in renal failure patients may exacerbate erythropoietin DIALYSIS
resistance.54 Splenectomy may be required in case of
erythropoietin requirement increases in a patient with Red cell production increases slightly in patients on
splenomegaly. hemodialysis, with small increases in hematocrit and
Anti-erythropoietin antibodies, including production decreases in transfusion requirement.58
of pure red cell aplasia, have rarely been reported.55 As a general rule, anemia is less severe in patients
• Darbepoetin (novel erythropoiesis-stimulating protein): receiving peritoneal dialysis, with consequently lower
The long-acting erythropoietin analog Darbepoetin erythropoietin and transfusion requirements.59,60
(novel erythropoiesis-stimulating protein) appears to
be safe and effective in the anemia of renal failure.56 Anemia in Cirrhosis and Other Liver Diseases
The recommended starting dose is 0.45 µg/kg/week. • Certain degree of anemia is commonly seen in patients
• Iron: Iron repletion and maintenance is the second with liver disease like alcohol-induced cirrhosis, biliary
pillar of anemia management in kidney disease. The cirrhosis,61 hemochromatosis, postnecrotic cirrhosis,
current NKF-KDOQI recommendation for targets of and acute hepatitis.62
iron therapy is to maintain serum ferritin at >100 ng/ • The term anemia of liver disease refers to mild or
mL and TSAT at >20 percent in pediatric HD, PD and moderate anemia associated with liver disease in the
non-dialysis CKD patients. absence of any complicating factors such as blood
loss, marrow suppression by exogenous agents or The hemoglobin level rarely falls below 10 g/dL in the
nutritional deficiency. absence of bleeding or severe hemolysis. Approximately,
• Some of the pathogenetic mechanisms of anemia 5 percent of liver disease patients, with severe liver disease
include: develop spur cell hemolytic anemia and hemoglobin
– Shortened red cell survival and red cell fragmen concentrations <10 g/dL.65 Episodic hemolysis when
tation (spur cell anemia) in cirrhosis. accompanied by jaundice and hyperlipidemia is known as
– Hypersplenism with splenic sequestration in the Zieve’s syndrome.
presence of secondary portal hypertension
– Iron deficiency anemia secondary to blood loss
from esophageal varices in portal hypertension HEMATOLOGIC FINDINGS
– Chronic hemolytic anemia in Wilson’s disease • PBF: Anemia of liver disease is mildly macrocytic.
secondary to copper accumulation in red cells Target cells and cells with increased diameters are
– Aplastic anemia resulting from acute viral hepatitis evident on blood smear (Figs 3A to C). The cells
(particularly hepatitis B) in certain immunologically appear hypochromic. These morphologic changes are
predisposed hosts accompanied by increased resistance to hemolysis
– Megaloblastic anemia secondary to folate defi in osmotic fragility tests.66 When spur cell hemolytic
ciency in malnourished individuals. anemia supervenes, characteristic acanthocytes—
Individuals with cirrhosis of any etiology are at erythrocytes covered with five to ten spike like
increased risk for hemorrhage. Blood loss occurs in projections are evident.
24 to 70 percent of patients with alcoholic cirrhosis. The • Reticulocyte count: The reticulocyte count often is
upper gastrointestinal tract is the major site of bleeding, increased, but sustained reticulocytosis of 15 percent
but loss of blood from the nose, hemorrhoids, and uterus or more is unusual in the absence of hemorrhage or
often occurs in association with coagulopathy of hepatic spur cell anemia.
origins. • Platelet count: Approximately 50 percent of patients
with cirrhosis have mild thrombocytopenia, but values
PREVALENCE AND CLINICAL less than 50 × 109/L are uncommon.63
MANIFESTATIONS • Total leukocyte count (TLC): A variety of leukocyte
Approximately 75 percent of patients with chronic liver abnormalities may be observed. Severe pancytopenia
disease develop anemia as defined by a reduction in associated with splenomegaly in liver disease is known
the hematocrit or hemoglobin level.63 The whole blood as Banti’s syndrome.
volume in liver disease averages 10 to 15 percent greater • Bone marrow aspiration: Bone marrow cellularity
than normal; thus, hemodilution tends to exaggerate the is normal or increased.63 Erythroid hyperplasia is
prevalence and degree of anemia.64 Patients with more observed. Red cell precursors have been described
severe liver disease and bleeding tend to have reduced as macronormoblasts, a term that implies their size
red cell mass. is increased, but their nuclear chromatin appears
The anemia is usually mild-to-moderate and hemo normal.63,67 Frank megaloblastosis is seen in <20
globin level averages 12 g/dL or the hematocrit 0.36.64 percent of patients.
A B C
Figs 3A to C Anemia of liver disease. (A) Crenated cells in renal failure; (B) Burr cells; (C) Macrocytes and target cells in liver disease
PATHOGENESIS Darbepoetin (also called novel erythropoiesis-stimula

ting protein) is an erythropoietin analog with modified
• Shortened erythrocyte survival: Red cell survival is glycosylation permitting a longer half-life. The results of
decreased in 33 percent of patients with alcoholic liver studies comparing Darbepoetin and erythropoietin in
disease. Various causes are congestive splenomegaly, anemic cancer patients vary depending on specific study
abnormal erythrocyte metabolism and hypophos endpoints, but both appear to be effective.73
phatemia with reduced erythrocyte adenosine tripho It is debated whether or not to administer iron routinely
sphate levels and consequent hemolysis. to patients receiving therapy with erythropoietin products.
Characteristic alterations in red cell membrane Although there are reports of correction of ACD by
lipids are found in patients with hepatitis, cirrhosis, intravenous iron without erythropoietin,74 normalization
and obstructive jaundice and may also be another of hemoglobin was only described in patients who were
contributor to shortened red cell survival.68 clearly iron-deficient. Anemic cancer patients treated
• In spur cell hemolytic anemia, the erythrocyte with concurrent intravenous iron and recombinant
membrane accumulates excess cholesterol without erythropoietin appear to have a better response than
a corresponding increase in lecithin, resulting in the those treated with no iron supplementation or with iron
characteristic morphologic abnormality. This change is supplementation alone.75
accompanied by pronounced reduction in erythrocyte
survival, probably because the distorted cells are less
ANEMIA ASSOCIATED WITH
deformable than normal and thus become trapped by
splenic macrophages.
ENDOCRINE DISORDERS
• Inadequate erythropoiesis: The marrow response A mild-to-moderate anemia commonly accompanies
to the anemia in patients with liver disease may disorders affecting the thyroid, adrenals, parathyroids,
be inadequate. Serum from cirrhotic patients can gonads, or pituitary. It is usually not associated with
suppress hematopoietic colony formation in vitro,69 symptomatology and in fact may reflect a physiologically
and cytokines implicated in inhibition of erythropoiesis appropriate hemoglobin concentration because the
have been found to be increased in patients with hormone deficiency often results in reduced oxygen
liver disease.70 Dyserythropoiesis with morphologic requirements. Most individuals present as referrals for
abnormalities and intramedullary hemolysis has also evaluation of moderate anemia with normal iron, B12, and
been reported in severe liver disease.71 folate studies.
ANEMIA IN PATIENTS WITH CANCER Hypothyroidism

Much of the anemia commonly observed in patients with A mild anemia with no other apparent etiology occurs
cancer can be attributed to the mechanisms involved in 10 to 25 percent of patients with hyperthyroidism.82,83
in ACD. Erythroid precursors may be displaced from Anemia is associated with severe or prolonged hyper
marrow by metastatic tumor, tumor-induced fibrosis, thyroidism. Hemoglobin value falls but remains within
or tumor-associated marrow necrosis. The treatment normal limits.82 MCV is either normal or modestly
of cancer can also produce or exacerbate anemia by a decreased. Hemoglobin A2 levels are slightly increased but
variety of mechanisms, including impaired erythropoietin not as much as in thalassemia.79 Both the anemia and the
production and cytotoxic effects of therapy on erythroid microcytosis are corrected when the hyperthyroidism is
progenitors. successfully treated. Erythropoiesis usually is accelerated
Typically, the serum transferrin is either low or low but ineffective along with increased plasma erythropoietin
normal. The major differential diagnosis is iron deficiency levels.
anemia. Anemia is observed in 21 to 60 percent of hypo
thyroid patients. The anemia may be normocytic and
Treatment normochromic, hypochromic and microcytic, or macro
cytic. Hypochromic microcytic anemia found in asso
Fewer than 30 percent of patients have anemia sufficiently ciation with hypothyroidism should be considered iron
severe to necessitate transfusion, and assessment of the deficiency.76,77 The microcytic anemia responds to iron
symptomatic state should always be considered before therapy, even if thyroid hormone is not administered, but
administration of blood products. does not typically respond to thyroid hormone without
Recombinant erythropoietin is effective and safe but iron.76 Hypothyroid individuals are more likely to become
expensive.72 iron-deficient because of predisposition to menorrhagia
and achlorhydria77 and because thyroid hormone itself of adrenal insufficiency cases associated with the
may be essential for normal iron absorption.76 Severely, polyglandular autoimmune syndrome type I.84
macrocytic anemia usually results from complicating
deficiency of vitamin B1277 or folate. Androgen Deficiency
Anemia usually affects children whose height is below
the third percentile. The anemia usually is mild, with the After puberty, values for the hematocrit, blood hemoglobin
hematocrit rarely falling below 0.35. The plasma volume concentration, and red cell count average 10 to 13 percent
often is decreased, which tends to make the reduction in higher in men than in women. In castrated men, these
hematocrit less than might be expected for a given decrease values fall to within the normal female range.85 This is due
in red cell mass.77 The degree of anemia is related to both to a difference in erythropoietin production. After the sixth
the severity and the duration of the hypothyroidism.78 The decade, male hemoglobin values fall back toward those
MCV may be increased in hypothyroid patients, even in observed in women.86 The anemia in these patients is
the absence of anemia.76 Acanthocytes are apparent in 20 corrected by androgen replacement.The differences in red
percent of patients. Usually, the leukocyte and platelet counts cell parameters between the sexes are accounted for chiefly
by the stimulating effect of androgens on erythropoiesis.
are within the normal range, although both may be slightly
The administration of androgens to castrated males restores
reduced.78 The bone marrow may be mildly hypoplastic, but
male values for hemoglobin concentration. Androgens can
the myeloid-to-erythroid ratio is not significantly altered.
also stimulate erythropoiesis in normal subjects. In normal
Hemoglobin A2 levels are reduced slightly.
men, testosterone enanthate induced an average red cell
mass increase of 1.7 to 2.3. The increase in hematocrit
Pathogenesis was of smaller magnitude (from 0.456 to 0.494), probably
The anemia of hypothyroidism results from decreased red because the plasma volume also increased. Androgens
cell production. Plasma iron transport and erythrocyte act by increasing renal synthesis of erythropoietin.85
iron turnover rates are reduced, indicating subnormal Estrogens produce anemia when given in large amounts
red cell production.79 Erythropoietin secretion is reduced which suggest that this effect results from suppression of
in hypothyroid patients,80 and 2, 3-DPG levels are not hepatic synthesis of erythropoietin, but it may also simply
increased81 as occurs in most anemic and hypoxic states. represent opposition to androgen effects in general.
The response of anemia of hypothyroidism to thyroid
hormone is gradual. No striking reticulocytosis occurs, and Hypopituitarism
the hematocrit returns to a normal value only gradually
Moderately severe anemia is seen as a feature of all
over approximately a 6-month period.76,77
types of pituitary insufficiency. Reduced hemoglobin
levels are seen in Simmonds’ disease, pituitary neoplasms
Hyperthyroidism and prepubertal pituitary dwarfs. The anemia usually is
A mild anemia with no other apparent etiology occurs in 10 normocytic and normochromic and the red cells appear
to 25 percent of patients with hyperthyroidism.82,83 Anemia normal morphologically. Studies demonstrate reduced
is associated with severe or prolonged hyperthyroidism. red cell production.87
Hemoglobin value falls but remains within normal limits.82 The anemia of hypopituitarism results chiefly from
MCV is either normal or modestly decreased. Hemoglobin deficiencies of the hormones of target glands controlled by
A2 levels are slightly increased but not as much as in the pituitary, especially the thyroid and adrenal hormones,
thalassemia.79 Both the anemia and the microcytosis are but also from deficiency of androgens. In addition, lack
corrected when the hyperthyroidism is successfully treated. of other pituitary factors, such as growth hormone,87,88
Erythropoiesis usually is accelerated but ineffective along prolactin, or factors characterized less clearly, may be of
with increased plasma erythropoietin levels. importance.
As suggested for the anemia of hypothyroidism,
panhypopituitarism produces its effects on erythropoiesis
Adrenal Insufficiency
chiefly by reducing tissue oxygen consumption.88 The
Although anemia is common and nearly universal in organism reacts to this decreased need for oxygen by
adrenal insufficiency, it may be masked by the dehydration secreting less erythropoietin and the red cell mass
characteristic of this syndrome.83 The red cells were diminishes until a new equilibrium between oxygen
normocytic and normochromic. Pernicious anemia is supply and demand is established.
observed in 3 to 16 percent of cases of nontuberculous Treatment with a combination of thyroxine, cortisone,
adrenal insufficiency and may complicate 13 percent and growth hormone corrects both the anemia and the
marrow hypoplasia88 and is more effective than any single 8. Durum SK, Schmidt JA, Oppenheim JJ. Interleukin 1:
hormone by itself. Administration of recombinant human an immunological perspective. Ann Rev Immunol.
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of the anemia. 9. Vreugdenhil G, Lowenberg B, van Ejik HG, et al. Anaemia
of chronic disease in rheumatoid arthritis: raised serum
interleukin-6 (IL-6) levels and the effects of IL-6 and anti-IL-6
Hyperparathyroidism on in vitro erythropoiesis. Rheumatol Int. 1990;10:127-30.
Anemia is a rare complication of primary hyperpara 10. Murray HW. Interferon-gamma, the activated macrophage,
and host defense against microbial challenge. Ann Intern
thyroidism.89 The anemia is normocytic and normochromic
Med. 1988;108:595-608.
and no evident reticulocytosis. Some authors conclude 11. Vilcek J, Gray PW, Rinderknecht E, et al. Interferon γ: a
that parathyroid hormone decreases proliferation of lymphokine for all seasons. Lymphokines. 1985;11:1-32.
erythroid precursors in culture. Marrow fibrosis may also 12. Balkwill F, Burke F, Talbot D, et al. Evidence for tumor
be a result of excess hormone levels. Myelofibrosis is a necrosis factor/cachectin production in cancer. Lancet.
common finding in bone marrow biopsy specimens, but 1987;2:1229-32.
the usual morphologic signs of myelophthisis are lacking.89 13. Fillet G, Cook JD, Finch CA. Storage iron kinetics. VII. A
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J Clin Invest. 1974;53:1527-33.
disease, it is difficult to ascertain the relative importance
14. Dallalio G, Fleury T, Means RT. Serum hepcidin in clinical
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C H A P T E R17
Thalassemia Syndromes
Mamta Vijay Manglani, Ambreen Pandrowala, Ratna Sharma, MR Lokeshwar
Thalassemia syndromes are a heterogeneous group of single gene disorders, inherited in an autosomal recessive manner, prevalent
in certain parts of the World posing a major health problem. In populations from the Mediterranean basin, Indian subcontinent
and South-East Asia, β-Thalassemia is the most common genetic variant associated with Thalassemia major. However population
migration these days has, no longer, restricted the gene frequency to above tropical areas in which it was first observed, and hence
it is seen all over the world.1- 4
HISTORICAL REVIEW1-6
Thalassemia was first described by Cooley and Lee in
1925,1 (Fig. 1) cases of severe anemia occurring in Italian
children with hepatosplenomegaly, growth retardation,
discoloration of skin and sclera, with peculiar bony
changes, during the Transactions of “American Pediatric
Society”. It was then called as Cooley’s anemia.1
The term thalassemia was first used in 1932, by
Whipple and Bradford.2 The word was taken from the
Greek language which means great sea (anemia around
the sea). As it was first described around Mediterranean
countries. It was also called as Mediterranean anemia.1,2
However, it was soon realized that it also occurs in South-
East Asia, Indian subcontinent and Middle-East and not
only around Mediterranean regions. The first case from
India was reported by Dr M Mukherjee5 from Campbell Fig. 1 Dr Thomas Cooley
Medical School Calcutta India in 1938.4 Dr PK Sukumaran5
from Mumbai did pioneering work in the field of diagnosis
of thalassemia syndromes in India.
Currently, as the life span of affected patients has been EPIDEMIOLOGY3,7-17
considerably prolonged by improvement in supportive
care, the age distribution of patient population has dra-
Thalassemia Incidence: World Scenario
matically altered. Furthermore, in many countries, the All over the world there are more than 250 million (1.5% of
use of DNA based prenatal diagnosis has substantially world population) carriers of b-thalassemia gene, and in
reduced number of births of affected individuals, accentu- South-East Asia, there are 40 million carriers of this gene
ating the trend of increasing age of thalassemia patients. (50% of these are in India alone, i.e. 20 million).1-4
Thalassemia syndrome: Indian scenario

The mean prevalence of the carrier status in India is 3.3%
(ranging from 1% to 17% in various communities). If you draw
a line between Mumbai (Bombay) and Kolkata (Calcutta) on
the map of India, the region above the line has an incidence of
3–17 %, where as the region below the line has an incidence of
less than 3%.
Thalassemia is more common in the following communities

North India Sindhis, Punjabis, Khatris, Kukrejas, migrants
from Pakistan
Gujarat Bhanushalis, Kutchis, Lohanas Mahars,
Maharashtra Chamars, Buddhas, Navabuddhas, Kolis,
Agris, Kunbis
Fig. 2 Thalassemia Belt—World Scenario
Andhra Pradesh Reddys, Gowdas, Lingayats, Kurgs
and Karnataka
Goa Goud Saraswats
Flow chart 1 Pathophysiology of β-thalassemia
Other Certain Muslim and Christian communities
It is estimated that every year approximately 100,000

children with thalassemia major are born world over.
With the birth rate of 22.8 per 1000 in India, it
is estimated that there are about 65,000 to 67,000
b-thalassemia patients in our country and about 9,000 to
10,000 cases being added every year.
The prevalence of thalassemia varies in different
communities, religions and ethnics groups.
The thalassemia belt stretches across the African
continent, the Mediterranean regions, Middle-east,
Indian subcontinent, South-east Asia, Thailand, Cambo-
dia, Laos, Vietnam, Malaysia, Singapore, Southern China,
and Melanesia1 which is the same as malaria belt. The
observation that the prevalence of thalassemia and falci-
parum malaria was similar, suggested the hypothesis that
nature developed genetic mutation to overcome mortality
and morbidity of malaria (Fig .2).
PATHOPHYSIOLOGY (FLOW CHART 1)18-21

Normally hemoglobin consists of two pairs of amino acid
chains:
• Adult hemoglobin HbA consists of two pairs of
α-chains and β-chains each. of production of one or more of the globin polypeptide
• HbA2 consists of two pairs of α-chains and δ-chains. chains of the hemoglobin molecule leading to imbalance
• HbF fetal hemoglobin is constituted by two pairs of in α and non-α chains of hemoglobin.
α-chains and γ-chains. In β-thalassemia, decreased (β+) or absent (β0)
Thalassemia is an inherited disorder of hemoglobin. chains lead to excess of unpaired α chains, that have no
Thalassemia syndromes refer to a group of blood complementary non-α-chains with which to pair, form
diseases characterized by defects in the synthesis of one insoluble inclusions that precipitate on red cell membrane
or more of the globin chains that form the hemoglobin to form insoluble inclusion bodies, leading to damaged
tetramer. This results in reduced or complete absence cell membranes, perturbed internal ionic environment of
Chapter-17 Thalassemia Syndromes 165
the cells, decreased red cell deformability and interference a-thalassemia trait, HbH disease and hydrops fetalis due
with egress from the bone marrow spaces. This leads to to 1,2,3 and 4 gene defects respectively.
premature destruction of RBCs in bone marrow (Ineffective The b-globin genes are represented one on each
erythropoiesis) and in peripheral circulation, particularly chromosome 11.
in reticuloendothelial system of spleen (Extravascular The b-thalassemias also include four clinical synd
hemolysis) resulting in progressive anemia. romes of increasing severity:
To compensate for the reduced hemoglobin, the 1. Silent carrier
synthesis of gamma chains persist after fetal life even 2. Thalassemia trait
beyond 6 months of age. The normal switch mechanism 3. Thalassemia intermedia
leading to reduction in β-chain synthesis does not occur. 4. Thalassemia major (Table 3)
This leads to higher fetal hemoglobin α2γ2(HbF) in The former two result from a single gene defect
postnatal life. (heterozygous) whereas the latter two from both genes
Increased fetal hemoglobin (HbF) with its high affinity affected (homozygous).
for oxygen leads to tissue hypoxia, which in turn stimulates
erythropoietin secretion leading to both medullary and Molecular Genetics
extramedullary erythropoiesis (expansion of bone marrow (Figs 3 and 4 and Table 4) 5,24-43
space) causing characteristic hemolytic faces with fronto-
parietal and occipital bossing, malar prominence and More than 200 mutations have been described that are
malocclusion of teeth and complications that include responsible for thalassemia. The β-globin genes are
distortion of ribs and vertebrae and pathological fractures clustered on chromosome 11 and are arranged over
of the long bones, splenomegaly and its complication— approximately 60,000 nucleotide bases (Fig. 3). These
hypersplenism, hepatomegaly, gallstones and chronic leg mutations occur in both introns and exons, and outside the
ulcers, etc. coding regions of the genes. Most types of β-thalassemia
The precise mechanism controlling the switch from are due to point mutation affecting the globin gene but
fetal to adult hemoglobin (α2b2) is not fully understood. some large deletions are also known. However, within
each geographical region, few common mutations are
CLASSIFICATION3,22,23 responsible for over 90 percent of β-thalassemia.
Thalassemias are classified depending on, which globin Table 1 Classification based on the chain affected
chain is defective—a(alpha), b(beta), g(gamma), d(delta),
• a-thalassemia • a-chain is affected
etc.
• b-thalassemia • b-chain is affected
Classification of α-thalassemia (Tables 1 and 2) • b0-thalassemia • b-chain is absent
• b + thalassemia • If b-chain is partially present
The gene for a-globin chains is duplicated on chromosome
16 with each diploid human cell containing four copies • D
ouble heterozygous • S ickle cell thalassemia, HbE
of the a-globin gene. The four a-thalassemia syndromes states thalassemia, HbD thalassemia,
with increasing clinical severity include silent carrier, etc.
Table 2 Classification of a-thalassemia syndromes18,22,23

Syndrome Clinical features Hemoglobin pattern α-globin genes affected
Silent carrier No anemia, normal red cells 1–2% Hb Bart’s (γ ) at birth
4
1
a-thalassemia trait Mild anemia, hypochromic 5–10% Hb Bart’s (γ4) at birth 2
microcytic red cells
HbH disease Moderate anemia, 5–30% HbH (β4) 3
hypochromic, microcytic, red
cells
Hydrops fetalis Death in utero caused by Mainly Hb Bart’s, small 4
severe anemia amounts of HbH
Table 3 Classification of b-thalassemia18,22,23

Syndrome Clinical features Hb pattern β-globin genes Inheritance
Silent carrier No anemia, normal, asymptomatic Normal 1 Heterozygous state
Thalassemia trait Mild anemia, hypochromic, microcytic red cells Elevated HbA2 1 Heterozygous state
Thalassemia Moderate anemia, requires some transfusion HbF elevated 2 Homozygous state/
intermedia occasionally and not dependent on blood Double heterozygous
transfusion for their survival
Thalassemia Severe anemia, dependent on regular blood HbF elevated 2 Homozygous state
major transfusion for survival
Table 4 Various mutations have been found in the Indian

population with β-thalassemia
5 most common mutations in Newer mutations
Indian population
1. 619 bp deletion • Codon 15 (TGG—TAG)
2. IVS 1-5(G-C) • C
odon 4/5 and 6 (ACT CCT
GAG—ACA TCTTAG)
3. IVS 1-1(G-T) • Codon 47/48 (+ATCT)
4. FS 8/9 (+G) and • Codon 55 (+A)
5. FS 41/42 (-CTTT) • IVS 2-837 (T to G)
• Codon 88 (+T)
Fig. 3 The globin gene cluster on short arm
• Codon 5 (-CT)
• IVS 1-110 (G-A)
Clinical Heterogenecity of Thalassemia due to

Diversity of Mutations
In populations in which thalassemia is prevalent, different
types of mutations, affecting genes of either a- or b- or
both globin clusters, may co-exist. Frequently, patients are
compound heterozygotes for these mutations. The relative
degree to which globin chain synthesis is impaired reflects
Fig. 4 Timing and normal development switching of this genetic heterogeneity and determines the clinical
chromosome human hemoglobin phenotype.
Mild b-thalassemia
Inheritance of Genes (Figs 5A and B) • Silent carrier: Silent carriers of b-thalassemia have
• Inherited by autosomal recessive pattern normal levels of HbA2. However, they often reveal mild
• If the child inherits one normal and one abnormal gene microcytosis, or a slight impairment in the b-globin
from each parent, child will have no disease (carrier/ synthesis in the peripheral blood reticulocytes.
minor) Several patients who are homozygous for the silent
• If both parents are carriers, i.e. thalassemia minor carrier b-thalassemia gene have been described.
(single gene affected), there is a 1 in 4 (25%) chance of These children rarely require transfusions. They have
having a thalassemia major child in each pregnancy. significant hepatosplenomegaly and have HbF of 10
This is depicted in Figures 5A and B. to 15 percent with an elevated HbA2 (as seen in traits).
A B
Figs 5A and B Inheritance of thalassemia
Two point mutations in the b-globin gene regions have Severe b-thalassemia
been linked to the silent carrier phenotype.
• The -101 promoter mutations seen in Italians, Thalassemia major suggests severe homozygous
b-thalassemia requiring regular transfusion to sustain life.
Bulgarians and Turkish population.
Patients who are homozygous for b-thalassemia mutations,
• +1 Cap site Inv mutation in an Indian family.
based on family studies, but maintain a hemoglobin
b-thalassemia trait: Individuals with thalassemia trait
concentration of 6 to 10 g% without transfusions are termed
are heterozygous for b-thalassemia, i.e. expression of
as thalassemia intermedia.
one b gene is impaired by mutation, whereas that of
Various factors affect the severity of b-thalassemia.
the other gene is normal. Individuals with thalassemia
But the most important factor is the imbalance between
trait exhibit mild microcytic, hypochromic anemia
a- and total non-a-globin synthesis.
with basophilic stippling, target cells and elliptical cells Four factors determine this ratio:
on peripheral blood smear. Characteristically, HbA2 1. The mutations (b0 or b+) in both globin genes.
and/or HbF are elevated in b-thalassemia trait, though 2. Abnormalities in the a-globin gene cluster that
various combinations may result depending upon the increase or decrease a-globin gene expression.
genetic mutation. 3. The genetic capacity to produce HbF.
• High HbA2 b-thalassemia trait: This is the most 4. Co-inheritance of Xmn1 polymorphism.
common form of b-thalassemia trait. HbA2 levels • Dominant b-thalassemia: Rarely, heterozygote state
vary from 3.5 to 8.0 percent whereas HbF from 1 to 5 of b-thalassemia may be associated with severe
percent.27 transfusion-dependent disease. Mutations involving
• Thalassemia trait: Individuals heterozygous for these the Exon 3 of the b-globin gene have been associated
mutations have increased HbF levels (5–15%) and low with such thalassemia.
HbA2 levels. • Severe b-thalassemia trait: Increased production
• High HbA2 high HbF b-thalassemia: This form of of a-chains may lead to severe expression of
thalassemia trait is associated with deletions of the b-thalassemia trait due to increase in unbalanced
globin gene that leave the d- and b-goblin genes intact. a-chains. Co-inheritance of triplicated a-globin gene
• Normal HbA2 b-thalassemia trait: This should be chromosomes may result in severe b-thalassemia trait
distinguished from the silent carrier state. Both have manifesting like thalassemia intermedia.25
normal HbA2, however, individuals with normal • Increased HbF synthesis: b-globin gene deletions
A2 b-thalassemia trait have red cells which are with mutations in g-globin promoters are associated
hypochromic, microcytic in contrast to near normal with increased HbF production and are shown to
red cells in a silent carrier.25 ameliorate the clinical course of thalassemia. This has
led to pharmacologic manipulation of HbF production remain undiagnosed and are not given red blood cell
to manage thalassemia patients. transfusions. Whereas at the other end of the spectrum
is a heterozygous form (Thalassemia minor) in which the
patient can lead a practically normal life except for a mild
Management of Thalassemia: Principles persistent anemia not responding to hematinics. They
of Therapy have a normal life span.
• Confirmation of diagnosis
In between these two extremes are forms with varying
• Transfusion therapy—packed red cell transfusions degrees of clinical manifestations of anemia, spleno
• Management of complications hepatomegaly and bony changes who maintain their
• Transfusion related complications life fairly comfortably and are not dependent on blood
• Iron overload: Removal of iron with iron chelating agents transfusions for their survival and are called thalassemia-
• Hypersplenism: Role of splenectomy intermedia (they are also homozygous).
• Transfusion transmitted infections: Hepatitis B and C HIV, Untreated or irregularly treated children develop signi
Yersinia spp, malaria, CMV ficant hemolytic faces including frontoparietal bossing
• Gallstones and leg ulcers with a hot-cross-bun appearance of the skull (caput
• Curative treatment: Bone marrow transplantation quadratum with “hair-on-end” appearance on X-ray
• Future treatment: Pharmacologic manipulation of HbF/Gene
skull), depressed bridge of nose, malar prominences and
therapy
• Prevention of the disease by antenatal diagnosis and
malocclusion of teeth with protrusion and malocclusion
genetic counseling of maxillary teeth.
LABORATORY DIAGNOSIS OF
Confirmation of Diagnosis b-THALASSEMIA SYNDROMES (TABLE 5)
Clinical manifestations of β-thalassemia:1-4,6,7,9,11-17 For a reliable diagnosis of thalassemia, it is advisable to
• Children with thalassemia major are generally correlate clinical profile and ethnicity of the individual
diagnosed between 6 and 18 months of life. In with various laboratory investigations to confirm the
India, many children born with thalassemia major diagnosis. Hemoglobin electrophoresis is the confirmatory
die undiagnosed due to lack of facilities or ideal test for diagnosis of most cases of thalassemia syndromes.
treatment. The spectrum of clinical manifestation of However, a complete blood count and examination of the
β-thalassemia varies widely. Severe b-thalassemia peripheral smear provide very vital information and are an
usually becomes manifest in the first year of life when important primary screen in thalassemia syndromes.
the fetal hemoglobin (a2,g2) starts declining and adult
stable Hb cannot be formed. Thalassemia Major/Intermedia (Figs 7A and B):
The clinical syndromes associated with thalassemia,
arise from the combined consequences of:
Complete Blood Count
• Inadequate hemoglobin production Complete blood count (CBC), examination of PBF and a
• Unbalanced accumulation of one type of globin chain. reticulocyte count can help in suspecting and identification
The former causes anemia with hypochromia and of hemoglobinopathies. CBC reveals generally severe
microcytosis whereas the latter leads to ineffective anemia, a high leukocyte count (due to immature myeloid
erythropoiesis and hemolysis. cells as well as nucleated red cells—also known as a
The high affinity of HbF for oxygen leads to tissue “leukoerythroblastic reaction”). WBC and platelets may
hypoxia, which in turn stimulates erythropoietin secretion decrease if there is accompanying hypersplenism.
leading to both characteristic hemolytic facies—with The red cell indices reveal a severe hypochromia
frontoparietal and occipital bossing, malar prominence with microcytosis. Patients in whom diagnosis is delayed
and malocclusion of teeth. there is significant macrocytosis due to relative folate
At one end of the spectrum is the serious homozygous depletion.
form (Thalassemia major) that presents in early infancy The red cell distribution width (RDW) in thalassemia
(6–18 months) with progressive pallor, failure to thrive, major is significantly high (ranging from 30 to 40% for a
irritability, intercurrent infections and bony changes normal value of 12–16%), suggesting a very high degree of
and hepatosplenomegaly. Ninety percent thalassemia anisocytosis. The peripheral smear shows a striking and
children do not survive beyond 3 to 5 years of age if they characteristic bizarre picture with hypochromic, microcytic
Table 5 Clinical and hematological features of the b-thalassemia syndromes1-4,6,7,9,11-17

Major Intermedia Minor
Severity of manifestations ++++ Homozygotes, double ++
genetics Homozygotes, double heterozygotes heterozygotes, rarely heterozygotes Heterozygotes
Splenomegaly ++++ ++, +++ +, 0
Jaundice ++ ++, + 0
Skeletal changes ++++, ++ +,0 +,0
Anemia (Hb, g/dL) <7 7–10 >10
Hypochromia ++++ +++ ++
Microcytosis +++ ++ +
Target cells 10–35% ++ +
Basophilic stippling ++ + +
Reticulocytes (%) 5–15 3–10 2–5
Nucleated red cells +++ +, 0 +
A B
Figs 6A and B Peripheral smear showing (A) Reticulocyte; (B) Target cell and normoblast
as well as macrocytic red cells, anisopoikilocytosis, target intermedia, it is increased to 3 to 6 percent. The reason
cells, polychromasia (more common in thalassemia for a low reticulocyte count in thalassemia major is
intermedia), basophilic stippling, nucleated red cells and significant ineffective erythropoiesis preventing the
sometimes immature myeloid cells (Figs 6A and B) . precursor red cells from maturing to reticulocyte stage
to be thrown into peripheral blood. In thalassemia
Thalassemia Minor intermedia, since the ineffective erythropoiesis is milder,
the reticulocytes are increased in peripheral blood due to
The CBC in thalassemia trait is associated with high red the anemia.
cell count relative to hemoglobin concentration and
hematocrit, resulting in a marked fall in mean cell volume
(MCV), mean cell hemoglobin (MCH) as well as mean cell Naked-eye Single Tube Red Cell Osmotic
hemoglobin concentration (MCHC). RDW is normal in Fragility Test (NESTROFT) for Thalassemia
thalassemia trait. Minor44-46
Reticulocyte count: Reticulocyte count is generally low Many investigators have studied naked-eye single tube red
to normal in thalassemia major, whereas in thalassemia cell osmotic fragility test. The test has a high sensitivity of
In children with thalassemia major, the values of ferritin are

proportionate to iron overload due to multiple transfusions.
Serum ferritin estimation is used as the most common test
for diagnosing iron overload. However, ferritin can be
affected by various clinical situations—it may be elevated
in acute infections as an acute phase reactant, in chronic
diseases and chronic inflammatory disorders, etc.
Quantitation of Various Hemoglobins (Figs 8 and 9)

Separation of hemoglobins either by electrophoretic mobility
or chromatographic separation is the confirmatory investi
gation for diagnosis of thalassemia syndromes.
A Quantitation of various hemoglobins can be done by the

following methods
• Isoelectric focussing
• Microcolumn chromatography
• High performance liquid chromatography
• Both anion and cation—exchange HPLC
• Cellulous acetate electrophoresis (Fig. 8A)
• Paper electrophoresis (Fig. 8B)
• In untransfused patients with thalassemia major, hemo
globin pattern reveals 20–100% HbF, 2–7% HbA2 and 0–80%
HbA, the quantities varying depending upon the genotype.
Thalassemia minor is characterized by elevated HbA2 of
more than 3.4% on paper electrophoresis, and more than
4% by certain other method.
B • Microcolumn chromatography and HPLC,48,49 by

auto mated machines (Fig. 9A) are now becoming
Figs 7A and B (A) Thalassemia major on irregular treatment; increasingly popular due to the ease of performing the
(B) Thalassemia intermedia test, less time consumption and greater reliability and
reproducibility. It has thus become the gold standard
for diagnosis of thalassemia syndromes and other
95 percent, but its poor precision, interobserver variability hemoglobinopathies. It generates graphs depicting
and low specificity has precluded it from becoming a various abnormal and normal hemoglobins with
robust test. quantification. HbA2 value of > 9 percent indicates
the presence of a co-eluting abnormal hemoglobin
Iron Studies47 such as Hb E, Hb D Iran and Hb Lepore. Microcolumn
chromatography and HPLC are currently used in most
Serum iron and transferrin saturation would be normal laboratories. Hemoglobins are separated graphically
to increased (increased especially in multiply transfused and quantified by photometer utilizing sophisticated
children) in thalassemia major, whereas the total iron computer (Fig. 9B).
binding capacity (TIBC) would be decreased. It is generally Radiological findings include widening of medulla due
normal to high even in thalassemia minor. Though iron to bone marrow hyperplasia, thinning of the cortex and
deficiency is extremely uncommon in thalassemia minor, trabeculations and fracture are seen in long bones, meta-
in our country, due to a high incidence of IDA, concomitant carpals and metatarsals. X-ray—skull AP and lateral views
iron deficiency may be present in children with thalassemia show “hair on end”appearance (Figs 10A to C).
minor. In such patients, serum iron level would be low with
reduced transferring saturation and a high TIBC.
Tests Used for Estimating Iron Overload50-53
Serum ferritin is high in children with thalassemia major
and normal to increased/decreased when concomitant • Liver iron concentration (LIC): This is done on a liver
iron deficiency associated in those with thalassemia minor. biopsy tissue collected on a filter paper. Any level above
A B
Figs 8A and B (A) Cellulose acetate electrophoresis; (B) Paper electrophoresis
A B
Figs 9A and B (A) BIO RAD variant machine; (B) Pattern of hemoglobin in thalassemia minor/trait
A B C
Figs 10A to C (A and B) Thalassemia major X-ray skull; (C) Widening of medulla, thinning of the cortex and trabeculations,
and fracture in the long bones
7 mg/g of dry liver tissue is indicative of significant liver iron and has proven to be more accurate than se-
iron overload. Levels above 15 mg/g is associated with rum ferritin in quantifying the total body iron overload.
cardiac iron overload. • T2 weighted cardiac magnetic resonance imaging56-60
• Superconducting quantum interference device helps in accurately determining the cardiac iron
(SQUID)54,55 is a non-invasive method of estimating overload.
• Bone marrow examination not indicated for the be done in all patients at regular intervals to detect these
diagnosis of thalassemia major but if done, it shows disorders and treat them appropriately.
normoblastic erythroid hyperplasia with excessive • Diabetes may be seen as early as 5 years of age
iron on iron staining with Prussian blue. • Short stature is commonly noted at 10 to11 years of age.
Periodic tests for organ dysfunctions. While diagnosis A glucose tolerance test, thyroid functions, serum
of thalassemia major can be achieved by the tests, it calcium and phosphorus should be monitored every
is also mandatory to do baseline screening of various year, beginning at the age of 5 years and evaluation of
parameters: height velocity, 10 years age onwards, goes a long way
Liver function tests (LFT) especially serum bilirubin, in early detection and treatment of these complications.
SGOT, SGPT, GGT, GTC (glucose tolerance curve). Management would depend upon the specific abnormality
• Baseline HBsAg, HIV and HCV antibody test found.
• Hormonal assays as and when required
• DEXA scan for density of the bones.
BONE DISEASE: OSTEOPENIA
Clinical Consequences, Diagnosis AND OSTEOPOROSIS (FIG. 11)65-68
and Management Osteopenia and osteoporosis are major causes of morbidity
in the aging thalassemic population, and it is suggested
Most of the complications of b-thalassemia are attributable that prevalence is higher in men than in women. It is more
to iron overload. Excess iron is toxic to the heart, liver, and severe in the spine than in the femoral neck.
various endocrine glands. In b-thalassemia, 70 percent of Osteoporosis as defined by WHO as a “progressive
deaths are due to cardiac complications. Growth failure systemic skeletal disease characterized by low bone mass
is seen in 30 percent of patients in the Western world and and microarchitectural deterioration of bone tissue, with
nearly all patients in our country, and is due to various consequent increase in bone fragility and susceptibility to
factors including endocrine dysfunction. The other fracture”.
factors responsible for growth failure include: anemia, • DEXA SCAN (Fig. 11) for assessing bone mineral
hypersplenism, desferrioxamine, and liver disease. density (BMD)—helps in identifying various bones
with osteopenia and osteoporosis.
ENDOCRINE DYSFUNCTION61-64 • Biochemical evaluation like serum calcium, serum
The commonly affected endocrine glands include pituitary phosphorus, serum alkaline phosphatase—help in
gland, thyroid gland, parathyroid gland, pancreas, gonads. identifying vitamin D deficiency as well as hypo
Clinically, they may remain latent. Investigations should calcemia, which may be due to hypoparathyroidism
Fig. 11 DEXA scan in a child with thalassemia
in addition to calcium deficiency related to vitamin D In a study done at LTMG Hospital in 28 children with
deficiency. thalassemia major above the age of 10 years, 3 children
Urinary calcium/Creatinine ratio >0.2 as well as urinary had evidence of overt cardiomyopathy. Abnormalities
Phosphorus/Creatinine ratio >0.6 are useful screening such as left atrial dilatation, aortic root dilatation, reduced
parameters for suspecting osteopenia and osteoporosis. left ventricular internal dimension in systole and diastole
Genetic factors also play a role in bone mineralization, were seen in 20 (71.42%), 11 (39.28%), 6 (21.42%) and 10
regulated by estrogen receptor gene, vitamin D receptor (35.7%) children respectively.
(VDR) gene, COLIA1 and COLIA2 genes. Furthermore, Ejection fraction was decreased in 16 out of the 28
TGF-b1 has also been implicated as a possible mediator (57.14%) patients. Of these, 33.3 percent were chelated
of coupling between bone resorption and formation. adequately using deferiprone and 84.6 percent were
Testosterone, estrogen and progesterone are involved in unchelated. Four (14.28%) of the 28 children had decrease
the regulation of bone mineralization and contribute to in fractional shortening.
the osteopenia and osteoporosis in thalassemic children. Kaya SB et al.80 also reported 3 out of 28 patients between
Though studies have demonstrated iron deposition the age group of 7 to 23 years with abnormalities on ECG.51-53
along the mineralizing perimeter of the bone, there are Since cardiac function has strong correlation with
other factors influencing, such as anemia, which affects chelation early institution of appropriate and regular
erythroid activity. chelation and regular transfusion therapy to maintain pre-
transfusion Hb above 10 gm% will lead to better cardiac
function in these children. Early detection of cardiac
Cardiac Complications69-71 involvement can be done by evaluation of ferritine level
Most of the complications of β-thalassemia are attributable regularly and then doing the various tests to evaluate the
to iron overload. Excess iron is toxic to the heart, liver, cardiac functions like ECG, 2D echocardiogram, stress
and various endocrine glands. Iron overload causes test, etc. However, these tests do not quantitate the cardiac
deposition of iron in the ventricular walls, mainly in the function. T2 cardiac MRI is the only method of accessing
left, relatively sparing the atria and the conduction system. accurately the severity of cardiac iron overloading.
In the ventricular wall, the epicardium contains most of
the iron, the endocardium moderate with least iron in the
Management of Thalassemia Major72-76
intermediate layer. When iron accumulation increases, The management of thalassemia has undergone tremen
free iron damages the cells of the layers of the heart, dous changes over the last 3 decades.
inducing lipid peroxidation and lysosomal rupture.When • If untreated, patients of thalassemia major die by the
iron accumulates in the cardiac tissue, free iron damages age of 3 to 4 years due to severe anemia.
the cells of the layers of the heart, due to lipid peroxidation • With transfusion therapy alone, patients with thalas
and lysosomal rupture. Cardiac failure and ventricular semia major children died due to cardiac compli
arrhythmias are the main cause of death in patients with cations (related to iron overload) as early as 10 to 12
b-thalassemia major and account for 70 percent of the years of age. However, with the advent of chelation
deaths. therapy as well as better screening procedures for
Cardiac iron overload related heart disease may be transfusion related infections as well as leukodepletion
divided into three stages: through prestorage/bedside filtration, the lifespan
1. Preclinical of these children have improved considerably. If no
2. Early clinical complications occur, they live for an almost normal
3. Advanced disease. span with an improved quality of life.
Early detection of cardiac involvement can be done Management of thalassemia involves a multidiscipli
by evaluation of serum ferritin level regularly and then nary therapeutic team approach and should be preferably
doing the various tests to evaluate the cardiac functions done at a comprehensive thalassemia children care center
like ECG, 2D echocardiogram, stress test, etc. However, with outdoor transfusion facilities (Table 6).
these tests do not quantitate the cardiac function. T2
weighted cardiac MRI weighted cardiac MRI is the only
Transfusion Therapy in Thalassemia77-79
method of assessing accurately the severity of cardiac iron Packed red cell transfusions remain the cornerstone of
overloading. In acute cardiac failure and arrhythmias, therapy in thalassemia major. The decision to initiate
continuous subcutaneous desferrioxamine can reverse lifelong regular transfusions in patients with β-thalassemia
the ventricular dysfunction, whereas intravenous should be based on the molecular defect, severity of
desferrioxamine can improve complicated arrhythmias symptoms, and clinical criteria such as failure of growth,
and progressive heart failure. development and bone changes.
Table 6 Thalassemia outdoor center without transfusions. If the hemoglobin drops to below
7 gm% without transfusion, in absence of any concurrent
Team Members Goals of Transfusion
illness, it is imperative to put the child on a regular
• Pediatric hematologist • To obviate anemia transfusion program. If the child maintains hemoglobin
• Pediatrician • To reduce above 7 gm%, the diagnosis of Thalassemia Intermedia
• Dedicated nurses hepatosplenomegaly
has to be considered.
• Transfusion medicine by reducing ineffective
Whenever possible, it is equally important to know
specialist erythropoiesis
• Physiotherapist • To reduce hemolytic faces the compete genotype of the red cells to prevent red
• Endocrinologist • To improve tissue cell alloimmunization following repeated transfusions.
• Psychologist and social oxygenation However, this is not feasible in India and the alternative
worker • To improve growth to this is Coomb’s cross-match for each transfusion to
prevent alloimmunization.
Progress in the Concept of Transfusion TYPE OF TRANSFUSIONS (FIGS 12 AND 13

Therapy for Thalassemia AND TABLE 7)
The most ideal way to transfuse thalassemics is using group
• In the 1960s, Wolman et al. proposed a palliative
and type specific packed red cells that are compatible
transfusion therapy77 for children with thalassemia
major. It was aimed at maintaining the hemoglobin at
the level of 8.5 gm%. This led to improved survival, but
the chronic illness, bone disease and cardiomyopathy
persisted.
• To overcome these problems, Piomelli and workers
suggested maintaining the hemoglobin.78 A above
minimum of 10 gm%. These vigorous regimens were
termed as hypertransfusion, although Normo-trans
fusion may be a more descriptive term. Hypertransfusion
promotes normal growth and development, prevents
the onset of severe hepatosplenomegaly and hemolytic
facies, lowers the absorption of gastrointestinal iron
and reduces the anemic cardiomyopathy changes.
• In 1980, Propper and colleagues introduced a further
improvised regimen79 called supertransfusion, and
main tained a pretransfusion hemoglobin of above
12 gm%. However this did not prove significantly Fig. 12 Cold centrifuge
superior to hypertransfusions and was given up.
Hypertransfusion remains the most accepted regimen
in most parts of the world. However, in Europe, a yet
newer regimen termed the “moderate transfusion
regimen” has been adopted and recommended by the
Thalassemia International Federation. In this regimen,
pretransfusion hemoglobin is maintained between
9 and 10.5 gm%.
INITIATION OF TRANSFUSION THERAPY

Before embarking on a lifelong transfusion therapy,
it is essential to establish the diagnosis firmly with
DNA analysis. This would help to know the severity of
thalassemia as well as would help in prenatal diagnosis for
future pregnancies.
One can ascertain the diagnosis of thalassemia
Intermedia by observing the rate of fall of hemoglobin Fig. 13 Leukodepleting filters at bedside
Table 7 Various transfusion regimens (Progress in transfusion therapy)

Year of regimen Transfusion regimen Pretransfusion Hb
1960s Palliative Hb to 8.5 gm% (Wolman et al.)
1970s Hypertransfusion Hb 10–12 gm% (Piomelli and workers)
1980 Supertransfusion Hb > 14 gm% (Propper and colleagues)
2005 Moderate transfusion Hb 9–10.5 gm% (European regimen)
by direct antiglobulin test. The hematocrit should be Advances in Transfusion Therapy

standardized to 65 to 75 percent. This maintains the desired
viscosity as well as aids in calculating the yearly requirement Daycare Transfusion Center
in a given patient. It is ideal to use prestorage leukodepleted • Transfusions are given on an outdoor basis and hence
blood, however, the next best is to use leukodepleting filters no hospitalization is required.
at bedside but they are also costly. An alternative to this is • Children are more comfortable with the familiar staff
use of triple saline washed red cells. The red cells should be members.
fresh, not more than 4 to 5 days old to maintain adequate • There is less school absenteeism and parents lose
levels of 2,3-DPG. Various other methods of leukodepletion fewer work days.
are available, including use of frozen red cells (highly • The cost of the hospital stay is almost 5 times less.
expensive and impractical in developing countries), filtra • There is no threat of contacting infections from other
tion in the blood bank, use of apheresis, etc. patients in the wards.
Neocyte transfusions to improve the survival of red cells • Parents and children are happy to be with other
after transfusion, have been tried but with limited success. children and parents and discuss common issues of
thalassemia management.
Amount and Rate of Transfusions
Approximately 180 mL/kg of red cells are required to be Management of Complications of
transfused per year in nonsplenectomized, nonsensi Transfusion Therapy
tized patients to maintain the hemoglobin above 10 gm%,
A major problem encountered in the management of
whereas splenectomized patients require 133 mL/kg per
thalassemia is iron overload. Regular red cell transfusions
year. Even without hypersplenism, the requirement is 30
to maintain hemoglobin and increased iron absorption
percent higher in nonsplenectomized patients (Table 7).
from GI tract due to ineffective erythropoiesis and conse
quent low hemoglobin in irregularly transfused children is
Rate of Transfusion responsible for iron overload.
These red cells should be transfused 10 to15 mL/kg at the
rate of 3 to 4 mL/kg/hour every 2 to 4 weeks to maintain Transfusion Related Complications
the hemoglobin. Patients with cardiac decompensation
should be given red cells at the rate of not more than 1 to • Iron overload
2 mL/kg/hour. • Transfusion transmitted infections.
Adequacy of Transfusions Iron Overload and Chelation Therapy

It is important to check the adequacy of transfusions to Clinical Consequences, Diagnosis and Management
achieve best results and manage thalassemics ideally.
• In the first decade of life, normal growth confirms Most of the complications of b-thalassemia are attributa-
adequate transfusions. ble to iron overload. Excess iron is toxic to the heart, liver,
• Also the number of normoblasts should be <5/100 and various endocrine glands. In b-thalassemia, 70 percent
WbCS in well-transfused children. This may be an of deaths are due to cardiac complications. Growth failure
indicator in older children. However, this is not is seen in 30 percent of patients in the western world and
applicable to those children who have not been nearly all patients in our country, and is due to various
initiated on therapy early and adequately in early life. factors including endocrine dysfunction.
Iron Chelation Therapy80-112 complications related to iron overload and may

require to be given high dose intravenous desferal,
The aims of chelation therapy are to reduce the serum through the port or central line. This again is beyond
ferritin to below 1000 ng/mL and hepatic iron concentra the reach of many children in developing countries.
tion below 268 mmol/g of dry weight of liver. In addition, The dose is 20 to 40 mg/kg/day given subcutaneously
iron chelation also promotes growth and gonadal function. over 8 to 10 hours for 6 nights a week with the help of
Established iron induced dysfunction of heart and liver subcutaneous desferal infusion pump. In recent times,
may improve with effective chelation therapy, however, methods of administration have improved with better,
pituitary failure does not reverse. Improvement in thyroid more convenient smaller, lighter infusion pumps with
and glucose tolerance is seen. LCD display. Balloon pumps, pre-filled syringes of
Chelation therapy should be started when: desferal are available though they are prohibitively
• Serum ferritin level > 1000 ng/mL or above. costly.
• More than 10 transfusions Sixty percent of DFO chelated iron is excreted in
• Hepatic iron concentration > 3.2 mg/g dry weight. urine and 40 percent in stool.
The standard available chelators used are: Adverse effects include local reactions, auditory
• Desferrioxamine (Desferal, DFO) (Figs 14A and and visual toxicity, growth retardation, severe Yersinia
B): This was introduced in the early 1960s,82-90 but spp. infections, etc. Rarely, pulmonary infiltrates and
its impact on the survival curves in thalassemic renal toxicity has been encountered.
patients was recognized 15 to 20 years later. This is • Intravenous desferal (Table 8): Intravenous desferal
a hexa-dentate chelator and cannot easily mobilize can be given particularly in those with very high iron
iron from intracellular compartment due to its overload through port-a-caths (central line). However,
high molecular weight. It slowly binds iron to form it is not easy to maintain the central catheter and
Ferrioxamine and does not bind with iron from infections are extremely common. However, high dose
transferrin. 1 g of desferrioxamine binds 93 mg of iron. desferal (100 mg/kg, 3–9 g/day) can be given in severe
Desferrioxamine (DFO) is the gold standard therapy hemosiderosis to prevent/reverse cardiac toxicity of
and is the most effective and safe iron chelator. Though, iron overload.
desferrioxamine is the gold standard it has not become The dose for intravenous desferrioxamine is 50
popular particularly in the developing countries and to 100 mg/kg body weight. With depot preparation
is preferred by only 10 to 15 percent of thalassemia of desferal there is slow release of desferrioxamine
patients in our country. This is mainly due to its leading to substantial plasma level of desferrioxamine
high cost and the need for continuous subcutaneous in the blood for longer period and hence urinary iron
injection over 6 to 8 hours with the desferal pump. excretion is sustained for longer period. This is more
Hence, many of the thalassemic children develop effective than the conventional subcutaneous infusion.
A B
Figs 14A and B Desferal infusion pumps
Table 8 Indication for intravenous desferrioxamine is indicated

in the following conditions
• Cardiac complications
• Very high ferritin level
• During pregnancy
• Prior to stem cell transplantation
• Rarely in patients with persistent local reaction at injection site
Toxicity of Desferrioxamine
• Minimal or no tachyphylaxis has been observed
• When given parenterally there may be liberation of
histamine leading to bradycardia, hypo/hypertension,
rigors, headache, photophobia, feeling cold and hot, Fig. 15 Slit-lamp examination of eye
etc.
• When given subcutaneously local pain, indurations,
irritability and redness may occur
• Visual abnormality may occur in 4 to 10 percent of
patients and includes decreased acuity of vi sion,
peripheral field vision defects
• Defective dark adaptation, thinning of retinal vessels,
retinal stippling, abnormal visual evoked responses
and cataract
• High frequency sensory-neural hearing loss has been
reported in 4 to 38 percent of patients
• Delayed linear growth accompanied by mild skeletal
abnormalities such as short trunk, sternal protrusion
and genu valgum.
As the auditory and visual toxicities are reversible, yearly
slit-lamp examination and audiometry are mandatory to
detect them early (Fig. 15).
Role of vitamin C: Ascorbic acid deficiency increases
insoluble iron90 (hemosiderin). Vitamin C helps in Fig. 16 Kelfer capsules
conversion of hemosiderin into ferritin, from which iron
can be chelated. High doses of vitamin C can lead to
increased free radical liberation and lipid peroxidation, and therefore, zinc supplementation is mandatory in those
resulting in tissue damage and rapid cardiac decompen receiving deferiprone.
sation and even death. Addition of 100 mg of vitamin C
daily, prior to DFO therapy increases iron excretion. Side-effects of Deferiprone
• GI symptoms like nausea and vomiting
Oral Chelator
• Pain in abdomen and diarrhea
Deferiprone (L1) (Fig. 16) or 1,2-dimethyl-3-hydroxypyri- • Arthropathy
din-4-one (Kelfer)91-98 is a water soluble, bi-dentate mol- • Neutropenia/Agranulocytosis.
ecule. It mobilizes iron from transferrin, ferritin and Patients on deferiprone should be monitored with
hemosiderin. It has been licensed for use in India since monthly CBC, LFT, RFT besides clinical monitoring for ar-
1995. It is used orally and is less expensive. The recom- thralgia/arthropathy. It should be stopped in children who
mended dose for deferiprone for effective iron chelation develop arthropathy and cytopenias. It may be restarted
is 75 to 100 mg/kg body weight/day. Deferiprone/L1 has once the counts recover. In children with arthropathy, it
a better protective effect on myocardial tissue as shown should be cautiously be restarted in a lower dose of 50 mg/
in various studies. It also chelates zinc in addition to iron kg/day. If symptoms and signs recur, it should be discon-
tinued permanently. There are some controversial reports Hydroxybenzyl-ethylenediamine-diacetic Acid103,104

of increased hepatic fibrosis due to L1.
Deferasirox99-101 or ICL670 (4- [3,5-bis (2-hydro Hydroxybenzyl-ethylenediamine-diacetic acid (HBED)
xyphenyl) -1,2,4-triazol-1-y1] benzoic acid), (Asunra, was able to clear radiolabeled iron when administered
Desirox, Deferijet, Desifer) is a new class of tri-dentate parenterally and that it remained active after oral adminis
chelators65-72 with a high specificity for iron. It is an orally tration. However, further evaluation in both iron-loaded
active chelating agent developed for the treatment of primates and humans revealed that the oral activity was
chronic iron overload. The compound is an N-substituted too small to be of value in the treatment of iron overload.
bis-hydroxyphenyl-triazole that was selected from more Recently Bergeron et al. continued the preclinical
than 700 compounds that were screened as part of a drug evaluation of the efficacy and safety of HBED monosodium
development program. Deferasirox is able to mobilize salt for the treatment of both transfusional iron overload
iron from both the hepatocellular and reticuloendothelial and of acute iron poisoning in animals. Na-HBED was
source. It has ability to prevent myocardial cell iron uptake as efficacious as DFO in iron-loaded monkeys, either as
and removes iron directly from myocardial cells, the liver a subcutaneous (SC) bolus or a 20-minute intravenous
cells, the intracellular labile iron pool and the surface of infusion (IV). Na-HBED was about twice as efficient
reticuloendothelial cells where iron is handed over to as DFO in excreting iron. Safety evaluation showed no
transferrin. Iron is excreted predominantly in the bile and systemic toxicity.
hence in the feces.
This novel agent is well tolerated, with no major safety
Pyridoxal Isonicotinoyl Hydrazone105
concerns at doses up to 80 mg/kg/day. Iron excretion Pyridoxal isonicotinoyl hydrazone (PIH) was identified as
is dose-dependent. The plasma half-life (11–19 hours) an effective iron chelator in 1979. PIH is a tridentate chelator
supports the once daily oral dosing regimen used in with a selectivity for iron comparable to that of DFO. Over
subsequent clinical studies. the years, various PIH analogs have been synthesized and
It is also approved by the US FDA for use in non- some of them, such as pyridoxal-benzoyl hydrazones, were
thalassemia related transfusional iron overload. The as much as 280 percent more effective than PIH. Studies
drug is given orally as a single dose preferably in the in iron-overloaded patients treated with 30 mg/kg/day of
morning on empty stomach. The recommended dose PIH have much less than required iron excretion. However,
varies based on the serum ferritin levels and ranges recently investigators have examined the chelating
from 20 to 40 mg/kg once daily and requires monthly potential of two additional PIH analogs, alone or in
monitoring of CBC, liver functions, renal functions, combination with DFO in hypertransfused rats. The latter
urine protein estimation and annual audiometry and studies have shown that the orally administered analogs
ophthalmic examination. are about 2 to 6 times more effective than intraperitoneally
administered DFO in mobilizing liver iron in rats.
Adverse Events
GT56-252106,107
• Include gastrointestinal disturbances causing abdo
minal pain, nausea, vomiting, diarrhea and occasio GT56-252 is a novel orally available iron chelator derived
nally constipation. from DFT that forms a 2:1 complex with Fe3+. Eighteen adult
• Headache, fever, anxiety and sleep disorders. patients with b-thalassemia received 3 to 8 mg/kg in 2 doses
• Hearing loss has been reported. with food or fasting. The compound was well tolerated,
• Nephropathy in patients with compromised renal with no related serious adverse clinical events, laboratory
function can be life-threatening. abnormalities or changes in the electrocardiogram (ECG).
Further studies are in progress to define the effect of GT56-
Newer Iron Chelators 252 on iron balance.
Desferrithiocin102 40SD02 (CHF1540)108,109

Desferrithiocin (DFT) caused kidney damage in rats on 40SD02 is a new entity synthesized by chemically attaching
prolonged oral DFT. The damage was believed to be due DFO to a modified starch polymer. The resulting high
to toxic effects of the Fe3+ complex, ferrithiocin. One of its molecular weight chelator has a prolonged half-life. A
analogs, 4-OH-desaza-desmethyl-desferrithiocin, appears Phase I study in 10 patients with thalassemia and chronic
to be less toxic while remaining biologically active as an iron overload showed that single doses of up to 600 mg/
orally administered iron chelator in animal studies. This kg of the compound were safe and well tolerated, and
analog may enter Phase I human trials shortly. stimulated a clinically significant amount of iron excretion.
Combination Therapy: Management of Cardiac Complications69-71

The Shuttle Hypothesis110,111 Investigations such as Holter monitoring, 2D-echo
Additive and synergistic effects of combination of iron cardiography and stress test assist in evaluating, but do
chelators have been explained by the shuttle hypothesis. not quantitate, the cardiac function. T2 weighted cardiac
The theory is that a bidentate (L1) or tridentate ligand with MRI58-62 is the only method of assessing accurately the
access to a variety of tissues acts as a “shuttle” to mobilize severity of cardiac iron overloading.
the iron from tissue compartments to the bloodstream, In acute cardiac failure and arrhythmias, continuous
where most exchanges with a larger hexadentate (DFO) subcutaneous desferrioxamine can reverse the ventricular
“sink”. The sink binds this iron irreversibly, promoting dysfunction, whereas intravenous desferrioxamine can
its excretion. Experiments using a DCI assay showed that improve complicated arrhythmias and progressive heart
simultaneous administration of L1 and DFO produced failure.
shuttling of iron from L1 (shuttle) to DFO (sink).110,111
Clinical studies using DFO and L1 in combination have Transfusion Transmitted Infections113-126
confirmed this hypothesis. Several other combinations
exhibiting shuttle mechanism have been tried with The common transfusion transmitted infections seen in
success—HBED and L1 as well as ICL670A and DFO (in these multiply transfused thalassemic children include
experimental cells). the following:
• Hepatitis B and C
• HIV
Management of Bone Disease in Thalassemia • Yersinia spp.
Major: Osteopenia and Osteoporosis (Intervention • Malaria
Recommended Based on BMD Result) (Table 9)65-68 • CMV.
Hormone replacement therapy with estrogen in females Hepatitis B has been reported in 5 to 30.6 percent
and hCG for males improves bone density parameters. amongst multiply transfused individuals in various
Calcitonin, an inhibitor of osteoclasts, can reduce osteo studies.
porosis and increase cortical thickness in thalassemic A study at LTMG hospital have reported hepatitis B
children. Hydroxyurea, oral biphosphonates or injectable infection—recent or past in 85 percent of cases at LTMG
pamidronate are other useful modalities. Hospital, Mumbai, Maharashtra, India.
• Stringent screening of donors and testing of all blood
bags for HIV, HBsAg and Anti-HCV has reduced the
Prevention of Osteopenia/Osteoporosis rate of these transfusion-transmitted infections.
Children with b-thalassemia should be encouraged to • Additionally, it has been possible to effectively control
indulge in moderate- and high-impact activities such hepatitis B through transfusions by vaccinating with
as walking, ballet dancing, aerobics, climbing, mild hepatitis B vaccine, all children at diagnosis, with
sports, jogging, running, etc. to prevent bone changes. double dose, intense regimen (0, 1, 2 and 12 months).
The diet should be rich in calcium and vitamin D may • Boosters may be given at 5-year intervals.
be supplemented. Hormone replacement therapy for
endocrine abnormalities needs to be administered. Hepatitis C
The prevalence of hepatitis C is quite high in thalassemics
the world over, and more so in our country. Hepatitis C has
Table 9 Management of osteopenia/osteoporosis been reported in 39 percent of transfused thalassemics
Category Treatment (before it was mandatory to screen all blood bags for
Normal Diet + exercise HCV antibodies), whereas Narang et al.115 from Delhi
Osteopenia Diet + exercise + calcium have reported it in 69 percent, on the other hand, Wonke
et al.116 reported HCV positivity in 11.1 percent and Sarin
Osteoporosis Diet + exercise + calcium +
et al.117 in 53.3 percent of cases. Agarwal et al.118 from
bisphosphonates
Mumbai, have reported hepatitis C in 16.7 percent of
Established osteoporosis Diet + exercise + calcium + cases.
bisphosphonates + treatment Treatment of these patients is difficult, as it is highly
of fractures
expensive. Interferons and Ribavirin are the commonly
used therapies. Pegylated interferons are obtained by Hypersplenism (Fig. 18)130-133

conjugating Intron A with polyethylene glycol. This pro
longs the activity of interferon allowing weekly dosing • Hypersplenism may occur due to inadequate trans
instead of the conventional thrice weekly dosing. fusions, alloimmunization, rarely autoimmune hemo
lysis complicating thalassemia major and chronic
liver disease. Splenectomy is recommended when the
Human Immunodeficiency Virus (HIV) transfusion requirement exceeds 200 to 250 mL/kg/
The occurrence of transfusion transmitted HIV has ranged year of packed red cells.
from 0 percent by Chaudhary et al.113,114 to 70 percent by • Splenectomy should be deferred till 5 years of age.
Currimbhoy et al. It has been reported in 10 percent of • Following vaccines should be given 3 to 6 weeks prior
children in one study by Manglani and Lokeshwar24 from to procedure
LTMG Hospital, Mumbai. Children with transfusion – Meningococcal
transmitted HIV progress to AIDS over a period of 5 to 10 – Pneumococcal
years unlike perinatally infected children, who progress quite – H. influenzae type b
rapidly. They can be managed with antiretroviral therapy, as • Lifelong penicillin prophylaxis should be advised.
per the national guidelines for children and adults.
Other infections commonly seen in these children
include malaria and CMV infections.123,124 CMV infection
can be prevented by reducing the leukocyte content of
packed red cells, either by pre storage filtration or using
bedside leukocyte filters. Also, use of red cells from CMV-
negative donors and frozen red cells are other alternatives
in the developed world. Malaria has to be treated as
and when it occurs, as despite screening blood bags for
malaria, it would not be possible to identify all donors
harboring the parasite due to a low sensitivity and inter-
observer variability of peripheral blood smear evaluation.
Yersinia spp. infection is known to occur in iron
overloaded125,126 and desferrioxamine-treated patients.
If a patient on desferrioxamine comes with symptoms of
abdominal pain, diarrhea and vomiting, desferrioxamine
should be stopped immediately, appropriate stool culture
or blood serology undertaken and treatment with co- Fig. 17 Leg ulcer in thalassemia
trimoxazole or aminoglycoside given.
Gall stones127,128: With better management of thalassemia
with regular and adequate transfusions, the incidence of
gallstones has considerably decreased. However, those
who do not receive optimal treatment, may develop gall-
stones. Prophylactic cholecystectomy during splenectomy
has been recommended as a safe, standard procedure for
these patients.
Leg ulcers (Fig. 17)129: Ulceration around the ankles
is commonly seen in thalassemia intermedia and
inadequately transfused thalassemia major. This occurs
due to venous stasis, vaso-occlusion, anemia, and
local trauma. Treatment includes bed rest, increased
transfusions, wound care, and the use of local tissue
factors. Local irrigation of the wound with granulocyte-
colony stimulating factor (G-CSF)—1 vial diluted in
normal saline has been found beneficial. Fig. 18 Splenectomy
• Hospitalize promptly whenever required for cultures Umbilical Cord Stem Transplantation140-142
and antibiotic sensitivity and for IV antibiotics.
• Treat the infection promptly with appropriate anti Umbilical cord stem cells collected from the cord of
biotics. unaffected fetus at delivery, can be used for transplantation,
even if partially matched. This has yielded better results in
some centers.
Curative Treatment
• BMT in utero (Fig. 20)143,144: Research is underway
Stem Cell Transplantation (Fig. 19)134-139 on BMT in utero, at 14 weeks of gestation. Since the
immune system is not developed at that time, there
Bone marrow transplantation offers the potential perma would be no rejection and mother’s purified stem cells
nent cure, if an HLA-matched sibling donor is available. could be used, although the risk of GVHD would have
Though expensive, it is cost-effective when compared with to be looked into.
the long-term ideal treatment.
• Pharmacologic manipulation of HbF145,146: Various
Multiple pricks, blood transfusion hospitalization, can
drugs, which induce production of HbF, have been
be averted. The outcome of this procedure depends upon
tried in hemoglobinopathies with variable success.
the three factors:
These include butyrates, hydroxyurea, 5-azacytidine,
1. Hepatomegaly
erythropoietin, etc.
2. Hepatic fibrosis
• Hydroxyurea147-150,154,155: Few studies on effect of
3. Irregular chelation.
hydroxyurea in b-thalassemia major have been
According to these factors, patients are classified into
published and have shown variable responses.
three classes as follows:
However, its efficacy in thalassemia intermedia has
1. Class I: None of the above factors.
2. Class II: One or two factors. been established. Similarly, in double heterozygotes,
3. Class III: All of the above. the usefulness has been established.
For those with none of these factors (Class I), the • Butyrates151-155: Similar results, as seen with hydroxyurea,
success rate is about 93 percent, with one or two factors were seen with butyrates in b-thalassemia. Though
(Class II), it is 85 percent and with all three factors (Class an increase in HbF has been documented, no sub
III), it drops to a very low figure of 60 percent. stantial increase in total hemoglobin or decrease in the
transfusion requirement has been reported.
Fig. 19 Stem cell transplantation (Courtesy: MR Lokeshwar)

lives for 50 years, then he would require 2000 units of

packed red cells, 15,000 desferrioxamine injections, which
translates into 1.5 lac hours of a needle in his body and ` 90
lacs for chelation alone (personal communication). This is
besides the cost incurred by the hospital where he receives
his regular treatment including packed red cell transfusions
and other medical care.
The birth of a thalassemic child thus places consi
derable strain not only on affected child and family but
on society at large. Therefore there is an emphasis for shift
from treatment to prevention of birth of such children in
future. This can be achieved by:
Population Education167
Fig. 20 BMT in utero
• Mass screening of high-risk communities for thalas
semia minor
• Genetic counseling of those who test positive for
• Erythropoietin156-158: It has shown no benefit when
thalassemia minor.
used alone, but in combination with hydroxyurea, an
additive effect has been found, resulting in increase in
HbF as well as total hemoglobin.40 Prenatal Diagnosis168,169
The question that arises in the mind is whether prevention
Gene Therapy159-161 at a national level is cost-effective.
Inherited disorders of hemoglobin remain desirable The answer to this is Yes, it is cost-effective and we
targets for genetically based therapies. These genetically should strive to prevent the birth of a thalassemic child.
based strategies aim at addition of a normal copy of the Prevention of thalassemia, is practical, feasible and
human globin gene along with key regulatory sequences the answer to the agony of so many children, families and
to autologous hematopoietic stem cells. But this approach nations.
has been impeded by a difficulty of attaining high- The methods would include creating awareness
titer vectors. Recent advances in vector construction amongst high-risk communities about the prevalence and
have circumvented some of the problems limiting gene the difficulties in management of this condition. Screening
transfer efficiency and regulation of transgene expression. young people amongst all high-risk communities before
However, it will be some time before clinical application of marriage is the right way to go. If screening is performed
this therapy becomes a reality. in childhood, it is often forgotten around the time, they get
married. Hemoglobin electrophoresis is the confirmatory
Prevention162-166 test to diagnose thalassemia minor or carrier status. All
at-risk couples need to be counseled about the prenatal
• The cost of treatment of an average weight 4-year- diagnosis to confirm the thalassemic status of the fetus.
old thalassemic child is around ` 90,000 to 100,000 Thus, every baby born to two carriers of thalassemia trait
annually in a private set-up. Therefore, not more than should be screened in utero and termination should be
5 to 10 percent of thalassemic children born in India advised for those fetuses who are found affected, so that
receive optimal treatment. Stem cell transplantation as no child or parent has to suffer the agony of management
a curative treatment, which costs between 6 and 16 lac of thalassemia.
rupees is out of reach for majority of children. Future research is directed at improving the prevention
• Besides bearing the cost of treatment, the psychological strategies by diagnosis before the embryo is formed,
stress to both the patient and the parents/family is to reduce the psychological trauma of termination of
phenomenal. pregnancy. These newer methods include:
It may be starling to know from a 15-year-old
thalassemic child the account of what he has undergone
so far. He has received around 250 units of packed red cells
Preimplantation Diagnosis170-174
and 4000 injections of desferrioxamine. He has had a needle Biopsy of blastula: By washing uterine cavity after in vivo
in his body for over 40,000 hours of his life. His family has fertilization. Analysis of a single blastomere from an eight
already spent ` 16,20,000 for chelation alone. If this child cell embryo after in vitro fertilization.
Preconception Diagnosis175 deferasiroxor, icl670(4-[3,5-bis (2-hydroxyphenyl) -1,2,4-

triazol-1-y1] benzoic acid), (asunra, desirox), with mini-
Analysis of the first polar body of an unfertilized egg and mal toxicity, can be given orally in multiple dose or single
then. Distinguish between eggs which carry the defective/ dose, thus improving compliance.
normal gene in vitro fertilization of normal egg. Stem cell transplantation is curative but out of reach
of many because of cost and nonavailability of matching
SUMMARY donor. Gene therapy for thalassemia is still under research.
During the approach of a case with thalassemia, it is
necessary to suspect thalassemia by clinical evaluation and
Until last few decades, thalassemia was regarded as a uniformly
doing simpler hematological parameter like CBC, Nestrof fatal disease and death was expected during the second
test and confirm the diagnosis by estimating HbF and HbA2 decade of life before adulthood. However, progress in the
and other abnormal hemoglobins by doing various test like understanding and management of the disease in last 3 decades
hemoglobin electrophoresis, column chromatography, has improved prospects of survival such that they survive now
isoelectric focusing or microcolumn chromatography into 3rd and 4th decades of life. This is possible provided, they
and high performance liquid chromatography using receive the ideal treatment with good compliance. Thalassemia
various instruments like Bio-Rad Variant. It is important should no longer be, therefore, seen as a disease of childhood.
to anticipate complication due to iron overload involving Better management and improved survival has opened a new
various organs and due to transfusion complications. It is chapter in the management of thalassemia beyond transfusions
and chelation therapy. Problems of adolescence, growth and
therefore necessary to evaluate organ functions at regular
development, attainment of puberty and full sexual potential,
intervals for early detection of complications.
bone mineralization, proper education, suitable employment,
Management of thalassemia involves a multi-disci marriage and parenthood are some of the concerns that
plinary therapeutic team approach and should be prefer require attention.
ably done at a comprehensive thalassemia children care
center with outdoor transfusion facilities..
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C H A P T E R 18
Sickle Cell Anemia in Children
Sickle cell disease is a group of commonly inherited hematologic disorders. It is a single gene disorder, caused by a single point
mutation in the β globin chain of hemoglobin leading to defective hemoglobin synthesis. Sickle cell disease involves the red blood
cells or hemoglobin and their ability to carry oxygen, hemolytic anemia, painful crisis, end-organ failure and various complications.
Sickle cell anemia is the first disease, described to be due to a molecular mutation. It is characterized by relentless pain and so the
African tribes chose different names for sickle cell disease; chwechweechwe, hemkom, nuidudim all describing the relentless pain.
HISTORICAL ASPECT Their clinical importance, arises from the fact that
some of them are associated with higher HbF levels, e.g.
Sickle cell anemia has been prevalent in Africa for the 5,000 Senegal and Saudi-Asian variants, and tend to have milder
years or more. It was first described in 1910, by Dr James disease.6
Herrick in a dental student from Grenada who noticed In Caribbean and in North American sickle cell
abnormally shaped RBCs under the microscope. These patients of African origin, 50 to 70 percent of chromosomes
cells looked like a sickle and so the name.1 He also coined are Benin, 15 to 30 percent of chromosomes are Bantu-
the term crisis to describe the acute episodes of pain. CAR and 5 to 15 percent are Senegal.7,8 In Los Angeles,
Sydenstricker described the first pediatric case and 38 percent of patients are Benin homozygotes (Benin/
recognized that this disorder was a hemolytic anemia.2 It Benin), 25 percent are Benin/Bantu CAR, 13 percent are
was only in 1952 that sickle cell anemia was reported in the Benin/Senegal, 5 percent are Bantu CAR homozygotes
tea garden laborers of Assam in India. and 3 percent are Bantu CAR/Senegal.
Sickle cell disease is more commonly found in people
Types of Sickle Cell Gene Mutations from tropical regions particularly sub-Saharan Africa,
Sickle-cell gene mutation probably arose spontaneously India and Middle East which are endemic for malaria.9
in different geographic areas, as suggested by restriction Disease is found with equal frequency in males and
endonuclease analysis. females.
There are five major mutations of the sickle cell gene.
In Africa, four of the major sickle haplotypes are associated
Incidence and Prevalence of Sickle Cell
with different geographical areas:3,4
1. “Senegal” is located in Atlantic West Africa (5–15%) Disease
2. “Benin” in central West Africa (50–70%) The prevalence of the disease in the United States is
3. “Bantu” (also known as CAR) is in central Africa (15– approximately 1 in 5000, mostly affecting Americans of
30%) Sub-Saharan African descent, according to the National
4. “Cameroon” Institutes of Health.10 One in 600 African-Americans
5. In India and parts of Saudi Arabia, the African will have homozygous sickle cell disease, one in 800 will
haplotypes are not seen, but a unique “Arabian- have hemoglobin SC disease and one in 1700 will have
Indian” haplotype is found.5 Sickle-β Thalassemia Syndrome. The sickle gene has an
Chapter-18 Sickle Cell Anemia in Children 191
incidence of 8 percent in African-American individuals. Hb S Polymerization

The frequency of the gene can be higher in certain areas of
Africa. In the US, it has been estimated that 1,000 children As the RBC passes through the microcirculation there is
are born each year with sickle cell disease. One in twelve deoxygenation which causes conformational changes
African-Americans has sickle cell trait.10,11 in the Hb molecule. Due to the hydrophobic valine, and
Sickle cell disease primarily affects those of African interactions between two valine molecules, a polymer
descent and Hispanics of Caribbean ancestry, but the trait is formed which later progress into helical fibers which
has also been found in those with Middle Eastern, Indian, group together, stiffen and give rise to the characteristic
Latin American, Native American and Mediterranean sickle shape. So, the polymerization of hemoglobin S is the
heritage. primary event in the molecular pathophysiology of sickle
Sickle cell disease is prevalent in many parts of India, cell disease and results in distortion of the shape of the red
where the prevalence has ranged from 9.4 to 22.2 percent blood cell and a marked decrease in the deformability of the
in endemic areas.12 red blood cell. The resulting loss of red blood cell elasticity
plays a key role in the clinico-pathologic manifestations of
Sickle Cell Anemia and Malaria sickle cell disease. These cells thus assume a rigid sickle
shape and are unable to regain the normal biconcave
Sickle cell anemia is closely related to malaria. It is shape even after reoxygenation. Consequently, these rigid
well known that sickle cell disease (SCD) is seen in blood cells are unable to deform while passing through
those regions of the world where malaria is endemic. narrow capillaries resulting in vascular occlusion and
The presence of a sickle trait in an individual provides ischemia and are also hemolysed. These rigid sickle cells
natural protection against malaria.13 In persons with AS are responsible for the vaso-occlusive phenomenon and
(heterozygotes), when the parasite infects the RBC, it hemolysis which are characteristic of this disorder.
causes the abnormal hemoglobin to sickle. This leads to Data regarding the initial trigger for vaso-occlusive
preferential phagocytosis of the parasitized RBCs by the crisis shows that there is contribution of the vascular
spleen14,15 leading to a decrease in the parasitemia and endothelium, complex cellular interactions and a global
therefore less severe disease, with a relative protection inflammation mediated cell activation. The presence
from dying of malaria. It is believed that this mutation of anemia is due to hemolysis of abnormally shaped
arose by the process of natural selection. red blood cells. The presence of other hemoglobins in
the red blood cell, such as hemoglobin F, hemoglobin
Pathophysiology A, hemoglobin C, and hemoglobin O, has an effect on
The sickle mutation is a GAG-GTC conversion. The the sickling phenomenon and on the polymerization of
resultant sickle hemoglobin differs from normal adult hemoglobin S. Hemoglobin F has the most profound anti-
hemoglobin by alteration of one amino acid in the β-globin sickling effect, followed in order by hemoglobin A, C, and
subunit at the sixth position of the b-globin chain.16 Sickle O. Elevated levels of hemoglobin F can modify the clinical
β chain has hydrophobic valine instead of hydrophilic and hematological effects of sickle cell disease and prevent
glutamic acid in the sixth position of the β-globin chain. polymerization of hemoglobin and thereby sickling of red
The properties of hemoglobin S result in the clinical cells.
manifestations of sickle cell disease.
Normal red blood cells, owing to their elasticity, can
Increased Adhesion of RBCs to Endothelium
deform while passing through the capillaries. In sickle cell
disease, presence of hypoxia, acidosis, deoxygenation, Abnormal adhesion of sickle RBCs is initiated by young
high percent of Hb S, high MCHC, dehydration, fever, RBCs called stress reticulocytes. These cells are thrown out
acidosis promote red cell sickling due to formation of gel- prematurely into the circulation from the bone marrow.
like substance containing Hb polymers called tactoids. By virtue of their surface receptors, alpha 4 beta1 integrin
Repeated episodes of sickling, damage the cell membrane or VLA-4 (very late antigen 4), these cells bind directly to
and decrease the elasticity of the red blood cell. the VCAM1 on the endothelial surface. CD 36 on the RBC
Thus, the pathophysiology of sickle cell disease can be as well as on the endothelium are bound via a molecular
based on: bridge the plasmatic thrombospondin.
1. Hb S polymerization It is not only the RBCs but the polymorphs and
2. Increased adhesion of sickle RBC to endothelium platelets also that participate in the slowing of blood in the
3. Hemolysis microcirculation.
Hemolysis risk for passing the gene on to a child—there is a 25 percent

chance of having sickle cell anemia, 50 percent chance of
In addition to causing anemia, it also has other deleterious being a carrier like the parents and a 25 percent chance
effects. Heme is the most powerful scavenger of NO or of being unaffected. This means that there is a three in
nitric oxide which is a vasodilator. Hemolysis also releases four chance or 75 percent chance for the child to not have
erythroid arginase in plasma, which degrades L arginine, sickle cell disease.
the substrate of NO producing enzyme endothelial NO
synthetase. Thus, NO levels are reduced by increase in
scavenging as well as a decrease in production all leading
Compound Heterozygotes
to vasoconstriction and slowing of blood. Hemolysis of Where one β-globin gene is affected with sickle cell
abnormally shaped red blood cells during their passage in mutation and the other gene includes mutations associated
the microcirculation results in anemia. The bone marrow with Hb C (HbSC), Hb β-thalassemia (Hb S-β°thal/Hb S-
production of red blood cells does not match the rate of β+ thal, Hb D, Hb O Arab, hereditary persistence of fetal Hb
destruction. The sickle cells have a shortened life span of with variable clinical manifestations.
10 to 20 days as compared to the normal RBC lifespan of
90 to 120 days. Sickle-β Thalassemia Syndrome
Has two forms, Sickle-β° thalassemia and Sickle-β+
Genetics of Sickle Cell Anemia
thalassemia. The sickle-β° thalassemia individual is not
Sickle cell disease is an autosomal recessive disorder able to produce any hemoglobin A due to a complete
caused by a point mutation in the sixth codon of the deletion of the beta-globin gene. In sickle-β+ thalassemia,
β-globin gene found on chromosome 11 coding for there is a partial deletion of the hemoglobin gene and the
valine instead of glutamine. The disease includes various patient is able to make a variable amount of hemoglobin A.
genotypes containing at least one sickle gene where Hb S The amount of hemoglobin A will be less than the amount
constitutes > 50 percent of all hemoglobin. of hemoglobin S.
In general, hemoglobin SS disease is the most severe
Sickle Cell Syndromes of the sickle syndromes followed by hemoglobin S-β0
thalassemia, hemoglobin SC and finally hemoglobin S-β+
The common sickle cell syndromes are: thalassemia. The typical hematological values of these
• Heterozygous sickle cell anemia (AS) sickling syndromes are shown in Table 1.
• Homozygous sickle cell disease (SS)
• Sickle-beta thalassemia (β0 or β+)
• Compound heterozygotes sickle cell disease
Diagnosis of Sickle Cell Anemia
– SD Punjab disease CBC and Routine Laboratory Testing
– SO Arab disease
– Sickle E syndromes • Mild to moderate anemia (5–9 g/dL) with decreased
– Sickle α syndrome hematocrit (20–30%). The anemia is usually normocytic
– Sickle C syndrome. normochromic unless associated with alpha or beta
thalassemia or iron deficiency in which case microcytosis
and hypochromia may be present. MCV may be on the
Sickle Cell Trait or Carrier State
higher side due to reticulocytosis.
Heterozygous form (HbAS), where only one β-globin gene • Peripheral smear: The peripheral blood smear may
is affected, i.e. one parent is a carrier of Hb S mutation and show sickled red cells, typically irreversibly shaped
the other is normal, all the children will be phenotypically sickle cells, polychromasia indicative of reticulocytosis,
normal, however 50 percent will have sickle trait or will be target cells and Howell-Jolly bodies (RBCs with nuclear
carriers. Sickle cell trait is usually asymptomatic with Hb S remnants) indicative of asplenia.
constituting 40 percent of the total hemoglobin. • Reticulocyte count is elevated (Fig. 1.1) (3–15%).
Table 1 Sickle cell syndromes hematological values
Homozygous Sickle Cell Disease
Type Hb (g/dL) MCV Severity
Sickle cell anemia is the homozygous form (Hb SS) where SS 7–8 nL ++++
both β-globin genes carry the mutation. It is the most Sβ°-thal 8–10 <70 +++
severe type and Hb S is as high as 90 percent of the total SC 10–11 nL ++
hemoglobin. Thus, if both parents are carriers, they are at Sβ± thal 11–12 <70 +
However both these tests cannot distinguish between

sickle cell disease and trait.
Hemoglobin Electrophoresis
It helps to differentiate individuals who are homozygous
for Hb S (Hb SS) from those who are heterozygous by
demonstrating either a single band of Hb S (in Hb SS) or
Hb S with another mutant hemoglobin (in compound
heterozygotes). This is the definitive test for sickle cell
anemia. HbePP on cellulose acetate electrophoresis is the
usual method for Hb electrophoresis. However Hb S, G
and D have the same electrophoretic mobility. On citrate
agar electrophoresis, at ph 6.2, Hb S is separated. The most
commonly used method is by the variant machine.
• Only Hb S with an HbF concentration < 30 percent—
Fig. 1 Peripheral smear showing reticulocytes Sickle cell anemia. A homozygous patient will have
(Courtesy: MR Lokeshwar, India) hemoglobin SS (80–90%), hemoglobin F (2–20%) and
hemoglobin A2 (2–4%).
• There is unconjugated hyperbilirubinemia.
• A carrier patient will have Hb S (35–40%) and
• Decreased serum haptoglobin.
hemoglobin A (60–65%).
• Increased plasma hemoglobin levels.
• Hb S is predominant, Hb F < 30 percent, Hb A2 is
• Increased serum lactate dehydrogenase levels.
elevated - Hb S–beta-0 thalassemia.
• Hb S > A and Hb A2 is elevated—HbS–beta+thalassemia.
Hemoglobin Solubility Test • Hb A2 level is normal—to consider the possibility of
Hb S is an insoluble hemoglobin molecule. It forms tactoids concomitant Hb SS and iron deficiency.
or crystals when in the reduced state in high phosphate • Hb S and Hb C concentration in roughly equal amounts
buffer solution. These crystals refract and deflect light rays Hb SC disease.
and produce turbidity. The test may be inaccurate in a patient who has
recently received blood transfusions.
Sickling Test
Addition of sodium metabisulfite induces sickling of red Newborn Sickle Cell Disease Screening
cells, on the blood film (positive sickling test). It is done to The introduction of newborn screening has been a great
diagnose sickle cell anemia or when there is an abnormal advancement in the management of sickle cell disease
electrophoretic or chromatographic hemoglobin fraction as infants with sickle cell disease are healthy at birth and
in the position of Hb S, e.g. Hb D or G. develop symptoms only after decline of fetal hemoglobin.
Method of sickling test: Sodium metabisulfite reduces the Such programs help in early recognition of affected
oxygen tension inducing the typical sickle shape of red infants, early intervention to reduce morbidity and
blood cells. mortality and to provide anticipatory guidance for parents.
The most commonly used techniques for newborn
The method is as follows: A drop of fresh anticoagulated diagnosis are thin layer/isoelectric focusing and high-
blood is mixed with 1 drop of 2 percent sodium performance liquid chromatography.
metabisulfite solution on a microscope slide. The solution Repeat hemoglobin electrophoresis, if found abnormal
is freshly prepared each time. A cover slip is placed and and again at six months, to confirm the hemoglobinopathy.
the edge is sealed with wax/vaseline mixture or with nail It is recommended to conduct a complete blood count and
varnish. It is allowed to stand at room temperature for hemoglobin analysis on the parents to confirm diagnosis
1 to 4 hours. Under the microscope, in positive samples and offer genetic counseling. In newborn screening, the
the typical sickle-shaped red blood cells are seen. False patterns of hemoglobin are reported in decreasing order
negative results may be obtained if the metabisulfite has according to the quantities detected.
deteriorated or if the cover slip is not sealed properly. It • FS pattern: Newborns with sickle cell anemia (Hb SS)
is important to examine the preparation carefully and in have this pattern with predominant Hb F and small
particular near the edge of cover slip. amount of Hb S and no Hb A. It may also be found in
newborns with sickle cell-beta-0 thalassemia, sickle anemia will have 90 percent hemoglobin S (Hb S), 2 to
cell-hereditary persistence of fetal hemoglobin. Family 10 percent of hemoglobin F (Hb F) and normal amounts
studies help confirm diagnosis, in case of sickle cell- of minor fraction of adult hemoglobin A2 (Hb A2). Adult
beta-0 thalassemia, one parent has sickle cell trait and hemoglobin A (HbA), which increases at 3 months of age,
the other beta thalassemia minor. is absent.
• FSA pattern: This pattern is supportive of sickle cell- The most common clinical manifestation of sickle
beta+ thalassemia. cell anemia is vaso-occlusive crisis. It occurs due to the
• FAS pattern: Newborns with sickle cell trait have this sickled red blood cells obstructing the capillaries causing
pattern. ischemic injury to the organ. Factors precipitating vaso-
• FSC pattern: This is supportive of a diagnosis of Hb SC. occlusive crisis include hypoxia, acidosis, dehydration,
• AFS pattern: This is suggestive of transfusion prior to infection, changes in body temperature.
the test or an error. The clinical features of sickle cell anemia occur
All patients screened to have either sickle cell disease secondary to the physiological changes in the RBCs leading
or trait must be started on penicillin prophylaxis until the to various acute and chronic complications. When Hb S is
final diagnosis is determined. Due to clinical implications deoxygenated the Sol (soluble) form of Hb changes to Gel
of a diagnosis of either sickle cell disease or trait, the need form of Hb to form rigid, sickle shaped tactoids, and they
for repeat hemoglobin analysis at a later age must be polymerise forming insoluble structure. RBC membrane
emphasized. becomes more fragile. Upon reoxygenation the sickle cell
initially resumes normal configuration, but with repeated
Imaging Studies cycles of sickling and unsickling, fixation of membrane
occurs leading to irreversible sickle cell formation and
• Radiological abnormalities and stroke evaluation are hemolysis.
carried out as and when required to evaluate extent of
the lesion. If a CT scan is ordered, it is preferable not
Manifestation of Sickle Cell Anemia
use contrast until the hemoglobin S concentration
can be reduced below 30 percent. If available, MRI is Anemia
preferable.
• Ultrasonography: This can be used to visualize stones Patients are well compensated due to chronic nature of
and detect signs of thickening gallbladder walls or anemia. Anemia may be complicated by secondary folate
ductal inflammation, indicating possible cholecystitis. deficiency. This is due to increased RBC turnover and
folate utilization. Periodic episodes of hyperhemolysis
may occur. All patients are universally anemic and are
Prenatal Diagnosis hemolytic in nature. Repeated cycles of deoxygenation
Parents with a child suffering from sickle cell disease or and morphologic sickling irreversibly damage the red
those at risk of having a child with sickle cell disease, often cell membrane and result in hemolysis. Bone marrow
seek prenatal diagnosis and termination of pregnancy in increases red cell production but is unable to compensate
case of an affected fetus. However, patients with sickle cell for the rate of hemolysis. This results in moderate-to-
disease have a great variation in the symptoms despite severe anemia. Thus patients may simply be found to be
having the same mutation and therefore, it is as yet not a jaundiced or pale. Patients may show few manifestations
very useful test. of anemia as they readily adjust by increasing their heart
rate and stroke volume (compensated). Though they may
be able to carry out daily activities in a normal fashion,
Clinical Features of Sickle Cell Disease their tolerance for exertion is limited.
There are great variations in the manifestations of sickle • Splenic sequestration is an anemic crisis. It is a life
cell disease (Fig. 2). Clinical features of sickle cell disease threatening medical emergency in children with
are due to the intermittent episodes of vascular occlusion, sickle cell anemia. It occurs due to pooling of blood
tissue ischemia/reperfusion injury and hemolysis, all and trapping of deformed RBCs in the narrow splenic
of which lead to multiorgan dysfunction as well as pain. vessels. This causes rapid, painful enlargement of
Hypercoagulability, hyposplenia and infections also spleen, precipitous fall in hemoglobin, hypovolemic
contribute to the clinical spectrum of sickle cell disease. shock and persistent reticulocytosis. It tends to
Symptoms do not develop in the first 6 to 12 months of occur with higher frequency in infants and has been
age due to elevated levels of hemoglobin F, in circulation. reported as early as few weeks of age. Spleen suddenly
After infancy, the red cells of a patient with sickle cell becomes enlarged and traps blood cells. The platelet
Fig. 3 Sickle cell anemia with hepatosplenomegaly

(Courtesy: MR Lokeshwar, India)
acute event.17 Parents should be educated about the

signs and symptoms of sequestration and to palpate
the spleen to recognize sudden increase in the size of
the spleen so that early treatment may be sought.
• Aplastic crisis is also an anemic crisis. This is a serious
complication leading to worsening of anemia resulting
in pallor, tachycardia and fatigue. Aplastic crisis is
precipitated by parvovirus B-19 infection which infects
the red blood cell precursors in the bone marrow
thereby affecting red blood cell production. Impaired
red blood cell production that lasts for a few days can
cause an abrupt life threatening crisis in patients with
sickle cell anemia due to decreased lifespan of RBCs
(10–20 days). Initial reticulocytopenia is followed by
brisk reticulocytosis as bone marrow spontaneously
recovers in a few days to a week. The spleen usually
is not enlarged over baseline. Management includes
packed red cell transfusion.
Fig. 2 Site of manifestations of sickle cell disease
(Courtesy: Swati Kanakia, India)
Vaso-occlusive Crisis
count often is slightly decreased. It occurs due to Pain resulting from vascular occlusion and ischemia and
pooling of blood and trapping of deformed RBCs in can occur abruptly. Pain may be accompanied by malaise,
the narrow splenic vessels. Hypovolemic shock occurs fever, and leukocytosis. It is the leading cause of emergency
if a large volume of blood is trapped or sequestered. department visits and hospitalizations, causing disruption
Hemoglobin levels dramatically drop from baseline of daily life with pain lasting for several hours to days and
values and the reticulocyte count is elevated. In occurs in any part of the body particularly extremities,
Figure 3, a child showing the sickle cell anemia with bones, chest, abdomen. Bone pain occurs due to bone
hepatosplenomegaly. marrow infarction particularly due to obliteration of
Treatment includes early diagnosis and aggressive nutrient arteries of bone. Hence the site of involvement
management in the form of intravenous fluids and changes with the site of maximum bone marrow activity.
blood transfusion. The mortality rate may be up to 10 Factors precipitating vaso-occlusive crisis include hypoxia,
to 15 percent before transfusion therapy. Sequestration acidosis, dehydration, infection, changes in body, bone
crisis may be recurrent in 50 percent of the survivors marrow infarction, etc. and since marrow activity changes
and hence splenectomy is recommended after the first with age, site of infarction also changes.
• Abdominal pain: Patients may present with acute

abdomen due to severe pain which may be due to
mesenteric vein thrombosis, referred pain or secondary
to underlying organ or soft tissue infarction.
Majority of the pain episodes are short lived and can
be managed at home with pain medications and other
comfort measures (heating blanket, relaxation technique
and massage). Specific therapies for pain include
acetaminophen and NSAIDs in conjunction with oral/
IV opioids and their derivatives. Optimal maintenance
oral/intravenous fluids to maintain hydration must
be initiated in hospitalized children. The 2003 BCSH
guidelines recommend use of oral analgesia for treatment
of pain although severe pain is best managed with
parenteral analgesics.18 Morphine is the drug of choice
and dosing must be individualized for each patient. It
should be given hourly followed by three hourly dosing
once effective analgesia is achieved. Patient-controlled
analgesia may also be helpful. Sleeping through the night
may be an indication to decrease dosing by 20 percent
the next morning. Decreasing analgesia dose at night is
not advisable as pain is often worse at night. After 24 to
Fig. 4 Dactylitis (Hand-foot syndrome)
48 hours, when pain is controlled, patient may be shifted
to sustained-release oral morphine and discharged from
the hospital with gradual tapering of dose over several
• Dactylitis (Hand-foot syndrome) (Fig. 4): The usual days. Pain must not be undertreated due to fear of opioid
presentation is symmetric or unilateral swelling of the addiction or dependence as they seldom occur due to brief
hands and/or feet which may be relieved with pain duration of painful episodes (5–7 days). Blood transfusion
medication such as acetaminophen and codeine and does not help to decrease intensity or duration of a painful
supportive measures. Osteomyelitis must be ruled episode and is indicated in patients with hemodynamic
out in all cases as they may have similar presentation instability due to decreased hemoglobin. Hydroxyurea
but require different line of management. It is one of may decrease frequency and severity of pain episodes.
the earliest clinical manifestations of pain in children
with sickle cell anemia. Up to 18 months of age,
Neurological Complications
the metacarpals and metatarsals are active part of
erythropoiesis and are involved presenting as dactylitis It is most prevalent in childhood and adolescence though
or hand-foot syndrome. Occurs in 50 percent of the it may be found as early as 1 year of age. Involvement of the
children by 2 years of age. Episodes are frequently nervous system due to sickle cell anemia may have a varied
recurrent. Clinical involvement may be limited to a presentation such as headaches, seizures, cerebral venous
single phalanx or metacarpal or may involve all small thrombosis and reversible posterior leukoencephalopathy
bones of all small extremities. syndrome of which stroke is the most serious manifestation
Radiologic changes become evident after a in 10 to 15 percent patients of SCD. Infarcts are usually
few days and include patchy areas of osteoporosis ischemic in children and hemorrhagic in adults.19 While
and sclerosis, periosteal new bone formation, and it is unusual for children to have strokes, approximately
occasionally apparent disappearance of a bone. 11 percent of patients with sickle cell anemia have strokes
Resolution is usually complete both radiologically before they reach the age of 20 years. Hemiparesis is the
and clinically although occasionally premature fusion usual presentation.
occurs causing permanently shortened, deformed Overt stroke, occurring in 11 percent of the children, is
small bones. The long bones of the extremities which defined as the presence of focal neurological deficit lasting
retain marrow activity as the child grows older are for > 24 hours and/or evidence of a cerebral infarct on T2
affected during childhood. During adolescence, as weighted MRI of the brain corresponding to the deficit.
the marrow activity recedes further, pain involves the The presence of a cerebral infarct on T2 weighted MRI
vertebral bodies of the lumbar region. of the brain in the absence of a focal neurological deficit
lasting for > 24 hours is defined as a silent stroke. These management. Patients with coexistent asthma should
silent infarcts occur in 20 percent of the children and tend be treated promptly with bronchodilators and steroids.
to cause progressive decline in cognitive function, affect Incentive spirometry and chest physiotherapy can help
learning and behavior and increased risk of epilepsy as to reduce the frequency of episodes of acute chest pain.
compared to general population.20 Prevention of recurrent episodes of ACS can be achieved
Management of a patient presenting with focal with chronic transfusions and hydroxyurea which reduces
neurological deficit includes: the rate of episodes by 50 percent.
• Oxygen administration to maintain saturation > 96
percent. Avascular Necrosis of the Femoral or Humeral Head
• Blood transfusion initiated within 1 hour of
presentation to increase hemoglobin up to 10g/dL. Avascular necrosis (AVN) of the femoral head presents a
• Transfusion therapy to maintain Hb S level < 30 greater problem due to weight bearing (Fig. 5). Vascular
percent is the mainstay of therapy for primary and occlusion can result in avascular necrosis and subsequent
secondary prevention of strokes.21 This strategy results infarction and collapse at either site. Subjects with high-
in 90 percent reduction in the rates of overt strokes. baseline hemoglobin are at increased risk. Approximately
Transfusion therapy must be continued indefinitely for 30 percent of all patients have hip pathology by age 30
there is increased risk of stroke on stopping.22 years.
• Convulsions frequently are associated with stroke.
• Primary prevention of strokes can be achieved by Infection
transcranial Doppler (TCD) measurement of the Infection is a major cause of morbidity and mortality in
blood velocity in the circle of Willis and values of patients with sickle cell anemia. These children are prone
> 200 cm/sec suggest a high risk of stroke even before to life threatening infections as early as 4 months of age due
lesions become evident on MRI. to hyposplenia or functional asplenia. These patients are
• Chelation therapy must be given after 2 to 3 years for more prone to infections by encapsulated organisms such
iron overload from repeated transfusions. as Streptococcus pneumoniae and Haemophilus influenzae
type b and Salmonella responsible for osteomyelitis.
Acute Chest Syndrome Sickling of red cells within the spleen results in multiple
Acute chest syndrome (ACS) is defined as combination episodes of splenic infarction, leading to functional
of respiratory symptoms, along with fever, cough, asplenia (autosplenectomy) which occurs in most
respiratory distress, chest and/or back pain, and new children by 5 years of age. In addition, they may also have
lung infiltrates.23,24 It is the second most common cause abnormal IgM and IgG responses, defects in the alternate
for hospital admission in children with sickle cell anemia pathway complement fixation and opsonophagocytic
and a common cause of death.23,25 In young children, it is dysfunction.27,28
usually due to infection. Older children and adults have The two major measures in preventing infections
infarction more often. ACS is most frequently preceded by in these children are penicillin prophylaxis and
a painful episode requiring opioids. immunization for all patients.
Emergency management of ACS includes supplemen
tal oxygen and simple or exchange transfusion.
Continuous pulse oximetry monitoring is required and
oxygen therapy is initiated when room air saturation is <
90 percent. The aim of blood transfusion is to reduce Hb S
level < 30 percent and when administered early may help
prevent further respiratory complications and need for
supplemental oxygen.
The etiology of ACS is multifactorial including pulmonary
infarction, fat embolism due to bone marrow infarction
and more commonly infection. Due to similar clinical
presentation of pneumonia and ACS and infection being
a common causative factor, it is essential to start empiric
antibiotic therapy with a third generation cephalosporin
and a macrolide. S.pneumoniae, Mycoplasma and
Chlamydia species are common organisms.26 Other Fig. 5 Avascular necrosis (AVN) of the femoral head
treatment measures include adequate analgesia and fluid (Courtesy: MR Lokeshwar, India)
• Oral penicillin V in a dose of 125 mg twice daily should

be started at 2 months and given till 3 years of age after
which it is increased to 250 mg twice daily till 5 years of
age.
• Most clinicians stop prophylaxis at 5 years of age
provided the child has not had prior pneumococcal
infection or splenectomy and has received pneumo-
coccal vaccine.29
• Patients who are allergic to penicillin should be given
erythromycin 10 mg/kg twice a day.
• Children with sickle cell anemia should receive all
the routine childhood immunizations including
those against Streptococcus pneumonia, Haemophilus
influenzae type b, Neisseria meningitidis, seasonal
influenza, hepatitis B. Fever may be the first sign of
bacterial infection in these children prone to fulminant Fig. 6 Unhealed ulcers
and life threatening infections. (Courtesy: MR Lokeshwar, India)
• Thus, fever must be considered a medical emergency
requiring prompt medical attention and antibiotic Rest, elevation and dry dressings with antimicrobial
therapy. The factors with high risk for invasive infection ointments are the best approach to this problem. Attempts
requiring inpatient management:30 at skin grafting are frequently not successful due to poor
• Children younger than 2 years with hemoglobin SS
blood flow to the affected region. Healing usually takes
(Hb SS) or hemoglobin S-β° thalassemia
weeks to months.
• Temperature >40°C
The area should be protected against trauma. Socks
• WBC > 30,000 mm³ or < 5000 mm³
or other clothing that cover the area should be avoided,
• Hemoglobin < 5g/dL
to reduce friction injury. A simple dry dressing provides
• History of previous bacteremia (due to increased risk
of recurrence) additional protection. Zinc supplementation has been
• Presence of indwelling catheter. traditionally thought to aid wound healing.
• Signs of systemic toxicity, hemodynamic instability
and/or meningitis Other Complications
• Prior treatment with vancomycin or clindamycin Priapism (Fig. 7)
instead of ceftriaxone due to short half life of these
medications. It is defined as sustained, painful and involuntary erection
All patients must be immediately started on empiric of the penis lasting longer than 30 minutes. Priapism is
antibiotic therapy with a 3rd generation cephalosporin and not uncommon problem in sickle cell anemia and most
may be discharged after a 24 to 48 hours afebrile period patients experience their first episode by 12 years of age
with duration of antibiotic therapy titrated as per culture and by 20 years of age as many as 90 percent of the patients
reports. Outpatient management may be considered in have experienced one or more episodes of priapism. Minor
those without risk factors. Another distinct infection in recurrent episodes are common during adolescence.
these patients is osteomyelitis, most commonly caused Occasionally, the problem is seen in pre-pubertal
by Salmonella spp. Hence all patients with persistent pain boys. Pain becomes severe if erection persists longer
and fever or bacteremia due to Salmonella spp. must be than 3 hours. Episodes may be described as stuttering or
evaluated for osteomyelitis with a definitive bone scan. refractory. Stuttering episodes last for a few minutes but
less than 3 hours and resolve spontaneously. Refractory
Unhealed Ulcers (Fig. 6) episodes are prolonged, lasting longer than 3 hours.
Acute therapy of prolonged episodes includes aspiration
Skin Ulcers (Unhealed Ulcers) of blood from corpus cavernosa followed by irrigation
Skin ulcers are relatively infrequent. The most common site with dilute epinephrine to sustain detumescence to be
of skin ulcers is over the lateral malleoli. The ulcerations done in consultation with pediatric urologist. Supportive
often have no clear-cut antecedent trauma and progress measures like sitz bath and pain medications may be tried.
over a period of weeks. Lesions in children occur most Hydroxyurea may help to prevent recurrent episodes. A
commonly around malleoli where poor circulation along sympathomimetic drug with mixed alpha and beta actions,
with sickling and microinfarcts leads to poor healing and etilefrine seems promising for secondary prevention of
infection. episodes.31 Evidence for the role of transfusion therapy for
occlusive episodes. Various studies have found that more

than 40 percent of adults with SCD have pulmonary
hypertension that worsens with age and is a major risk
factor for death.
Cholecystitis
Due to chronic hemolysis, cholelithiasis is common in
children and 40 percent of adolescents with sickle cell
anemia are affected with cholecystitis or common duct
obstruction can occur. Consider cholecystitis in a child
who presents with right upper quadrant pain, especially if
associated with fatty food.
Consider common bile duct blockage when a child
presents with right upper quadrant pain and dramatically
Fig. 7 Priapism elevated conjugated hyperbilirubinemia.
Retinopathy
acute or preventive therapy is lacking.32,33 Prolonged and/
or recurrent episodes of priapism may lead to fibrosis and The retina is primarily affected with manifestations such as
impotence. proliferative retinopathy, retinal artery occlusion, retinal
detachment and hemorrhage.36,37 The vaso-occlusions
Renal Disease may begin in childhood and progressively lead to loss of
visual acuity. Prophylactic photocoagulation may have a
Renal involvement is common in sickle cell anemia and role in the treatment of proliferative sickle retinopathy.
contributes to the morbidity of the disease with renal Regular eye checkups are recommended.
failure in up to 18 percent of the patients. The primary
event is occlusion of vasa recta capillaries in renal medulla Growth and Development
where there is low oxygen concentration and high
osmolarity thereby increasing the concentration of Hb S. Growth failure and sexual maturation are delayed in
The renal manifestations of sickle cell anemia: patients with sickle cell anemia. Normal height may be
• Enuresis secondary to hyposthenuria. achieved by adulthood but weight remains lower for age
• Painless hematuria due to papillary infarcts. as compared to controls. Neurodevelopment and skeletal
• Proteinuria and hypertension. Asymptomatic maturation are also delayed.38,39
albuminuria is a precursor of progressive renal
disease.34,35 Cardiac Involvement
• Renal infarction, papillary necrosis, and renal colic. The heart is involved due to chronic anemia and
• Nephrogenic diabetes insipidus that can lead to microinfarcts. There is chamber enlargement due to
polyuria. compensatory increase in cardiac output. Arrythmias may
• Focal segmental glomerulosclerosis that can lead to be an important cause of death in older patients. As per
end-stage renal disease; dialysis is well tolerated and a study, coronary artery dilatation is common in children
increasing numbers of patients are being treated with with sickle cell anemia.
renal transplantation. Cardiac complications are usually related to high
• Renal medullary carcinoma is a malignancy found output stress due to anemia and manifest in the form
almost exclusively in black patients with Hb SC disease of congestive cardiac failure and hemosiderosis due
or sickle cell trait. to iron overload because of chronic blood transfusion.
Chelation therapy must be given after 2 to 3 years for iron
Pulmonary Hypertension overload from repeated transfusions. There is no specific
cardiomyopathy in sickle cell anemia.
Pulmonary hypertension occurs due to formation of
microinfarcts and microthrombi in the pulmonary
vasculature due to circulation of deoxygenated blood
Pregnancy
which promotes sickling. Depletion of nitric oxide may Pregnancy in patients with sickle cell anemia is associated
also contribute to the pathogenesis independent of vaso- with fetal and maternal complications.
Fetal complications are related to compromised • History of other severe vaso-occlusive events
placental blood flow and include spontaneous abortion, • Severe symptomatic anemia
intrauterine growth restriction, fetal death in utero, and low • Severe unremitting chronic pain that cannot be
birth-weight. Maternal complications occur in as many as controlled with conservative measures
one-half of pregnancies, including acute chest syndrome, • History of stroke or a high risk for stroke.
bacteriuria, urinary tract infection, pyelonephritis,
endometritis, pre-eclampsia, thromboembolic events, Transfusion Therapy
and the use of cesarean section.
As a result of these complications, careful monitoring Chronic transfusion with red blood cells decreases the
during the antenatal period to ensure maternal and fetal concentration of HbS in patients with sickle cell disease by
well-being is essential. three mechanisms:42,43
• Addition of Hb A from the blood of normal donors
dilutes the Hb S in the blood
Treatment • The rise in hematocrit following blood transfusion
Comprehensive medical care must be provided to these suppresses erythropoietin release thereby reducing
patients with the team effort of physician, disease specific the production of new RBCs containing Hb S
specialist and pediatric hematologist to treat the clinical • Longer circulating lifespan of Hb A containing normal
symptoms and complications. RBCs as compared to sickle RBCs decreases the levels
of Hb S.
Hydroxyurea Therapy Transfusion therapy for individuals with sickle cell
disease can be categorized as therapeutic or prophylactic.
Sickle cell disease (SCD) is a potentially devastating Accepted indications for transfusion therapy in individuals
condition, which results in the vaso-occlusive phenomena with SCD include:44,45
and hemolysis. The severity of the complications that
occur with this disorder are widely variable, but overall Therapeutic: Acute use of transfusions for acute stroke,
mortality is increased and life expectancy decreased when acute chest syndrome, acute multi-organ failure, sudden
compared to the general population. severe drop in hemoglobin (splenic sequestration, aplastic
Hydroxyurea is a chemotherapy agent (myelosup crisis, hyperhemolytic crisis), acute symptomatic anemia
pressive agent) currently approved by US Food and Drug (e.g. onset of heart failure, dyspnea, hypotension, marked
Administration (FDA) for the treatment of sickle cell fatigue).
disease. It acts by increasing the fetal hemoglobin as its Prophylactic: Use of periodic red cell transfusions for
metabolism leads to NO related increase in cGMP which primary or secondary stroke prevention.
increases gammaglobin synthesis.40 This in turn decreases
sickling of red blood cells. These effects have been found Transfusion: Related complications include alloimmuni-
to reduce the incidence of pain episodes, acute chest zation, infection and iron overload. Matching for minor
syndrome episodes and blood transfusions by 50 percent.41 antigens such as C, E, Kell, JKB (Kidd) and Fya (Duffy) an-
tigens can significantly reduce alloimmunization.
Starting dose: Hydroxyurea can be started at a dose of 10 A decision of simple versus exchange transfusion must
mg/kg orally, on a daily basis. The patient’s hematological be made on case to case basis.
status should be monitored to rule out fall in the neutrophil Simple blood transfusion is used when the aim is
count to less than 2,500 per cubic millimeter or platelet to restore blood volume or oxygen carrying capacity in
count to less than 80,000 per cubic millimeter. an acutely ill child. Partial exchange transfusions are
Dose escalation: The dose of hydroxyurea can be increased recommended when an acute or chronic reduction in
at a rate of 5 mg/kg/week as long as the hematological the concentration of Hb S is required without an increase
values remain in an acceptable range, and the patient in viscosity and iron burden (e.g. for acute emergencies
shows no other evidence of side-effect from the HU. such as multi-organ failure, suspected stroke, acute chest
Maximum dose is 25 mg/kg/day. Higher doses have been syndrome, primary and secondary prevention of stroke
given at some institutions 35 mg/kg/day. and prevention of recurrent painful episodes). The upper
limit of hemoglobin should be kept at 10g/dL to prevent
Indications for Hydroxyurea Therapy hyperviscosity and decreased oxygen delivery.
• Patients who are hospitalized more than 4 or 5 times Transfusion and surgery: In patients with sickle cell
per year with painful vaso-occlusive crises disease undergoing surgery, events like hypoxia,
• History of acute chest syndrome dehydration, hypothermia may result in intra or post
operative complications. Blood transfusion with the aim chromatin structure and transcription rates of
of maintaining hemoglobin concentration at 10g/dL and γ-globin gene.48
HbS concentration < 30 percent given preoperatively may – Trichostatin A: HDAC inhibitor which significantly
help to reduce complications. An exchange transfusion inhibits pulmonary vein expression of vascular cell
may be required in cases with Hb > 10g/dL. adhesion molecule (VCAM) and tissue factor (TF)
in mouse models.49
Chelation Therapy – Pomalidomide: Thalidomide derivative that
stimulates erythropoiesis, F cell production, total
Iron overload occurs as a result of repeated transfusions hemoglobin and Hb F synthesis in human CD34+
in patients with sickle cell disease resulting in heart and cells.50
liver failure along with other complications. MRI is a • Prevention of red blood cell dehydration: Senicapoc is
more accurate and non-invasive method of estimating a potent and selective blocker of the Gardos channel
tissue iron load and response to chelation. The chelating (potassium efflux pathway) which prevents the loss
agents commercially available and approved for use are: of solutes and water maintaining the hydration of
desferrioxamine, deferasirox. sickle RBCs which is critical to the rate and degree of
Desferrioxamine needs to be given parenterally or polymerization.51
subcutaneously by prolonged infusion and nearly every • Nitric oxide (NO): Sickle cell anemia is characterized
day (5 days a week), which has limited its effectiveness in by a state of NO resistance, NO inactivation and
many patients. Deferasirox is an effervescent tablet that is reduced NO availability. Options under investigation
dissolved in liquid and taken orally daily. to increase the supply of NO include direct
administration of inhaled NO, increasing the substrate
Erythrocytapheresis availability with arginine supplementation, treatment
of phosphodiesterase-5 inhibitor sildenafil to prevent
This technique involves automated red cell exchange that
breakdown of endogenous NO.
removes blood containing Hb S from the patient while
• Anti-inflammatory agents: Statins have potent anti-
simultaneously replacing that same volume with packed
proliferative and anti-inflammatory actions and
red cells free of Hb S. Transfusion usually consists of sickle- stabilize endothelial barrier function. Steroids have
negative, leuco-reduced, and phenotypically matched been reported to reduce the duration of severe pain
blood for minor red cell antigens C, E, K, Fy and Jkb. episodes and severity of acute chest syndrome in
The procedure is performed on a blood cell processor children and adolescents, however more studies are
(pheresis machine) and is a better technique than manual required to assess the benefits and risk of this therapy.
exchange transfusion. This technique has the advantage of • Treatment with tinzaparin (LMWH) may help in
minimum iron accumulation; however it is less commonly painful episodes as it decreases coagulation activation
performed due to requirement of expertise and equipment. and adhesion of cell to vessel wall. Use of eptifibatide
(glycoprotein IIb/IIIa inhibitor) in acute pain episodes
Bone Marrow Transplantation are currently being studied.
• Other agents: Nix-0699 (Niprisan), a phytomedicine
Allogenic bone marrow transplantation (BMT) is the has shown to inhibit RBC sickling and produce a
only known cure for sickle cell disease. Many risks are left shift of the oxygen dissociation curve of HbS on
associated with BMT, and the risk-to-benefit ratio must in vitro studies.52 Safety and efficacy of intravenous
be assessed carefully. The lack of availability of a matched immunoglobulin (IVIg) in these patients with acute
donor may limit the utility of BMT. pain episodes is being studied. Studies to evaluate
the use of Bosentan (endothelin receptor antagonist)
Novel Therapies which have shown results in mouse models must be
considered.
New agents have been developed which are now in clinical
trials based on the pathophysiology of sickle cell anemia.
Supportive Therapy
• Induction of hemoglobin F
– Decitabine: It is a nucleoside analoge that Supplementation with folate (1 mg/day), multivitamin
hypomethylates cellular DNA without cytotoxicity. without iron, oral calcium and vitamin D along with
This results in re-expression of γ-globin genes and nutritional management are recommended.
induces γ-globin synthesis.46,47 Addressing psychological issues like depression,
– Butyrate: It is a short chain fatty acid that inhibits scholastic backwardness, neurocognitive dysfunction
histone deacetylase (HDAC) thereby affecting with appropriate therapy and rehabilitation.
Morbidity and Mortality 6. Green NS, Fabry ME, Kaptue-Noche L, Nagel RL. “Senegal
haplotype is associated with higher HbF than Benin and
The following three prognostic factors have been identified Cameroon haplotypes in African children with sickle cell
as predictors of an adverse outcome:53 anemia”. Am J Hematol.1993;44:145-6.
• Hand-foot syndrome (dactylitis) in infants younger 7. Schroeder WA, Powars DR, Kay LM, et al. Beta-cluster
than 1 year haplotypes, alpha-gene status and hematological data
• Hb level of less than 7 g/dL from SS, SC and S-beta-thalassemia patients in southern
• Leukocytosis in the absence of infection California. Hemoglobin. 1989;13:325-53.
The mortality rate in infants and young children who 8. Hattori Y, Kutlar F, Kutlar A, et al. Haplotypes of beta S
have access to comprehensive health care has decreased chromosomes among patients with sickle cell anemia
from Georgia. Hemoglobin. 1986;10:623-42.
dramatically. Due to routine use of antibiotic prophylaxis
9. Weatherall DJ, Clegg JB. “Inherited haemoglobin disorders:
and immunization, acute chest syndrome and multi-organ
an increasing global health problem”. Bull World Health
failure are replacing bacterial sepsis as the leading cause of Organ. 2001;79:704-12.
death. In regions where comprehensive care is available, 10. National Institutes of Health. Introduction to Genes
the disease has shifted from a fatal pediatric illness to and Disease: Anemia, Sickle Cell. National Center for
a chronic disease often associated with progressive Biotechnology Information. Available at http://www.ncbi.
deterioration in quality of life and organ function. nlm.nih.gov/books/NBK22238/. Accessed April 29, 2014.
11. Centers for Disease Control and Prevention. Sickle Cell
Patient Education Disease: Health Care Professionals: Data & Statistics.
Centers for Disease Control and Prevention. Department
Patient education regarding the nature of the disease is of Health and Human Services. Available at http://www.
essential. They must be taught to recognize early signs of cdc.gov/ncbddd/sicklecell/hcp_data.htm. Accessed April
complications to obtain prompt treatment and identify 29, 2014.
environmental factors that may precipitate a crisis. 12. Awasthy N, Aggarwal KC, Goyal PC, Prasad MS, Saluja S,
The importance of hydration, prophylactic penicillin, Sharma M. “Sickle cell disease: Experience of a tertiary
immunization and drug therapy and regular follow-up care center in a non endemic area”. Annals of Tropical
must be emphasized. Patients (including asymptomatic Medicine and Public Health. 2008;1:1-4.
heterozygous carriers) must be explained the genetic basis 13. Wellems TE, Hayton K, Fairhurst RM. “The impact of
of the disease and made aware of genetic counseling and malaria parasitism: from corpuscles to communities”. J
Clin Invest. 2009;119:2496-505.
prenatal diagnosis. Genetic testing can identify parents
14. Luzzatto L, Nwachuku-Jarrett ES, Reddy S. Increased
at risk for having a child with sickle cell disease. Families
sickling of parasitized erythrocytes as mechanism of
must be encouraged to join support groups for information resistance against malaria in the sickle cell trait. Lancet.
of newer drugs and therapy such as of gene therapy, bone 1970;1:319-21.
marrow transplantation and the usage of cord blood stem 15. Roth EF Jr, Friedman M, Ueda Y, et al. Sickling rates of
cells and psychosocial support. human AS red cells infected in vitro with Plasmodium
falciparum malaria. Science. 1978;202:650-2.
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C H A P T E R 19
Antenatal Diagnosis of
Hemoglobinopathies
BACKGROUND acid substitutions in one of the globin chains are known.

Many of them are associated with hemoglobin disorders
The hemoglobinopathies and thalassemia syndromes of different types and severity. Interaction of various
are a group of autosomal recessively inherited disorder thalassemia mutations and abnormal hemoglobins
resulting from either the qualitative (structurally abnormal produces complex genotypes and an extremely wide
globins-hemoglobinopathies) or quantitative defects spectrum of clinical and hematological phenotypes.
(abnormal synthesis of one or more of the globin chain- The three main categories of these interactions that are
thalassemia syndromes). It is one of the common group associated with severe disease states and require further
of single gene disorder with a carrier frequency of >5%. In genetic counseling and prenatal diagnosis are as follows:
India, the carrier frequency varies from 3% to 17%, highest • b-thalassemia syndromes (includes δb thalassemia and
being in Pakistani Sindhi and Punjabi populations. E/b mutations)
Thalassemia is an example of a best studied disease of a • Sickle cell disease (HbSS and variant interactions e.g.
known molecular mechanism, and one of the first human Hb S/C, Hb S/b thalassemia, Hb S/D Punjab, Hb S/O
genes cloned and sequenced. It is classified according to Arab, HbS/Lepore, Sbd)
the abnormal synthesis of the globin chain. It is known • Hb Bart’s and HbH hydrops fetalis syndrome.
as α-thalassemia or α-thal if the α chain is affected or the
b-thalassemia/b–thal if b chain is affected. b-thalassemia
affects HbA whereas α-thalassemia affects both HbA and Alpha Thalassemias
HbF. Hemoglobins with any of the four identical globin The α thalassemias are a group of disorders characterized
chains are completely unstable and incompatible with by α reduction in a globin synthesis. They can be divided
life for example HbH disease with four b chains (b 4) or into the severe types (α1 or α° thalassemias) with typical
Hb Bart’s disease with four γ chains (γ4). Until 1970s, microcytic hypochromic blood picture in heterozygotes,
prenatal diagnosis for hemoglobinopathies was possible and the milder form (α2 or α+ thalassemias) which is
by analyzing globin chain levels on fetal cord blood, but usually silent. Deletion mutations are the common in α
with gradual advancements in DNA based technology the thalassemia, although α+ thalassemia can also be caused
testing gradually shifted to the more reliable and efficient by point mutations (nondeletional α thalassemia or αT).1
PCR based studies on chorionic villi DNA to provide earlier Deletion of all 4 globin chains results in the most severe
fetal diagnosis. Recent technological advancements have type of α0 thalassemia which is known as Hb Bart’s hydrops
lead to the possibility of fetal diagnosis on maternal blood fetalis syndrome. In the absence of α globin, HbF and HbA
as well. are not synthesized and fetal blood contains abnormal
hemoglobin Bart’s (γ4) leading to severe anemia, hydrops
MUTATIONS IN GLOBIN GENES fetalis and fetal death. Alpha0 thalassemia is particularly
Although globin gene is one of the small gene, till date common in South East Asia whereas α+ thalassemia
more than 1000 hemoglobin variants with single amino predominates in Africa and India. The compound
Chapter-19 Antenatal Diagnosis of Hemoglobinopathies 205
heterozygous condition of α°/α1 thalassemia (- - / - α), α0

Box 1 Disorders of globin chain synthesis warranting
thalassemia and nondeletion α+ thalassemia (- - / αTα) or
prediction in a fetus and prospective parents
homozygous nondeletion α+ thalassemia (αTα/αTα) results
in moderately severe to severe HbH (β4) disease. In India, Disorders of globin chain synthesis that should be predicted in a
HbH traits have been reported among Bengali’s, Malayalis, fetus
and Tamils, Gujaratis and Sindhis in Singapore. Other • b-thalassemia major (including E/b0 mutations)
• b-thalassemia intermedia (including E/b0 mutations)
common structural hemoglobin variants are hemoglobin
• Sickle cell disease
Constant Spring and Koya Dora from the populations of
• Hb Bart’s hydrops fetalis syndrome (Homozygous α0
Southeast Asia2 and Andhra Pradesh3 in India respectively. thalassemia) and (rarely) HbH hydrops fetalis syndrome (α0/
High prevalence of hemoglobin Constant Spring (both αT α)
heterozygotes and homozygotes) have also been reported • Compound heterozygous sickle cell states (including Hb
among the coastal people of Orissa.4 S/C, Hb S/b-thalassemia, Hb S/D Punjab, Hb S/O Arab, HbS/
Lepore).
b-Thalassemia5
The b-thalassemia are a heterogeneous group of disorder. HbD, deletional or nondeletional hereditary persistence
These can be classified either as b0 thalassemia with the of hemoglobin and δb trait or Hb lepore trait.
absence of b globin chain synthesis or b+-thalassemia • Inheritance of hemoglobinopathies: In most of the
with reduced rate of beta chain synthesis. More than 200 common hemoglobinopathies like alpha, beta
mutations have been identified majority of them being thalassemias and sickle cell anemia the mode of
the point mutations. About 10% of mutations are deletion inheritance is autosomal recessive. Autosomal
mutations of varying size. Homozygous or compound recessive disorders manifest if both the parents are
heterozygous state of most b+ or b0 type of severe carrier of the mutant gene. The disease usually occurs
mutations result in b-thalassemia major, a transfusion in one generation only. The risk of occurrence and
dependent anemia. That usually present in later part of recurrence is 25% in such cases (Fig. 1). Identification
infancy or second year of life. Thalassemia intermedia of mutation in such couples or proband is the
is the milder clinical condition that present usually after prerequisite for prenatal diagnosis. There is no risk of
second year of life. The type of mutation in the b globin having an affected child if only one partner is a carrier
gene determines the phenotype. Thalassemia intermedia or is affected (Figs 2 and 3). If only one partner is a
too can be of varying severity depending upon the type of carrier there is a 50% chance of the baby being a carrier
mutations and its interaction with other variant forms. In and all babies will be carriers if one partner is affected
authors experience, due to the complex gene interactions with an autosomal recessive hemoglobinopathy.
and unpredictability of the phenotype prenatal diagnosis Identification of carrier status is easy and is possible by
is often requested by the parents. measurement of HbA2 which is almost always high in
carriers except in a rare situation of silent carrier status
when HbA2 may be normal and carrier status can only
Sickle Cell Disease5
be confirmed by molecular studies.
The sickle cell disease (SCD) is widely prevalent in India • Diagnosis of hemoglobinopathies5-7: Hematological
and has a variable clinical course. It is characterized by and biochemical investigations are basic screening
hemolytic anemia with acute crises due to infection or vaso investigations that are widely available for diagnosis
occlusive episodes. An A to T substitution in codon 6 of the as well as carrier screening. DNA studies are important
beta globin gene is responsible for SCD. Variable severity for fetal diagnosis and making genotype phenotype
in SCD occurs due to the interaction with beta thalassemia correlation.
and other structural hemoglobins. Homozygosity for – Basic hematological parameters:
HbS, HbS/HbD–Punjab, HbS/HbO–Arab; and HbS/β- i. Complete blood count including Hb, RBC,
thalassemia (severe mutations) results in moderate to MCH, MCV, and RDW
severe clinical disease. ii. Hemoglobin electrophoresis at pH 8.6 using
Box 1 summarizes the various globin chain disorders cellulose acetate membrane for common
associated with severe disease states that requires prenatal hemoglobin variants i.e. HbS, C, DPunjab, E,
diagnosis and genetic counseling. Clinically asymptomatic OArab and Lepore Hb
states are b thal trait, α thal trait (α -/α – or α α/--) or silent iii. Hemoglobin electrophoresis at pH 6 using
carrier state (α α/α-), HbE trait, homozygous HbE, HbC, acid agarose–Distinguishes between C, E, and
result of a broad spectrum of mutations (N>1000).

These include point mutations, frame shift
mutations, large deletions and rearrangements.
Various techniques such as oligonucleotide
specific hybridization, oligonucleotide specific
amplification, oligonucleotide specific ligation,
gap-PCR, restriction endonuclease analysis
of amplified product, and real-time PCR are
being used to identify these alterations. Each of
Fig. 1 Twenty-five percent risk of affected child if both parents these techniques has its inherent advantages
are carrier and disadvantages. Every laboratory selects its
approach of mutation detection on the basis of
the mutation that is prevalent in that particular
population, ethnicity of that particular family and
running cost, suitability to routine diagnosis and
reliability of the technique.
Some laboratories have started using direct sequencing
as the first approach to identify a large number of different
mutations in at-risk populations. Indirect mutation
tracking using restriction enzymes having polymorphic
sites linked to mutations becomes the second choice
where mutations are not identified in previous affected
Fig. 2 No risk of affected child if only one parent is carrier child and family has requested for prenatal diagnosis in
next pregnancy.
Source of DNA: For molecular diagnosis in parents and
in affected child, preferable source of DNA is blood. The
5 mL blood in EDTA can be transported to the laboratory at
room temperature within 24–48 hours for further testing.
However blood spots and buccal swabs can also be used
in case the child is very young or parents are apprehensive
of venipuncture.
On the other hand, for prenatal diagnosis fetal DNA
can be obtained from chorionic villi sample, amniotic fluid
and cord blood. Chorionic villi sample is usually preferred
over amniotic fluid and cord blood because it can be
done at earlier gestation and gives better yield of DNA.
Fig. 3 No risk of affected child if one parent is affected The 20–25 mg of tissue in heparinized saline provided by
laboratory/RPMI culture medium can be transported to
the laboratory within 24–48 hours at room temperature,
OArab from each other and also HbS and, where fetal tissue is carefully separated from maternal
DPunjab from each other. tissue by looking under inverted microscope.
iv. HPLC for simultaneous detection and quanti Antenatal screening followed by prenatal diagnosis
tation of hemoglobin fractions. in carrier couples and selective termination of affected
– Supplementary hematological methods pregnancies is the only modality available which could
i. S, Fe and Ferritin, transferrin saturation significantly reduce genetic load of hemoglobinopathies
ii. Globin chain synthesis in various countries. Cyprus for instance, has reduced
iii. Immunological measurement of fetal cells for their incidence of thalassemia and thus stands as an ideal
HPFH. Distinguishes between heterocellular for other countries. It has progressed from reporting the
and pancellular distribution highest occurrence of thalassemia in the world to nil
– DNA analysis: An analysis or the research within an impressive span of just 11 years (1991–2002).8 In
conducted at various places across the globe reveals addition to the incidence, the carrier frequency has also
that hemoglobinopathies are caused due to the been reduced by 1.89% in 24 years.9 Today, in India also
many centers have opened up which are actively involved partners. If the family has a previously affected child then
in providing prenatal diagnosis. the most important step in prenatal diagnosis is to first
determine the mutation status of the affected child in the
Carrier screening and prenatal diagnosis: The best way
pretransfusion state along with the parents genotyping. If
to decrease the burden of hemoglobinopathies is to
proband is not available then, parental DNA can be tested
perform carrier screening followed by prenatal diagnosis
for thalassemia mutation followed by prenatal diagnosis.
and selective termination of affected pregnancies.
There are five common mutations reported in Indian
Before prenatal diagnosis was available, premarital
population along with about 12 rare mutations. According
screening and counseling did not affect the marriage and
to our experience mutations are identified in 95–97% of
reproductive behavior in Greece and Cyprus, countries
North Indian families.
with very high carrier frequency. With the introduction
The technique used for identification of mutations
of prenatal diagnosis along with mandatory premarital
is based on allele specific amplification and is called
screening had a tremendous effect on reducing the birth
amplification refractory mutation system (ARMS) PCR in
of affected babies to almost zero. This was only possible
most centers in India and abroad and is very reliable.10
by mass education program with involvement of religious
If the mutations are identified chorionic villus sampling
leaders. The screening protocol which is usually followed
(CVS) is offered around 10-12 weeks of pregnancy after
is outlined in the Flow chart 1.
counseling. CVS is the most widely used sample as it gives
Prerequisites for prenatal diagnosis:
good DNA yield and can be done early in pregnancy. The
• Confirmation of carrier status in both partners and
risk of fetal loss with CVS is around 2-3%. It usually takes
identification of disease causing mutations/informa
about a week for initial mutation screening and the same
tive linkage
time for CVS reporting. In situations when the mutations
• Counseling about recurrence risk of disease (25% in
are not identified, use of linkage studies or cord blood
each pregnancy) and medical termination of affected
analysis for globin chain synthesis or HPLC can be useful
pregnancy
for prenatal testing.
• Explanation about procedural risk (i.e. chances of fetal
Flow chart 2 shows the strategy being followed in our
loss).
laboratory for prenatal diagnosis.
Prenatal diagnosis is increasingly becoming available
world over. Across India, many centers are now offering
prenatal diagnosis using molecular techniques.6,7 Once Flow chart 2 Thalassemia prenatal diagnosis protocol
the carrier status of the partners is confirmed, it is followed
by DNA studies to identify the mutant allele in both the
Flow chart 1 Thalassemia carrier screening protocol for

thalassemia
DNA Techniques Required for nucleotide at the -2/-3 position from 3’ end to increase the
Hemoglobinopathies specificity of these primers. Second modification is addition
of another set of primers amplifying a different gene locus
Detection of Known Mutations which acts as an internal control. This modification is
introduced while setting up an ARMS PCR reaction. To
There are numerous PCR based techniques which are being
detect five common Indian mutations the general practice
used to detect known mutations, other than large deletions
is to set up four different reactions containing one mutant
and rearrangements causing hemoglobinopathies.
primer for each mutation naming IVS1-5(G>C), IVS1-
Commonly used techniques are amplification refractory
1(G>T), Cdn8-9(+G) and Cdn41-42(-CTTT), a common
mutation system, restriction enzyme PCR (RE-PCR) and
reverse primer and also a set of primer specific to 5th
reverse dot blot analysis with allele specific oligonucleotide
common mutation (619 bp deletion). This primer set for
probes.
619 bp deletion serves dual purpose. First it serves as an
internal control by amplifying a region of 861 bases and
Reverse Dot Blot Analysis second it also detects patients having this deletion of 619
Reverse dot blot analysis is a nonradioactive technique bases from that region by showing an additional band
in which allele-specific oligonucleotide (ASO) probes of 242 bp size. Figure 4A depicts the principle of ARMS
are immobilized on a nylon membrane. Separate probes PCR and Figure 4B shows an agarose gel photograph of a
complementary to each known mutant normal allele common beta-thalassemia mutation.
are spotted on a nylon membrane and subjected to ARMS PCR is mostly preferred to detect known point
hybridization with patient DNA. Signals are generated mutations, small insertions and deletions over RFLP
only for those particular mutations which are present in and direct gene sequencing due to its simplicity, cost
patient DNA. These strips are commercially available and effectiveness and less labor intensive nature.
can be customized according to mutation spectrum of any
population. Prior knowledge of common and uncommon Restriction Enzyme PCR
mutations in a population and simplicity of the technique
Restriction enzyme PCR (RE-PCR) is less preferred
make it affordable and suitable for routine diagnostic labs.
technique for molecular diagnosis of hemoglobinopathies
In India, this technique is commonly used to detect five
common mutations and other variants like HbS, HbD and because consumption of restriction enzyme for a large
HbE in HBB gene. Whereas in other countries like Sicily set of samples make it comparatively expensive than
it is also used to detect α-thalassemia and δ-thalassemia ARMS PCR and there are very few mutations in the
point mutations. list of hemoglobinopathies which creates or abolish
a pre-existing restriction site. It was previously used
very commonly for the detection of HbD, HbS and HbE
Amplification Refractory Mutation System
mutations. Flanking sequence of a particular mutation is
Amplification refractory mutation system (ARMS) is first amplified by PCR before cutting a mutant DNA with a
the most commonly used technique throughout the restriction enzyme resulting in a different restriction map.
world to detect known common and few rare mutations For example, a missense mutation (A > T) at codon 6
causing beta-thalassemia. Its principle is based on the of HBB gene causing the substitution of glutamic acid by
quality of perfectly matched primer over mismatched valine resulting in sickle cell anemia the most common
primer resulting more efficiency in annealing and primer hemoglobinopathy of tribal population abolishes a
extension. The 3’ terminal nucleotide of these primers specific recognition site for the restriction endonucleases
is specific to the desired allele. Hence annealing of this Dde I and Mst II.
primer is possible only if the desired (Mutant/normal)
allele is present in the tested DNA. So, for every patient
Gap PCR
two parallel reactions in two separate tubes containing
normal and mutant primer each are set up. A homozygous Detection of large genomic rearrangements, gross
mutant and wild sample shows amplification only in the deletions causing deletional HPFH, δβ-, γ δβ-, εγ δβ –
tube containing mutant and normal primer respectively thalassemia and α thalassemia was previously based on a
whereas a heterozygous sample shows amplification in nonPCR technique called southern blot. But with time and
both tubes. technical advancements this technique has been totally
Two modifications are generally practiced in most of replaced by Gap PCR.
ARMS PCRs. First modification is done at the time of primer If the deletion is less than 1 Kb then only two primers
designing. This includes the incorporation of a mismatched flanking deleted sequence are needed which generate
B
Figs 4A and B (A) ARMS PCR; (B) gel photograph of ARMS
two fragments of different sizes in heterozygous patients. and rearrangements in addition to the previously reported
The smaller one is due to deletion. But in case of larger in literature. But its high cost limits its use in populations
deletions one more primer complementary to a part of where frequency of large deletions and rearrangements is
deleted sequence is needed which gives an amplification very low.
in normal individuals, as distance between primers
flanking deleted sequence is too large to amplify normal Detection of Unknown Mutations
allele. On the other hand, in the presence of deletion these
flanking primers produce an amplified product. Detection of unknown mutations was previously based
Gap PCR is much faster, simpler, cost effective and less on few screening techniques like denaturing gradient
labor intensive in comparison with southern blot hence gel electrophoresis/single stranded conformation
most suitable for a routine diagnostic set up. polymorphism/heteroduplex analysis followed by
MLPA is another technique which is gaining in sequencing of that particular exon where a change
reputation day by day. The technique has an advantage over was suspected by any of above mentioned screening
gap PCR because of its ability to detect unknown deletions technique.
However, these days, most laboratories resort to heterozygosity screening of different intragenic markers
direct gene sequencing as a first hand test since the rapid like AVA II, BamH I, etc. in family members including
advancement and competition within the biotechnology, parents, previous affected child/normal child. By
sector has led to a significant fall in the cost of gene establishing heterozygosity of these markers and following
sequencing. This has made the process of reporting their patterns in parents and affected child mutated allele
faster and high throughput. Direct sequencing analysis can be tracked indirectly in fetal DNA with no knowledge
is particularly applicable to the globin genes which are of causal mutations. The β-globin gene cluster is known
compact and relatively small (1.2–1.6 kb) with the majority to characterize at least 18 restriction fragment length
of the point mutations within the gene or its flanking polymorphism (RFLP). But nonrandom association of
sequences. Mutations in the HBB gene are not limited to these sites result in only few haplotypes. Application of
the exons and their direct splice sites so the primers are this technique is limited to only those families where at
designed in the manner that they cover deep intronic least two intragenic markers are informative and previous
region and regulatory sequence. affected child/normal child’s sample is available. The
diagnostic error of this technique is a little higher (0.3%)
Diagnosis Using Indirect Methods than direct mutation detection methods due to chance of
recombination between RFLP site and mutation locus.
Restriction Fragment Length Polymorphism (Fig. 5) Laboratories having no access to relatively advanced
Linkage analysis can be an approach for prenatal and expensive techniques of direct mutation analysis
diagnosis in families at risk where mutations could not (e.g. gene sequencing and MLPA) can make use of this
be identified by using direct mutation detection methods. technique in families at risk of producing children with
Before offering PND to these families it is mandatory to do hemoglobinopathies.
Cord blood high performance liquid chromatography
or globin chain synthesis in fetal blood may be useful in
rare situations if the mutation is not identified.
PRECAUTIONS TAKEN WHILE DOING DNA

ANALYSIS
A lab following reasonably good standards can also result
in 1-2% diagnostic error. This can arise due to several
technical reasons such as maternal DNA contamination,
cross contamination causing false amplifications
leading to false positive results, false paternity, genetic
recombination, deficient endonuclease digestion, allele
drop out, undiagnosed hemoglobinopathy in compound
heterozygote state in parents leading to misdiagnosis of
fetus and also due to human errors like sample exchange,
mislabeling of samples, etc.
Following guidelines can help in minimizing these
errors:
• Chorionic villus sample should be washed in saline to
get rid of maternal blood and it should also be dissected
under microscope to get rid of maternal decidua before
sending to the fetal diagnostic laboratory
• Appropriate positive and negative controls should
always be analyzed with fetal sample
• Fetal sample should always be run in duplicate
• PCR program should have a limited number of cycles
to minimize amplification contaminating DNAs
• VNTR analysis using different polymorphic markers
should always be run in parallel to rule out maternal
Fig. 5 Use of linkage studies for prenatal testing contamination
• Every sample should have more than one identification mutations. To overcome this problem now focus is
code e.g. full name, lab number, date of birth, sample towards finding new SNPs which are linked to known
date, etc. beta-thalassemia mutations and can be used later as a
• The test that involves gene sequencing should always marker for NIPD of beta-thalasssemia in couples carrying
be done in both directions same mutations. Papasava, et al. used this approach
• The type of DNA analysis method used and its inherent in combination with arrayed primer extension (APEX)
risk of misdiagnosis should be clearly mentioned in method in genetically homogenous population of Cyprus
prenatal diagnosis report. and screened 34 families to know the most informative
paternally inherited single nucleotide polymorphisms
Noninvasive Methods (SNPs). Only 11 families were infomative for more than
two SNPs.16
Owing to abortion risk to the fetus and the anxiety experi More recently a group in Netherland assessed
enced by mothers undergoing any prenatal diagnostic the possibility of using pyrophosphorolysis-activated
procedure done to rule out hemoglobinopathies, focus polymerization (PAP) technique for noninvasive prenatal
has been shifted towards development of noninvasive diagnosis (NIPD) of β-thalassemia major and sickle-
tests which are not only accurate but rapid, and can be cell disease (SCD). Phylipsen, et al. developed this assay
performed early in pregnancy for prenatal diagnosis. to detect SNPs specifically inherited from the father by
There are two noninvasive approaches which have linkage to the normal or mutant allele to determine the
been followed till today. One involves analysis of cell free risk of having an affected fetus. This approach can provide
fetal DNA in maternal plasma and serum and the other NIPD to the families where both partners carry same
approach utilizes fetal cells within maternal circulation as mutation.17
a source of fetal DNA. Determination of fetal gender and Tracking associated SNP to the paternal allele is
RhD status of the fetus with in RhD-negative pregnant an indirect approach of knowing presence of father’s
women by using maternal plasma and serum has already mutation and has its own limitations. For instance, the low
been established in European countries.11,12 heterozygosity of these markers make the analysis more
This approach, however is not suitable for the analysis time consuming and allows only few families suitable for
of fetal loci that do not differ largely from the maternal allele NIPD. Secondly, it does not give any direct information of
(e.g. beta-thalassemia), due to the vast predominance of the genotype of the fetus.
cell-free-maternal DNA in maternal samples. The introduction of massive parallel sequencing in the
To overcome this problem researchers have used field of NIPD and recent demonstration of deep sequencing
size fractionation as a possible enrichment technique to of maternal plasma genome augmented the applicability of
enrich fetal DNA molecules. Use of peptide nucleic acids CFF DNA to many more diseases unlike before. Applicability
followed by allele specific real time PCR to suppress the of this technique in screening aneuploidies has already
amplification of wild type maternal allele is another been established but in the field of hemoglobinopathies
approach which has been used extensively and has given research is limited to very few reports and world is still
promising results in detection of paternally inherited waiting for an accurate, safe, rapid, noninvasive tests
fetal point mutations of beta-thalassemia and sickle cell for prenatal diagnosis of hemoglobinopathies. More
disorder.13 recent technologies such as next generation sequencing
MS-SABER (mass spectrometry-based single- (NGS) and whole genome sequencing (WGS) although
allele base extension reaction) and MS-ASBER (mass have potential to replace currently practiced invasive
spectrometry-based allele-specific base extension procedures for both pre- and postnatal diagnosis in the
reaction) have also been used in this field and have enabled near future. Although highly useful, these techniques will
sensitive differentiation of fetal specific alleles down to a not be suitable to all laboratories performing prenatal
single nucleotide level and has shown to be useful for the diagnosis of hemoglobinopathies given their high cost and
detection of certain beta-thalassemia mutations and HbE sophistication.
disease respectively.14 Full range of genetic disorders including
Moreover Gaibiati, et al. explored the applicability hemoglobinopathies can be diagnosed noninvasively
of COLD PCR (coamplification at a lower denaturation through examination of intact fetal cells circulating with
temperature polymerase chain reaction) to enrich in maternal blood. But due to scarcity of fetal cells within
paternally inherited mutated allele in maternal plasma maternal blood, procedures to enrich the cells and enable
which was then detected on a sequencer.15 single cell analysis with high sensitivity are the complexities
Applicability of above mentioned techniques is limited associated with this technique. Recent advancements like
to only those families where partners carry different lectin-based method to separate fetal cells from maternal
blood and autoimage analysis have reported better 7. Arora S, Kabra M, Maheshwari M, et al. Prenatal diagnosis
sensitivity. The noninvasive prenatal testing is already in of hemoglobinopathies. Natl Med J India. 2001;14:340-2.
clinical practice for aneuploides and the progress in the 8. Bozkurt G. Results from the north cyprus thalassemia
field of single gene disorders is promising. prevention program. Hemoglobin. 2007;31(2):257–64.
9. Kyrri AR, Kalogerou E, Loizidou D, et al. The changing
epidemiology of β-thalassemia in the Greek-Cypriot
COUNSELING
population. Hemoglobin. 2013;37(5):435–43.
Counseling for hemoglobinopathies include exact 10. Old J, Petrou M, Varnavides L, et al. Accuracy of prenatal
determination of parental genotypes, proper understanding diagnosis for haemogloin disorders in the UK: 25 years’
of their interaction and assessment of clinical severity of the experience. Prenat Diagn. 2000.pp.986-99.
disease in the fetus. It should also be accompanied with the 11. Devaney SA, Palomaki GE, Scott JA, et al. Noninvasive fetal
sex determination using cell-free fetal DNA: a systematic
available treatment and the overall prognosis. One should
review and meta-analysis. JAMA J Am Med Assoc. 2011;
offer the prenatal diagnosis by available methods along
306(6):627–36.
with the adequate pretest and post-test counseling. 12. Finning K, Martin P, Daniels G. A clinical service in the UK
to predict fetal Rh (Rhesus) D blood group using free fetal
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23.
1. Old JM. Prenatal Diagnosis of the Hemoglobinopathies. In 13. Li Y, Di Naro E, Vitucci A, et al. Detection of paternally
Genetic disorders and the fetus. 6.pp.646–79. inherited fetal point mutations for beta-thalassemia using
2. Weatherall DJ, Clegg JB. The alpha-chain-termination size-fractionated cell-free DNA in maternal plasma. JAMA
mutants and their relation to the alpha-thalassaemias. J Am Med Assoc. 2005;16:293(7):843–9.
Philos Trans R Soc Lond B Biol Sci. 1975;271(913):411-55. 14. Ding C, Chiu RWK, Lau TK, et al. MS analysis of single-
3. De Jong WW, Meera Khan P, Bernini LF. Hemoglobin Koya nucleotide differences in circulating nucleic acids:
Dora: high frequency of a chain termination mutant. Am J Application to noninvasive prenatal diagnosis. Proc Natl
Hum Genet. 1975;27(1):81–90. Acad Sci USA. 2004;101(29):10762–7.
4. Mishra RC, Ram B, Mohapatra BC, et al. High prevalence 15. Galbiati S, Brisci A, Lalatta F, et al. Full COLD-PCR protocol
and heterogenicity of thalassaemias in Orissa. Indian J for noninvasive prenatal diagnosis of genetic diseases.
Med Res. 1991;94:391-4. Clin Chem. 2011;57(1):136–8.
5. Weatherall DJ. The thalassaemia syndromes, 4th edition. 16. Papasavva TE, Lederer CW, Traeger-Synodinos J, et al. A
General Haematology Task Force of the British Committee minimal set of SNPs for the noninvasive prenatal diagnosis
for Standards in Haematology. Guideline: The Laboratory of β-thalassaemia. Ann Hum Genet. 2013;77(2):115–24.
diagnosis of Haemoglobinopathies. Br J Haematolo. 1998; 17. Phylipsen M, Yamsri S, Treffers EE, et al. Non-invasive
101:783-92. prenatal diagnosis of beta-thalassemia and sickle-
6. Saxena R, Jain RK, Thomas E, et al. Prenatal diagnosis of cell disease using pyrophosphorolysis-activated
b-thalassemia: experience in a developing country. Prenat polymerization and melting curve analysis. Prenat Diagn.
Diagn. 1998;18:1-7. 2012;32(6):578–87.
C H A P T E R 20
Red Cell Membrane Disorders
(Spherocytosis, Elliptocytosis,
Stomatocytosis)
Normal RBC survival span is 110 to 120 days (half life, 55–60 days). The plasma membrane of the red blood cell (RBC) consists of a
complex ordered array of lipids and proteins stretched over the outer surface of the cell in the form of a lipid bilayer that is dotted by
penetrating or surface proteins. Specialized interactions occur between specific membrane proteins or lipids, or both, to maintain the
stability of the membrane. Various red cell membrane proteins are necessary to maintain the normal shape of an erythrocyte, which
is a biconcave disc.1,2 A deficiency of any of the components of this membrane can lead to distorted red cell morphology (Fig. 1),
increased breakdown of red cells and anemia, i.e. intracorpuscular (intrinsic) hemolytic anemia (Table 1).
Common red cell membrane defects include: Table 1 Major human erythrocyte membrane proteins
• Hereditary spherocytosis SDS gel band Protein Location of
• Hereditary elliptocytosis protein in red cell
• Hereditary stomatocytosis membrane
1 Alpha spectrin Peripheral
HEREDITARY SPHEROCYTOSIS 2 Beta spectrin Peripheral
It is a genetically-transmitted form of spherocytosis, an 2.1 Ankyrin Peripheral
autohemolytic anemia characterized by the production of 2.9 Alpha adducing Peripheral
red blood cells that are sphere-shaped rather than donut- 3 AE1 Integral
shaped, and therefore more prone to hemolysis.
4.1 Protein 4.1 Peripheral
4.2 Peripheral
Prevalence 4.9 Demantin Peripheral
It is the most common red cell membrane defect and P55 Peripheral
is especially common in people of North European or 5 Beta actin Peripheral
Japanese descent, where the prevalence may be as high as Tropomodulin Peripheral
1/5000. 6 G3PD Peripheral
7 Stomatin Integral
Etiology Tropomyosin Peripheral
Hereditary spherocytosis is an autosomal dominant trait, 8 Protein 8 Peripheral
although sometimes the mode of inheritance can be PAS-1 Glycophorin A Peripheral
recessive, and an estimated 25 percent of cases are due to
PAS-2 Glycophorin B Peripheral
spontaneous mutations. A patient has a 50 percent chance
of passing the disorder onto his/her offspring, presuming PAS-3 Glycophorin C Peripheral
Glycophorin D Peripheral
that his/her partner does not also carry the mutation.
Glycophorin E Peripheral
Fig. 1 The structural composition of the red cell membrane in vertical and horizontal interactions
Table 2 Common gene mutations in hereditary spherocytosis of hereditary spherocytosis. The degree of anemia is
extremely variable and may be absent, mild, moderate, or
Mutation Protein-coded Inheritance
severe to the point of threatening life. The clinical severity
ANK1 Ankyrin Dominant/Recessive of this condition is generally classified into three forms, as
AE1 (SL4A1) Band 3 Mostly dominant shown in Table 3.
SPTB β-Spectrin Dominant Mild: It occurs in 20 to 30 percent of cases. These
SPTA1 α-Spectrin Recessive patients have no anemia, modest reticulocytosis, and
EPB42 Protein 4.2 Recessive little splenomegaly or jaundice, and may not be detected
until adolescence or adult life. Increase in erythropoiesis
is maintained via erythropoietin despite accelerated
• Hereditary spherocytosis is caused by a variety of hemolysis. The stimulus for increased production of
molecular defects in the genes that code for red cell erythropoietin is not known but does not appear to be
membrane. The protein that is most commonly hypoxia.
defective is ankyrin (dominant and recessive defects). When detected in the neonatal period, it is commonly
A recessive defect has also been reported in α-spectrin; accompanied by jaundice, requiring.
dominant defects have been reported in β-spectrin Treatment with phototherapy or exchange transfusion
and protein 3. Hemolysis can be marked in the neonate due to an
Table 2 depicts the common gene mutations seen increased level of Hemoglobin F. Hemoglobin F binds
in hereditary spherocytosis. These defects lead to loss 2,3-diphosphoglycerate poorly, consequently increased
of membrane surface area while the mean corpuscular levels of 2,3-diphosphoglycerate destabilizes spectrin-
volume is normal, and a consequent sphering of red blood actin-protein 4.1 interactions in the red cell membranes.
cells is seen. An increased permeability of the red cell However, most neonates have little or no anemia,
membrane to sodium occurs, which is compensated by an reticulocytosis, or spherocytosis on the peripheral blood
active transport of sodium out of the cell by a cation pump smear; this is followed by a reduction in the hemo
mechanism. There is an increased glycolysis to generate globin concentration over the ensuing three weeks
the adenosine triphosphate needed for the active transport that is transient, but may be severe enough to require
of sodium out of the red cells. The spherocytes are also less transfusions.5
deformable and hence are destroyed in the spleen during In infancy and childhood the presentation is variable.
passage through the splenic cords to the splenic sinuses.3,4 Some children present with pallor, acholuric jaundice,
icterus, exercise intolerance, and progressive splenomegaly
(Minkowski-Chauffard syndrome). Pigmentary gallstones
Clinical Features
appear starting from 4 to 5 years of age. Hemolytic facies
It has a varied clinical presentation, ranging from may be seen but are less marked than in thalassemia
asymptomatic to severe hemolysis.3,4 Anemia, jaundice major. Radiography may show widened diploe of skull
and splenomegaly are the common clinical features bones and oxycephaly. Chronic leg ulcers may be seen.
Chapter-20 Red Cell Membrane Disorders (Spherocytosis, Elliptocytosis, Stomatocytosis) 215
Table 3 Clinical severity of hereditary spherocytosis

Trait Mild Moderate Severe
Hemoglobin (g/dL) Normal 11–15 8–12 6–8
Reticulocyte count (%) ≤3 3.1–6 ≥6 ≥10
Bilirubin (mg/dL) ≤1 1.0–2.0 ≥2.0 ≥3.0
Spectrin per erythrocyte 100 80-100 60–80 40–60
Osmotic fragility:
• Fresh blood Normal Normal/slightly increased Increased Increased
• Incubated blood Slightly Increased Increased Increased

increased
Autohemolysis
without glucose (%) <10 ≥10 ≥10 ≥10
Correctability (%) >60 >60 0–80 50

Splenectomy Not Usually not needed during Necessary during school Necessary, delay till 6 years,
needed childhood and adolescence age before puberty if possible
Symptoms None None Pallor, erythroblastopenic Pallor, erythroblastopenic
crisis, hyperbilirubinemia, crisis, hyperbilirubinemia,
gallstones gallstones
These patients are particularly prone to aplastic crisis due • The red blood cells appear sphere shaped and lack the
to parvovirus infections. central pallor (Fig. 2).
Peripheral blood smear shows microspherocytes and
Diagnosis polychromasia. The percentage of microspherocytes usu
ally correlates with the severity of hereditary spherocy
The diagnosis of hereditary spherocytosis is established tosis. The mean corpuscular volume is normal, and the
by a combination of clinical examination, detailed history mean corpuscular hemoglobin concentration is raised
including family history, and laboratory tests. (36–38 g/dL). The red cell distribution width (RDW) is
• A positive family history is seen in up to 75 percent of increased.
patients. Evidence of hemolysis is in the form of reticulocytosis
(3–15%), decreased haptoglobin, indirect hyperbiliru
binemia and ultrasonic detection of gallstones may be
seen. Coomb’s test is negative. Increased red cell osmotic
fragility is seen. Spherocytes lyse in higher concentrations
of saline than normal red cells. This feature gets
accentuated when RBCs are deprived of glucose for 24
hours at 37°C (Incubated osmotic fragility test). However,
this test is not specific for hereditary spherocytosis, but may
be positive in hereditary elliptocytosis. Also, the test may
be negative in presence of iron deficiency, during recovery
from aplastic crisis or in presence of obstructive jaundice.
Also osmotic fragility cannot differentiate the immune
and non immune causes of spherocytosis. The eosin-5-
maleimide (EMA) binding dye test, which requires a flow
cytometer is also positive, and may be used as a screening
test in addition to cryohemolysis test. Gel electrophoresis
analysis of erythrocyte membrane proteins may be used as
Fig. 2 Spherocytes, lacking, central pallor confirmatory diagnostic test in selected cases.
Complications functions of spleen. Usually the lower pole is removed

• Hemolytic crisis while the upper pole is left in situ.7 Sometimes, there is a
• Erythroblastopenic crisis failed splenectomy, when the child continues to be pale
• Folate deficiency
and transfusion dependent even after splenectomy. This
is usually due to the presence of accessory spleens in the
• Gallstones
body.
• Hemochromatosis
• Leg ulcers
• Growth retardation
Two to Three Weeks Prior to Splenectomy
• Children should be vaccinated against pneumococcal,
Treatment meningococcal, and Halmophilus influenza B infec
tions.
Transfusion: Continued transfusion dependence is unu • Children should receive prophylaxis with penicillin
sual and it is important to avoid repeated transfusion (age < 5 year: 125 mg BD, age ≥ 5 year: 250 mg BD).
whereas possible. Many older children with Hb levels of Following splenectomy.
5 to 6 g/dL do not require transfusion. Children who • There is disappearance of jaundice, anemia, and
require one or two transfusions early in life frequently reticulocytosis.
become transfusion independent. • Peripheral smear shows the presence of Howell-Jolly
• A regular follow-up is to be done once a child is bodies, target cells, acanthocytes and siderocytes
diagnosed to have hereditary spherocytosis (HS). An • Mean corpuscular volume of RBCs rises, while the
annual visit to the physician is recommended even in mean corpuscular hem oglobin concentration falls.
the absence of symptoms. A hemogram is unnecessary • Pigment gallstones usually develop in patients of HS. In
in the absence of symptoms but a clinical examination case of symptomatic gallstone disease, it is preferable
including a general assessment, measurement of to remove the gallbladder at the time of splenectomy.
splenic size, growth, and exercise tolerance is needed.
• An ultrasonogram of abdomen, every 3 to 5 years is HEREDITARY ELLIPTOCYTOSIS
needed to look for gallstones, starting from the age of
Hereditary elliptocytosis are a heterogeneous group
5 years.
of inherited erythrocyte disorders, most of which are
• In the presence of chronic anemia, there may be
autosomal dominant that have in common the presence
increased iron absorption in these patients, necessita
of elongated, oval, or elliptically shaped red blood cells
ting estimation of iron load during follow-up visits.
(15–50% of RBCs) on the peripheral blood smear. Some
• Folic acid supplementation (2.5 mg/day up to 5 years
conditions like thalassemias, and iron deficiency anemia,
age and 5 mg/day thereafter) is needed to meet the
are also characterized by the presence of elliptocytes on
increased bone marrow requirements. In presence
peripheral smear but they constitute less than 10 percent
of erythroblastopenic crisis, leukocyte-depleted red
of RBCs.
blood transfusions are needed. Erythropoietin may be
of benefit in reducing or avoiding transfusion, and can
usually be stopped by the age of 9 months. Splenectomy
Prevalence
is needed in moderate and severe cases.4–6 The prevalence of hereditary elliptocytosis in the United
States is not greater than 2.5 to 5 per 10000. However,
Indications for Splenectomy in West Africa and South-east Asia, where malaria is
endemic, it may reach 1.6 and greater than 30 percent of
• Severe anemia the population, respectively. The hereditary pyropoikilo-
• Reticulocytosis > 10 percent cytosis variant of hereditary elliptocytosis is more frequent
• Repeated hypoplastic or aplastic crisis among black population, while the spherocytic elliptocy-
• Faltering growth tosis variant is reported only in Caucasians.
Following splenectomy, there are chances of septi
cemia, and thrombosis. Therefore, where possible,
splenectomy should be deferred till six years. Even in severe
Etiology
cases of HS who are transfusion dependent, it should not It is usually inherited as, however, hereditary pyropoi
be done till 3 years of age to avoid risk of septicemia. Some kilocytosis is a severe variant where there are two defective
people recommend partial splenectomy, where around alleles. Usually, there is a defect in spectrin leading to
85 to 90 percent of spleen is removed, while 10 to 15 defective spectrin heterodimer self-associations. Rarely,
percent of spleen is left behind to preserve the immune there may be abnormalities in protein 4.1 or glycophorin C.
Chapter-20 Red Cell Membrane Disorders (Spherocytosis, Elliptocytosis, Stomatocytosis) 217
jaundice with presence of poikilocytes and pyknocytes

on peripheral smear.
• The bone changes appear later.
South-east Asian Ovalocytosis is characterized by
presence of ovalocytes in the peripheral smear, which are
less elongated than elliptocytes. It is due defective protein
3. This condition offers protection from Plasmodium falci
parum malaria. Hereditary pyropoikilocytosis is the most
severe form characterized by presence of microspherocytes
in blood smear whose mean corpuscular volume is
decreased (MCV: 50–60 fL). This condition as the name
suggests is characterized by increased thermal lability of
RBCs and they lyse at 45 to 46°C, instead of 49 to 50°C.
Treatment
Fig. 3 Stomatocytes showing a slit-like gap in center of red cells • Treatment is needed only if there is chronic hemolysis.8,9
• Folic acid supplementation (1 mg/day) is needed.
• Splenectomy may be indicated, if the hemoglobin is
Elliptocytic RBCs are the characteristic finding on
below 10 g/dL or there is reticulocytosis >10 percent.
the peripheral smear (Fig. 3). The elliptocytic shape is
conferred by the red cell membrane skeleton, as shown
by persistence of the shape change in red cell ghosts and HEREDITARY STOMATOCYTOSIS
in skeletons prepared from ghosts by removal of the lipid Hereditary stomatocytosis is a group of autosomal
bilayer. Elliptocytes form as the mature RBC ages in vivo, dominant conditions which are characterized by the
since RBC precursors in the HE syndromes are round and presence of cup-shaped red blood cells (Fig. 4). The red
do not exhibit morphologic abnormalities. The elliptocytic cell membrane leaks sodium and potassium ions. There
shape change is thought to result from repeated episodes is a defective protein 7.2 or stomatin on chromosome 9.
of elliptocytic deformation that all RBCs experience during A variety of variants of this condition have been described:
each circulatory cycle, as they pass through capillary beds.
Whereas normal RBCs regain a discocytic configuration Overhydrated Hereditary Stomatocytosis
by a process of elastic recoil, hereditary elliptocytosis
RBCs appear to have disruption of the connections • Most severe variety, stomatin gene may be defective.
between the various cytoskeletal components, followed • Dehydrated stomatocytosis (Hereditary Xerocytosis/
by the formation of new contacts that lock the cell into the Hereditary hyperphosphatidylcholine hemolytic ane-
elliptocytic configuration. mia): It is the most common variety of stomatocytosis
caused by defective protein 7.2 or stomatin gene.
Types of Hereditary Elliptocytosis
The clinical features in generally fall into one of five
categories:
1. Silent carriers
2. Common HE
3. Hereditary pyropoikilocytosis
4. Spherocytic elliptocytosis
5. South-east Asian ovalocytosis.8,9
Hemolytic anemia in these disorders ranges from
absent to life-threatening. Severe hemolysis is usually
a consequence of homozygosity or compound hetero
zygosity for one or more of the various membrane protein
mutations associated with this disorder.
• Usual features of hereditary elliptocytosis like anemia,
splenomegaly, cholelithiasis and In the newborn,
hereditary elliptocytosis may manifest as hemolytic Fig. 4 Elliptocytes seen on peripheral smear
• Dehydrated stomatocytosis with perinatal ascites. 3. Bolton-Maggs PHB. Hereditary spherocytosis: New
• Cryohydrocytosis: It is the mildest type where the Guidelines. Arch Dis Child. 2004;89:809-12.
RBCs lyse on cooling in vitro and it is associated with 4. Bolton-Maggs PHB, Stevens RF, Dodd NJ, Lamont G,
pseudohyperkalemia. Tittensor P, King MJ. Guidelines for the diagnosis and
• Blackburn variant. management of hereditary spherocytosis. Br J Hematol.
2004;126:455-74.
Treatment 5. Trucco JI, Brown AK. Neonatal manifestations of hereditary
spherocytosis. Am J Dis Child. 1967;113:263.
Usually no treatment is needed. Splenectomy is not 6. Bolton-Maggs PHB. The diagnosis and management of
indicated as there is a greater tendency to life-threatening hereditary spherocytosis. Baillieres Best Pract Res Clin
thrombosis following splenectomy due to thrombocytosis Haematol. 2000;13:327-42.
post-splenectomy coupled with abnormal adherence of 7. Tracy ET, Rice HE. Partial splenectomy in hereditary
stomatocytic RBCs to vascular endothelium.8,9 spherocytosis. Pediatr Clin N Am. 2008;55:503-19.
8. Martin PL. Hemolytic anemias. In McMillan J, Feigin RD,
DeAngelis CD, Jones MD (Eds). Oski’s Pediatrics, 4th
REFERENCES end. Lippincot Williams and Wilkins, Philadelphia; 2006.
1. Delaunay J. Genetic disorders of the red cell membrane. pp.1700-7.
Crit Rev Oncol Hematol. 1995;19:79-110. 9. Gallagher PG, Forget BG. Hereditary spherocytosis,
2. Gallagher PG, Lux SE. Disorders of erythrocyte membrane. elliptocytosis and related disorders. In: Beutler E,
In: Nathan DG, Orkin SH, Ginsburg D, Thomas Look A (Eds). Lichtman MA, Coller BS, Kipps TJ, Seligsohn U (Eds).
Nathan and Oski’s Hematology of Infancy and Childhood, William’s Hematology, 6th edn. Mc Graw Hill: New York.
6th edn, volume 1, Saunders, Philadelphia. 2006.pp.560-682. 2002.pp.501-16.
C H A P T E R 21
Red Cell Enzymopathy
The hereditary hemolytic anemia resulting from altered red blood cell (RBC) metabolism due to defects in various enzymes associated
with glycolytic pathway, hexose monophosphate shunt or pentose phosphate pathway are described as red cell enzymopathy or
erythro-enzymopathy (EEP). These hereditary anemias are distinguished from hereditary spherocytosis by absence of spherocytosis
in the peripheral blood. The most well known and widely distributed EEP is the deficiency of G6PD, which is involved in the initial
reaction of pentose phosphate pathway.1,2 Deficiency of pyruvate kinase and other enzymes of glycolytic pathway also result in
hemolytic anemia but the magnitude of clinical problem resulting from deficiency of these enzymes is considerably less compared
to G6PD deficiency.
G6PD DEFICIENCY
The main role of pentose phosphate pathway is related
to metabolism of glutathione (GSH) through production
of reduced form of nicotinamide adenine dinucleotide
phosphate (NADPH). GSH is important for preservation of
sulfhydryl group in many proteins including hemoglobin
and to prevent the damage from oxidative radicals in
general. Thus, GSH should be constantly available in the
reduced form which is effected by, GSH reductase through
NADPH, the later is provided by G6PD.1 G6PD catalyses
nicotinamide adenine dinucleotide phosphate (NADP)
to its reduced form, NADPH (Fig. 1). NADPH protects Fig. 1 Pentose phosphate pathway
cells from oxidative damage. As red blood cells (RBCs)
do not generate NADPH in any other way, they are more
Prevalence
susceptible than other cells to destruction from oxidative
stress. Therefore deficiency of G6PD in red cells leads to G6PD deficiency is the most common EEP affecting
various clinical manifestations in human beings. The level approximately 400 million people worldwide.3 The dis
of G6PD activity in affected RBCs is lower than in other order is transmitted as a x-linked recessive trait. Though
cells in the body. the distribution of G6PD deficiency is worldwide, highest
Majority of mutations cause this enzyme deficiency prevalence is observed in Mediterranean countries,
in RBC by decreasing enzyme stability. The polymorphic Africa and Asia. In south-east Asia, the prevalence varies
mutations affect amino acid residues throughout the widely in different ethnic groups—10 to 20 percent in
enzyme and decrease the stability of enzymes in the RBC, certain Combodian groups to 1 to 3 percent in Vietnamese
possibly by disturbing protein folding.2 population groups.4
Table 1 Frequency distribution of G6PD deficiency in India Of them, 299 have been characterized with the methods
recommended by WHO.3,18 Molecular characterization
S. Authors Area/Caste/Tribes Frequency
is considered more important as different variants based
no. studied
on biochemical studies alone have been found to be
1. Thakur and Bastar (Central India), M12.3% same on molecular characterization. From India, 13
Verma,19928 Muria Gond tribes F–3.7%
different biochemically characterized variants have been
2. Handa et al, 19929 Punjab, Bania 2.8 % reported.3 A large series with biochemical and molecular
3. Ramadevi et al, Bengaluru (neonates) 7.8% characterization reported G6PD-Mediterranean to be
199410 the most common variant in India (60.4%) followed by
4. Kaeda et al, 199511 Odisha 3–15% G6PD-Kerala-Kalyan (24.5%) and G6PD Odisha (13.3%).
5. Balgir et al, 199912
Odisha (Mayurbhanj) 7.7–9.8%
Frequency distribution of various biochemical variants
is different in tribal and urban population. Kaeda et al.
6. Sukumar et al, Mumbai 5.7–27.9% observed that G6PD-Odisha is responsible for most cases
20047
in tribal population but is not found in urban population
7. Gupte et al.13 Gujarat, Surat, Vataliya 22% groups where most of the G6PD-Mediterranean is the
Prajapati most prevalent variant. G6PD-Chatham with undetected
enzyme activity and G6PD-Insuli with normal G6PD
In India, G6PD deficiency was first reported almost 40 activity are very rare among Indian population groups. 3,7,11
years ago.5 Prevalence in India varies from 0 to 27 percent
in various castes, tribes and ethnic groups.6 A recent series Clinical Features
from Mumbai including a large number of individuals from
various population groups reported overall prevalence of WHO has provided a classification of G6PD deficiency
10.5 percent the variation in various castes and linguistic which is based on residual activity of the enzyme and other
groups being 5.7 to 27.9 percent.6 characterization and clinical presentation (Table 2).19 The
Table 1 shows the prevalence rates of G6PD deficiency patients with G6PD deficiency can present clinically in
in various reports over the last 15 years from different parts following ways: acute hemolytic anemia (AHA), neonatal
of country and in different ethnic groups.7-13 jaundice (NNJ) and chronic non-spherocytic hemolytic
anemia (CNSHA).
MALARIA HYPOTHESIS
Variation in prevalence of G6PD deficiency has led to the Acute Hemolytic Anemia
hypothesis that G6PD deficiency is a polymorphism that Children with certain variants of G6PD deficiency are
confers protection from falciparum malaria. This hypo clinically in a steady state till they develop anemia of
thesis has been called G6PD/malaria or simply “malaria sudden onset under the effect of some oxidative stress.
hypothesis”.1,3 Malarial parasite grows less well in red Within 24 to 48 hours of exposure to such stress, the child
cells deficient in G6PD. Decreased parasitemia has been suddenly becomes pale and has discoloration of urine
documented in these individuals.14-16 A recent series by which is described as cola colored, strong tea colored
Mohanty et al. has reported good correlation between or simply brown in color. Other symptoms and clinical
prevalence of Plasmodium falciparum malaria and G6PD findings correspond to degree of anemia and hypoxia.
deficiency.17 Thakur and Verma have also observed inc
reased prevalence of antimalarial antibodies and higher
titers among those with normal G6PD levels compared to
Table 2 Categorization of G6PD variants
G6PD deficient persons.8 Mohanty et al. have referred to an
interesting observation that among Parsees in India who Class Clinical expression Residual G6PD Variants
migrated from Iran approximately 1300 years ago, preva activity (% of normal) reported
lence of G6PD deficiency is 15.7 percent which is considerably I Severe (CNSHA) < 20%* 94
higher as compared to prevalence among Zorasstrians in II Mild < 10% 114
Iran who belong to the same community. An explanation
III Mild 10–60% 110
offered is that at the time of migration, Gujarat and Mumbai
were endemic for malaria where Parsees settled.3 IV None 100% 52
V None >100% 2
Clinical and Biochemical Variants *Severe enzyme deficiency with CNSHA. The enzymes levels are usually
Based on biochemical and other characterization, less than 20% of normal. To classify in this group, the variant must be
442 different variants of G6PD have been identified. associated with CNSHA
Chapter-21 Red Cell Enzymopathy 221
Mild jaundice, breathlessness and tachycardia are often Table 3 List of drugs known to cause hemolysis in G6PD
present. Occasionally features of frank congestive cardiac deficient patients (In alphabetical order)
failure, backache and abdominal pain are observed.20,21
Actyl salicylic acid Ascorbic acid Chloramphenicol
Urinary discoloration is on account of intravascular
hemolysis but hemolysis is entirely not intravascular. Chloroquine Ciprofloxacin Colchicine
Depending upon degree of extravascular hemolysis, Dapsone Diphenhydramine Dopamine
splenic enlargement may be noticed. Hemolysis occurs Doxorubicin Furazolidone Isobutyl nitrite
after exposure to stressor but does not continue with Isoniazid Menadiol sod. Menadione
continued exposure. This is thought to be on account of sulfate
older RBCs being damaged first as they have most severe
Menadione sod. Mepacrine Nalidixic acid
deficiency of enzyme. Once the population of deficient bisulfate
RBC are hemolyzed, the juvenile RBC and reticulocytes
Naphthelene Niridazole Nitrofurazone
withstand the stress as they have typically higher levels
of enzyme.2 Hemoglobinemia and hemoglobinuria may Nitrofurantoin Norfloxacin Paracetamol
result in azotemia and/or acute renal failure. In one Indian Para amino-benzoic Phenacetin Phenytoin
series on acute renal failure in children, G6PD deficiency acid
accounted for 6 percent cases. Azotemia occurred in 20 Phenylbutazone Phytomenadione Primaquine
percent of 35 patients with G6PD deficiency in another Probenecid Procainamide Proguanil
series.20,22
Pyrimethamine Quinidine Quinine
Laboratory findings during intravascular hemolysis
and AHA include moderate to severe anemia which is Streptomycin Sulfadiazine Sulfadimidine
usually normocytic-normochromic. RBC morphology Sulfaguanadine Sulfmethoxazole Sulfanilamide
shows anisocytosis due to increased number of juvenile Trimethoprim
red cells and contracted cells. Poikilocytosis with New Drugs added in
presence of ‘bite cells’ (as if some portion of red cell has 2002
been bitten away) may be seen. Intense reticulocytosis
Astemizole Azatidine Cetirizine
is present. Plasma hemoglobin level is increased and so
is unconjugated bilirubin level. Haptoglobin and other Chlorpheniramine Cyproheptadine Diphenhydramine
hemoglobin binding proteins are decreased.1 Loratadine Promethazine Terfenadine
Pathogenesis of such events involves oxidation
of glutathione (GSH) to GSSG. On account of G6PD
deficiency, the red cells of these patients have limited NEONATAL JAUNDICE
capacity to regenerate GSH and the reserve gets depleted Other than AHA, neonatal jaundice (NNJ) is a common
soon. Exhaustion of GSH allows oxidation of sulfhydryl manifestation of G6PD deficiency. Almost one-third
group of hemoglobin (and other proteins) resulting of male neonates have been described to have NNJ.
in denaturation of hemoglobin. Coarse precipitates of NNJ resulting from G6PD deficiency has worldwide
hemoglobin lead to damage of red cell membrane and distribution occurring in Mediterranean countries, Africa
hemolysis. As is evident, the prerequisite for such a series and Asia. In an Indian series, in 12 out of 100 neonates
of events is oxidative stress which occurs in the form of with jaundice, G6PD deficiency was the cause, of them10
exposure to various drugs and “triggers”. In fact, G6PD being G6PD Mediterranean type.28 In another large series
was first described during investigation for ‘primaquin of 551 cases of NNJ, G6PD deficiency was the largest
sensitivity’. Since then many drugs have been incriminated single identifiable cause accounting for 17.1 percent.28
to result in intravascular hemolysis in individuals with Among neonates with severe jaundice requiring exchange
G6PD deficiency. Table 3 lists the drugs which can cause transfusion, G6PD deficiency has accounted for a large
such hemolysis.23-25 Other than drugs, ingestion of fava number of cases.29,30 In a review of cases of kernicterus
beans (favism) and various infective agents have been in world literature, G6PD deficiency was the cause in 13
identified as triggers. In an Indian series bacterial sepsis, out of 88 cases which is next in frequency to only Rh and
malaria and hepatitis were identifiable triggers other ABO incompatibility.31 Severe intrauterine hemolysis and
than drugs.20 Hepatitis results in very high levels of serum hydrops fetalis have been reported following maternal
bilirubin and increased morbidity.20,26 Mehta et al. have ingestion of oxidative agents and hemolysis has been
reported AHA following ingestion of soft drink containing observed in breastfeeding neonates following maternal
ascorbate.27 ingestion of fava beans.32
Why only some and not all neonates with G6PD transfusion, frequency of infectious episodes, number of
deficiency develop NNJ is not easily explained. It was chronic complications, disturbances on patient’s activity
postulated that NNJ is associated with only certain and total index severity.34 In an earlier report on this
variants of G6PD (just like CNSHA) but occurrence of association from India, decreased prevalence of painful
cases from various parts of the world does not support crisis was observed possibly due to poor survival of RBC
this hypothesis. Similarly, correlation with residual with HbS due to associated G6PD deficiency leading to
level of enzyme activity has also not been proven.1,30 decreased chances of sickling of cells. Hemolytic crisis
NNJ occurring due to some other insult or trigger is was observed to be more when sickle cell disease was
another explanation offered and is supported by higher associated with G6PD deficiency.35
rates of exposure to naphthalene observed in neonates
with NNJ compared to those without NNJ.1 Infants with Diagnosis
G6PD deficiency and associated mutation of uridine
diphosphoglucoronate glucoronosyltransferase-1 gene Various tests are available for diagnosis of G6PD deficiency.
promoter (UDPGT-1) have been found to be particularly Fluorescent spot test and dichlorophenol indophenol
susceptible to hyperbilirubinemia secondary to impaired (DPIP) de-colorization method and quantitation of
hepatic clearance of bilirubin. UDPGT-1 is the enzyme enzyme are suitable methods for routine use.
affected in Gilbert disease.33 The tests are likely to be negative during episodes
of AHA as the neocytes are rich in G6PD which are in
abundance during AHA compared to steady state. Thus,
CHRONIC NONSPHEROCYTIC a negative test does not exclude but a positive test will
HEMOLYTIC ANEMIA confirm the diagnosis. If negative, it is recommended
The term CNSHA in relation to deficiency of G6PD and to repeat the test after three months of acute episode.3,21
other enzymes is used to describe chronic anemia with G6PD genotyping can be performed using PCR but the
normal/near normal red cell morphology, particularly test is not routinely available. Quantitative methods are
to differentiate it from hereditary spherocytosis. CNSHA available for estimating enzyme levels.
develops in a minority of cases with G6PD deficiency (Class ELISA based method have been developed for field use.
I variants). Clinical picture is quite variable. Unlike NNJ A recent article reported a good sensitivity and specificity
which can affect female children, CNSHA affects only male of this test and recommended for use in resource limited
patients. Generally patients have had NNJ which might have settings.
required therapeutic intervention. The patient presents Another test for field studies is NADPH fluorescence
later with anemia and jaundice. Splenomegaly initially is test on paper (NFP test). This test was compared with
small but later it may increase in size. Anemia is normocytic polymerase chain reaction (PCR)-based G6PD genotyping
normochromic, slight macrocytosis may be observed due also using blood samples on filter papers. There was good
to reticulocytosis. Unconjugated hyperbilirubinemia, inc agreement between the NFP test results and the PCR
reased levels of lactate dehydrogenase and decreased findings. The estimate of the sensitivity of the NFP test
levels of haptoglobin are present. As most of hemolysis was 98.2 percent (95.8–99.6%) and the specificity was 97.1
is extravascular, hemoglobinemia and hemoglobinuria percent (94.2–99.2%).36,37
is not present. Continuous hemolysis in these patients is
thought to result from red cell membrane damage due to
oxidation of sulfhydryl group of hemoglobin resulting in
PREVENTIVE STRATEGIES AND TREATMENT
its precipitation. This is supported by observation of high Management issues in cases with G6PD deficiency
molecular weight aggregates in the red cell membrane of include prevention of AHA and NNJ and treatment of
patients with CNSHA. This phenomenon is not seen G6PD acute and chronic anemia and NNJ. Prevention of NNJ
deficient individuals without CNSHA. G6PD deficiency is based on neonatal screening for enzyme defect and
has been found to be associated with sickle cell disease then taking precautions in cases with enzyme deficiency.
and other hemoglobinopathies. Diop et al.34 found that Such a screening strategy has to be considered taking
prevalence of G6PD deficiency was higher in sickle cell into account the prevalence of G6PD deficiency in the
disease patients (21.6%) than in normal subjects (12.3%) population group as is being followed in Sardinia and
(p = 0.001). Will this association influence the severity of some of the Mediterranean countries.1 WHO recommends
sickle cell diseases is an obvious question because of the screening all newborns for G6PD deficiency I population
nature of the two diseases. However, no difference was groups with prevalence rates of 3 to 5 percent or more in
found in the two groups of male sickle cell disease patients males. Prevention of AHA centers around avoiding the
concerning number of vaso-occlusive crisis, number of known trigger drugs in these patients.
The list in Table 3 shows that many antihistaminic care. Fluid therapy during such episodes is important for
drugs are incriminated in AHA in G6PD deficiency, hence prevention and treatment of acute renal failure.22,22
it would be useful to avoid the cough and cold medicines Patients having CNSHA require meticulous monitor
in general which otherwise are of unproven efficacy in ing. In most cases occasional exacerbation will require
treating the upper respiratory infections. Some of the blood transfusion. During steady state, administra
known offending drugs have been successfully used in tion of folic acid is recommended as the requirement is
certain population groups.38 increased due to increased red cell turnover. Very few cas
Management of NNJ is like any other cause of uncon es will need to be started on chronic transfusion therapy
jugated hyperbilirubinemia including phototherapy and like patients with thalassemia and hemoglobinopathies.
exchange transfusion. Phototherapy in these neonates Splenectomy may be required due to large size, devel
may be started at a lower level than otherwise recom opment of hypersplenism or to decrease the transfusion
mended.31,39 requirement.1
A novel approach is use of heme-oxygenase inhibi
tor-tin-mesoporphyrin (Sn-MP) which reduces bilirubin Deficiency of Pyruvate Kinase and Other
production. It has been found to be extremely useful in Enzymes of Glycolytic Pathway
preventing the development of significant hyperbiliru As compared to G6PD deficiency, the defects of glycolytic
binemia in G6PD deficient neonates.40-42 pathway are very uncommon. Of them, pyruvate kinase
Treatment of acute episode of intravascular hemolysis deficiency is most well recognized and along with G6PD
includes transfusion support for anemia and supportive deficiency it is the most common cause of CNSHA.43,44 In
Table 4 Deficiency of enzymes of glycolytic pathway

S. Enzyme, inheritance Clinical features Hematological/other Treatment
no. laboratory findings
1. Hexokinase deficiency, AR 22 cases reported, NNJ, anemia, Red cell morphology Transfusions
splenomegaly, CNSHA, gallstones, unremarkable Folic acid
hyperhemolytic episodes Splenectomy
2. Glucose phosphate isomerase, 46 cases from 34 pedigree, 30% NNJ, Very high reticulocyte Same as above
AR hydrops, CNSHA, hyperhemolytic episodes count, MCV increased
3. Phosphofructokinase Involves red cells, muscle related symptoms No lactate production Unsatisfactory
deficiency, AR predominate—exertional myopathy, easy in “ischemic arm test”,
fatiguability (Type VII Glycogen storage muscle biopsy
disease), mild hemolytic anemia
4. Aldolase deficiency, AR Very few cases, severe CNSHA, mental Normal red cell Undefined
retardation morphology
5. Triose phosphate isomerase Moderate to severe CNSHA, neonatal Very high reticulocyte Transfusions, folic
deficiency, AR anemia, progressive neurological disease count acid, splenectomy
(unrelated to kernicterus)
6. Glyceraldehyde-3-phosphate Need not result in anemia, associated with Nonspecific -
dehydrogenase deficiency, AR other defects like hereditary spherocytosis
(Autosomal recessive)
7. Phosphoglycerate kinase CNSHA, NNJ, seizures, movement disorders, Reticulocytosis ? Splenectomy
deficiency, X-linked recessive psychomotor retardation, aphasia,
tetraplegia
8. 2,3-Bisphosphoglycerate Complete absence may be associated with Phlebotomy for
mutase deficiency, AR polycythemia, neonatal onset of progressive symptomatic
anemia described with 50% activity polycythemia
9. Enolase deficiency Shortened red cell survival not necessary, Spherocytosis Undefined
nitrofurantoin induced hemolysis
10. Pyruvate kinase deficiency, AR See text
11. Lactate dehydrogenase Decreased levels not associated with anemia - -
deficiency
Indian population, the deficiency of these enzymes has only Table 5 Clinical characteristics in pyruvate kinase deficiency
sparingly been studied.45,46 In contrast to G6PD deficiency,
patients with deficiency of enzymes of glycolytic pathway Clinical feature Number
usually have CNSHA with onset in neonatal period. Drug Consanguinity 4/56
induced hemolytic anemia- is not a common problem Anemia 55/61
in them. Most cases benefit from splenectomy. Pyruvate
Jaundice 43/61
kinase deficiency is described in the following section.
Rest of the conditions with their common clinical findings Neonatal jaundice 33/56
are listed in Table 4. Splenomegaly 47/58
Splenectomy 18/61
PYRUVATE KINASE DEFICIENCY Cholecystectomy 14/56
Deficiency of pyruvate kinase (PK) is the most common Aplastic crisis 1/61
enzyme deficiency of glycolytic pathway with over 350 Transfusions 18/59
cases reported.47 Exchange transfusions 25/56
It is transmitted as autosomal recessive trait and is
Desferrioxamine treatment 16/58
seen in patients of north European descent. PK enzyme
is a tetramer with four tissue specific subunits—R (RBC),
L (liver), M1—muscle and M2 platelets and leukocytes. Clinical Approach to a Child Suspected to
Genetic control is separate for RL subunit and M1 M2 have Enzyme Deficiency
subunits.43,47 Prevalence of PK deficiency has varied from
0.14 to 6 percent.38 Clinical diagnosis of erythroenzymopathy requires a high
The hallmark of PK deficiency is CNSHA Cases with index of suspicion. In cases presenting with acute intravas
neonatal anemia and hydrops have occurred. NNJ requiring cular hemolysis, history of exposure to trigger drugs helps
exchange transfusions have been described. Patients in the diagnosis. Family history of jaundice, anemia,
with CNSHA have unconjugated hyperbilirubinemia splenectomy and cholelithiasis may also point towards
and splenomegaly which at times may become massive. such a disease.2,50 In absence of such history, the diagnosis
Chronic leg ulcers are a complication in some individuals. may be difficult. Other causes like autoimmune hemolytic
Aplastic crisis due to parvovirus infection can occur. Gall anemia and malaria (blackwater fever) can be excluded by
stones are increasingly seen after first decade.43,47,48 appropriate tests.
Table 5 describes the clinical and laboratory features Cases with chronic hemolytic anemia pose a diagno
in a large series of 61 patients (all ages). stic problem. In our country, common causes of hereditary
Red cell morphology is normocytic normochromic. hemolytic anemia include b-thalassemia syndrome and
Macrocytosis due to active regeneration and acantho sickle cell disease. Both these conditions can be diagnosed by
cytosis may be observed. Reticulocyte count is increased hemoglobin electrophoresis. The diagnosis of enzymopathy
but not proportionate to hemolysis as in other hemolytic should be suspected in all cases with chronic hemolysis
anemias. This is on account of selective sequestration of and unexplained unconjugated hyperbilirubinemia parti
young RBCs and reticulocytes by spleen. A paradoxical cularly if red cell morphology is unremarkable. Intense
rise in reticulocytes after splenectomy therefore is a reticulocytosis supports the diagnosis of various enzymo
known phenomenon. On account of hepatic PK defi pathies (see Table 4). The diagnosis can be confirmed by
ciency, liver enzymes are elevated and coupled with appropriate enzyme assay. Unfortunately, the tests are
presence of hyperbilirubinemia may appear to be the not widely available. Demonstration of accumulation of
clinical picture with chronic liver disease. Iron overload proximal or depletion of distal intermediary compounds of
disproportionate to transfusions is an important observ glycolytic pathway supports the diagnosis.
ation in certain cases and has been explained on the
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11. Kaeda JS, Chhotray GP, Ranjit MR, et al. A new glucose- bilirubinemia. American Academy of Pediatrics-Technical
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Al -- >Gly), is the major polymorphic variant in tribal 32. Steiner LA, Gallager PG. Erythrocyte disorders in perinatal
populations in India. Am J Hum Genet. 1995;57:1335-41. period in adverse pregnancy outcome and the fetus/
12. Balgir RS, Murmu B, Dash BP. Hereditary hemolytic newborn. Semiar Perinatol. 2007;31:254-61.
disorders among the Ashram school children in Mayurbhanj 33. Edward CQ. Anemia and the liver. Hepatobiliary mani
district of Orissa. J Assoc physicians Ind. 1999;47:987-90. festations of anemia. Clin Liv Dis. 2002;6:891-907.
13. Gupte SC, Patel PU, Ranat JM. G6PD deficiency in Vataliya 34. Diop S, Sene A, Cisse M, Toure AO, Sow O, Thiam D,
Prajapati community settled in Surat. Indian J Med Sci. Diakhate L. Prevalence and morbidity of G6PD deficiency
2005;59:51-6. in sickle cell disease in homozygotes. Dakar Med.
14. Usanga EA, Luzzatto L. Adaptation of Plasmodium 2005;50:56-60.
falciparum to G6PD deficiency host red cells by production 35. Jain D, Jaju A, Khan T, Ughade SN, Hussain ZF. Co-existence
of parasite encoded enzyme. Nature. 1985;313:793-5. and interaction of G6PD deficiency with sickle cell disease: a
15. Roth J, Schulman S. The adaptation of Plasmodium study in central India. Abstract HO/14. Conference abstract
falciparum to oxidative stress in G6PD deficient human 40th national conference of IAP. 2003.pp.92-3.
erythrocytes. Br J Haematol. 1988;70:363-7. 36. Enevold A, Vestergaard LS, Lusingu J, et al. rapid screeing
16. Allison AC, Clyde DF. Malaria in African children with for glucose-6-phosphate dehydrogenase deficiency and
deficient erythrocyte G6PD. Br Med J. 1961;1:1346-9. haemoglobin polymorphisms in Africa by a simple high-
17. Mohanty D, Sukumar S, Mukherjee MB, Colah RB. throughput SSOP-ELISA method. Malar J. 2005;15:61.
G6PD deficiency and malaria in India. Am J Hematol. 37. Meissner PE, Coulibaly B, Mandi G, et al. Diagnosis of red
2003;72:150-1. cell G6PD deficiency in rural Burkino Faso: comparison
18. Betke K, Beutler E, Grewer GJ, et al. Scientific group on of a rapid fluorescent enzyme test on filter paper with
standardization of procedures for the study of glucose-6- polymerase chain reaction based genotyping. Br J
phosphate dehydrogenase. WHO Tech Rep Ser No. 366;1967. Haematol. 2005;131:395-9.
19. Beutler E, Yoshida A. Genetic variation of glucose-6- 38. Mandi G, Witte S, Meissner P, et al. Safety of combination
phosphate dehydrogenase: a catalog and future prospects. of chloroquine and methylene blue in healthy adult men
Medicine. 1988;67:311. with G6PD deficiency from rural Burkina, Faso. Trop Med
20. Sarkar S, Prakash D, Marwaha RK, et al. Acute intravascular International Health. 2005;10:325-8.
hemolysis in glucose-6-phosphate dehydrogenase defi 39. Management of hyperbilirubinemia in the newborn
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pediatrics; clinical practice guideline (subcommittee on 45. Baxi AJ. Red cell isoenzymes with a note on rare variants in
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40. Kappas A, Drummond GS, Valaes T. A single dose of 46. Mehta BC, Bapat JP, Baxi A. Two cases of PK deficiency
Sn-mesoporphyrin prevents development of significant (Unpublished). Quoted by Baxi A.
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nase-deficient neonates. Pediatrics. 2001;108:25-30. enzymes of glycolytic pathway. In: “Nathan and Oski’s
41. Dennery PA. Metalloporphyrins for treatment of neonatal Hematology of Infancy and Childhood”. Vol I. Nathan DG,
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42. Valaes T, Drummond GS, Kappas A. Control of hyperbili 1993.pp.665-703.
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43. Zanella A, Fermo E, Bianchi P, Valentini G. Red cell 49. Zanella A, Bianchi P, Iurlo A, et al. Iron status and HFE
pyruvate kinase deficiency : molecular and clinical aspects. genotype in erythrocyte pyruvate kinase deficiemcy: study
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44. Glader B. Hereditary hemolytic anemias due to enzyme 653-61.
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C H A P T E R 22
Autoimmune Hemolytic Anemia
Rajiv Kumar Bansal
Immune hemolytic anemia (IHA) is the clinical condition in which IgG and/or IgM antibodies bind to RBC surface antigens and initiate
RBC destruction via the complement system and the RE system.1 Autoimmune hemolytic anemia (AIHA) refers to a collection of
disorders characterized by the presence of autoantibodies that bind to the patient’s own erythrocytes, leading to premature red cell
destruction. The antigens targeted often have a high incidence so that both native and transfused RBCs are destroyed. In contrast
to AIHA, in alloimmune hemolytic anemia exposure to allogenic RBCs leads to formation of alloantibodies which do not react with
autologous RBCs. A positive direct antiglobulin test (DAT, also known as the Coombs test) is essential for diagnosis.
Specific characteristics of the autoantibodies, especially to RBC membranes in the cold and activates the hemolytic
the type of antibody; its optimal binding temperature; complement cascade when the cells are warmed to 37°C
and whether complement is fixed, influence the clinical (99°F).2 All three can occur as an idiopathic (primary)
picture. In all cases of AIHA, however, the autoantibody disorder or can coexist with another disease (secondary)
leads to a shortened red blood cell survival (i.e. hemolysis) (Table 1).
and, when the rate of hemolysis exceeds the ability of the
bone marrow to replace the destroyed red cells, to anemia EPIDEMIOLOGY
and its attendant signs and symptoms. Subjects of all ages
It is a relatively uncommon but certainly not rare disorder,
are affected by AIHA: from infants in the first few months
with an estimated incidence of 1 to 3 cases per 100000
of life to the elderly. Although, it occurs less frequently population per year. There is no evidence that AIHA is
than in adults, AIHA in the young is not a rarity. AIHA in confined to any particular race. It is less common than
children presents some differences from those of adults. immune thrombocytopenia. In teenagers and adults,
Three types of AIHA can be distinguished based on AIHA is more common in women than in men. The peak
their serologic properties and clinical characteristics. incidence in pediatric patients occurs in preschool-age
Most patients with AIHA (80%) exhibit warm-reactive children.2 Boys are 2.5 times more likely to be affected than
antibodies of the immunoglobulin IgG isotype on their girls.3 In children, occurrences in patients younger than
red cells. Most of the remainder of patients exhibit cold- 2 years and older than 12 years are more likely to have a
reactive autoantibodies. Two types of cold-reactive chronic unremitting course.2 However, the majority of
autoantibodies to RBCs are recognized: cold agglutinins pediatric cases are acute in onset and self-limiting. Often
and cold hemolysins. Cold agglutinins are generally of these cases resolve within 6 months without treatment,2
IgM isotype, whereas cold hemolysins usually are of IgG and the decision to treat is based on the degree of anemia
isotype. IgG warm autoantibodies bind to erythrocytes at and physiologic compromise. Some reports suggest
37°C (99°F) but fail to agglutinate the cells; cold agglutinins, that in childhood, secondary cases are more common
almost always of the IgM isotype, clump RBCs at cold than idiopathic forms. Viral and bacterial agents are
temperatures and occasionally lead to hemolysis; and the frequently the only recognizable stimuli; in fact, AIHA
IgG Donath-Landsteiner antibody (cold hemolysins) binds follows viral infection or vaccination much more often
Table 1 Classification of autoimmune hemolytic anemia (AIHA)*

Warm active antibodies: Autoantibody maximally active at body temperature (37°C)
Warm autoimmune hemolytic anemia:
• Primary or idiopathic
• Secondary (autoimmune disorders, lymph proliferative disorders)
• Drug-induced immune hemolytic anemia
– Autoimmune type
– Drug adsorption type
– Neoantigen type
– Nonimmune type (first generation cephalosporins)
Cold active antibodies: Autoantibodies optimally active at temperature < 37°C
Mediated by cold agglutinins:
• Primary or idiopathic chronic cold agglutinin disease
• Secondary cold agglutinin hemolytic anemia
– Acute transient (infections, e.g. Mycoplasma pneumonia or infectious mononucleosis)
– Chronic (lymphoproliferative disorders)
Mediated by cold hemolysins:
• Primary or idiopathic: Paroxysmal cold hemoglobinuria
• Secondary
– Donath-Landsteiner hemolytic anemia: Acute transient (various viral infections)
– Chronic (syphilis)
Mixed-type (cold and warm) autoimmune hemolytic anemia
• Primary or idiopathic
• Secondary (autoimmune disorders, lymphoproliferative disorders)
*Modified from Gehrs and Friedberg. Autoimmune hemolytic anemia. American Journal of Hematology. 2002;69:258-71.
in children than in adults. When it is associated with in this study. Associated diseases were found in 27 patients
infections in the former, AIHA is usually acute and of short and included infectious diseases, immunodeficiency, auto-
duration. Immunodeficiency or malignancy (especially immune related disorders.
malignancies of the lymphoreticular tissues), systemic
lupus erythematosus (SLE), and other types of collagen Lymphoproliferative Disease and
vascular diseases are most commonly associated with Rh-hemolytic Disease
immune hemolysis in children. Drugs are less commonly
Oliveira et al 2006 evaluated 17 patients younger than
associated with AIHA. The reason for this finding is
15-years-old admitted from 1988 to 2003 in Brazil.6 The
probably that the common inducers of immune hemolysis,
median age at diagnosis was 10.5 months. The direct
such as α-methyldopa in the past, are not normally Coombs polyspecific test was positive in 13 patients
prescribed for children. When drug-induced AIHA occurs, and negative in four patients. Monospecific testing was
it is usually due to immunoglobulin G (IgG) antibodies and performed for 14 patients. The most frequent red cell
it is associated with antibiotics such as penicillin. Biphasic autoantibody was IgG (five patients), followed by IgM in
hemolysin (historically related to syphilis) is, nowadays, two. Thirteen patients had severe anemia and needed
associated with viral infections such as measles, rubella, blood transfusions. Underlying diseases were identified
and chickenpox.4 in four patients: systemic lupus erythematosus, Hodgkin’s
deLuca et al. reported 29 children with autoimmune lymphoma, autoimmune hepatitis and Langerhans cell
hemolytic anemia (AIHA) in 1979 from Rome, Italy. Patients histiocytosis. The remaining patients were classified as
were divided into two groups, i.e. patients with transient AIHA having primary disease. The median follow-up period
(15 cases) and patients with the chronic form of the disease was 11 months (5–23 months). Three children died, two
(14 cases).5 The criterion for this distinction was based on after splenectomy and one with complications of the
the episode of increased hemolysis which was either shorter underlying disease.
or longer than 3 months. If a relapse of AIHA occurred, the Vaglio et al. published one of the largest studies of
case was considered to be chronic by them. There were no AIHA in children in 2007.4 A retrospective review of 100
patients with cold autoagglutinins or biphasic hemolysins cases of childhood AIHA (age range 6 months–16 years)
Chapter-22 Autoimmune Hemolytic Anemia 229
Table 2 Distribution of 100 patients with AIHA based on disease association and serologic findings (Vaglio et al)4
Condition Warm AIHA Cold AIHA PCH* Mixed AIHA
Idiopathic AIHA 38 6 0 2
Autoimmune diseases 0 0
Idiopathic thrombocytopenic purpura 9
(ITP)
Systemic lupus-erythematosus (SLE) 1 2
Infectious diseases 6 6 6
Neoplasia 0 5 0 0
Hematologic disorder
Sickle cell anemia 1 0 0 0
Thalassemia major 3 1
Myelodysplasia 4 0
Non-Hodgkin’s lymphoma 1 3
Chronic myelogenous leukemia 0 1
Acute myelogenous leukemia 0 1
Acute lymphoblastic leukemia 0 3
Liver and kidney transplant 1 0 0 0
Total 64 26 6 4
* PCH: Paroxysmal cold hemoglobinuria.
by them, diagnosed over a 20-year-period revealed a Drug-induced immune hemolytic anemia (DIIHA)
peak incidence in the first 4 years of life (Table 2). No sex is rare.9 The incidence is around 1 in 1 million of the
predilection was observed. The majority of patients (64%) population. The number of drugs and the suggested
were diagnosed with warm AIHA, whereas cold agglutinin mechanisms associated with DIIHA has changed over the
disease and PCH accounted for 26 percent and 6 percent last 40 years. In 1967, only 13 drugs were implicated; in
of children, respectively. A mixed type of AIHA was 1980, 32 drugs were reported and in 2007, 125 drugs were
observed in four subjects. Overall, 54 percent of children reported.10 Three groups of drugs predominated: 42 percent
had coexisting disease, including hematologic disorders, were antimicrobials; 15 percent were anti-inflammatory; 11
autoimmune disease, infection, and neoplasia. All cases of percent were anti-neoplastics.10 The specific drugs mainly
PCH were associated with a recent viral illness. implicated have changed dramatically. In the 1970, the
There are no large studies on AIHA in Indian literature. most common drug, by far, to cause DIIHA was methyldopa,
Sporadic case reports have been published from time to which caused a true AIHA with no drug antibody involved
time. Naithani et al. 2007 published one of the largest and accounted for 67 percent of all DIIHA. High-dose
pediatric series from India.7 A series of 26 cases were seen intravenous penicillin accounted for 25 percent of DIIHA.
by them over a period of 5 years in Delhi, India. Of the 26 When these therapies became less commonly used,
patients, 11 were males and 15 females. Patients were in the most common causative group of drugs became
the age group of 2 months to 17 years (median; 11 years). the cephalosporins, which, from the 1990s, account for
Nine (35%) children had secondary AIHA in this series. 70 percent of the DIIHA. The drugs most frequently
Of them 5 had autoimmune conditions, 2 had Evans associated with DIIHA at this time are cefotetan (more than
syndrome, 2 had SLE, 2 had JRA, 1 had nephritis, 1 had 50 percent cases of DIIHA), ceftriaxone, and piperacillin.
endocrinopathy and 4 had various infections. Ceftriaxone is the second most common drug to cause
In a study of twelve children diagnosed with auto- DIIHA.9 Some children have dramatic HA; 50 percent are
immune hemolytic anemia over a period of four years reported as fatal HA.11,12 Analysis of 21 patients (15 children
Gupta et al. (2008) from BHU, Varanasi found 9 had and 6 adults) showed that 40 percent of the children
primary disease and 3 had secondary disease.8 Tubercular started hemolyzing ≤ 1 hr after receiving ceftriaxone.
infection was seen in 2 patients with secondary disease. Hemoglobin levels fell to ≤ 5 g/dL in 62 percent and to
≤ 1 g/dL in 20 percent of the patients. Fatal HA occurred secondary to a generalized defect in immune regulation.
in 38 percent of the patients. The children have always Rather, these patients may develop warm-antibody AHA
received ceftriaxone previously, the DAT is usually through an aberrant immune response to a self-antigen or
positive (all have RBC-bound complement and most have to an immunogen that mimics a self-antigen. In patients
IgG in addition), and ceftriaxone antibodies are detectable with secondary AIHA, the disease may be associated with
in the patient’s serum. The HA is usually not as dramatic a fundamental disturbance in the immune system.
in adults. The fall in hemoglobin is much less and does not During fetal life, developing lymphocytes that come
occur in a few hours; fatalities are less common. into contact with antigen are eliminated or silenced.
Piperacillin can cause DIIHA and/or positive DATs. This effect is one of the mechanisms of immunologic
Although a semi-synthetic penicillin, unlike other semi- tolerance of endogenous antigens. The extreme rarity of
synthetic penicillins (e.g. ampicillin), it reacts differently autoimmune hemolytic anemia secondary to anti-A or
than penicillin G. In contrast to penicillin, the in vivo RBC anti-B antibodies indicates the deletion from the immune
destruction can be complement-mediated; most of the repertoire of B cells with the capacity to produce anti-A or
DATs are positive due to RBC-bound complement and IgG.13 anti-B antibodies. Such clones are probably eliminated
Hydrocortisone can also cause DIIHA.14 This adds or inactivated early in ontogeny because the embryo
another possible explanation for poor responses to steroid can synthesize A and B substances within 5 weeks of its
therapy in some cases of AIHA, where steroid-induced implantation in the uterine wall.
DIIHA may be masked by the autoimmune process. A population of CD4+/CD25+ T cells that express the
transcription factor Foxp3 restrains immune responses
PATHOGENESIS against autoantigens in adults. There is evidence that a
deficiency of these regulatory T cells plays a role in the
AIHA is an autoimmune disease in which there is loss of self
pathogenesis of autoimmune hemolytic anemia.
tolerance. Self tolerance refers to a lack of responsiveness to
an individual’s own (self) antigens. In the case of AIHA, the Warm antibodies-pathogenic effects: IgG anti–red cell
antibodies are directed against self RBC antigens, leading autoantibodies mediate the destruction of red blood
to their enhanced clearance through Fc-receptor–mediated cells outside the circulating blood in a process called
phagocytosis (extravascular hemolysis) or complement- extravascular hemolysis (Figures 1A to D). By contrast,
mediated breakdown (intravascular hemolysis). There is when lytic components of the complement system enter
some evidence that AIHA may be in large part due to self- the mechanism, destruction of red cells occurs directly
reactive antibodies against erythrocyte band 3, an anion within the circulating blood (intravascular hemolysis).
transporter found in erythrocyte membranes. In AIHA, The participation of lytic complement components in IgG-
autoantigenic T-cell epitopes have recently been mapped mediated autoimmune hemolytic anemia is, however,
for the RhD autoantigen. The degree of hemolysis in AIHA rare. IgG antibodies are relatively poor activators of the
depends on the characteristics of the bound antibody (e.g. classical complement pathway, but they, especially the
quantity, thermal amplitude, specificity, complement IgG1 and IgG3 antibodies are recognized readily by Fc
fixing ability and the ability to bind tissue macrophages) receptors on various phagocytic cells. The IgG sensitized
and also the characteristics of target antigen (density, RBCs generally are eliminated by the phagocytes of the
expression, patient age).15,16 R-E system. Presence of complement factors C3 (C3b
The cause of autoimmune hemolytic anemia is and iC3b) potentiates extravascular hemolysis by the
unknown. In about one-third of cases, the autoantibodies R-E cells in these patients as they have receptors for
have specificity for an antigen in the Rh system. In these complement components. Autoantibody-coated
another third, the antibodies target proteins in membrane RBCs are trapped by macrophages in the Billroth cords
glycoproteins (glycophorins) of the red cell; in other cases, of the spleen and, to a lesser extent, by Kupffer cells in
the antibodies have specificity for antigens in the Kell or the liver. The process leads to generation of spherical
Duffy blood group system (very rarely for ABO antigens) RBCs (Spherocytes) and fragmentation and ingestion of
or for structures in the membrane that are not blood group antibody-coated RBCs. Spherical RBCs are more rigid and
antigens (e.g. band 3, an anchor point in the membrane for less deformable than normal RBCs. As such, spherical
the red cell cytoskeleton). In all these cases, the patient’s RBCs are fragmented further and eventually destroyed
own erythrocytes display the relevant antigen.17 In primary in future passages through the spleen. Spherocytosis is
AIHA, the autoantibodies of any one patient often are a consistent and diagnostically important hallmark of
specific for only a single RBC membrane protein. The AHA, and the degree of spherocytosis correlates well with
narrow spectrum of autoreactivity suggests the mechanism the severity of hemolysis. Direct complement-mediated
underlying AIHA development in such patients is not hemolysis with hemoglobinuria is unusual in warm-
and occur in two forms: (1) Cold agglutinin disease (CAD)-

associated with IgM antibodies usually directed at the RBC
I antigen, typically occurs in adult patients and may be
primary or secondary to another disease process, usually
infectious, and (2) Paroxysmal cold hemoglobinuria
(PCH)-caused by the so-called Donath-Landsteiner
A B
antibody, an IgG hemolysin.
The great preponderance of cold agglutinin molecules
are IgM antibodies. Most cold agglutinins are unable to
agglutinate RBCs at temperatures higher than 30°C. The
highest temperature at which these antibodies cause
detectable agglutination is termed the thermal amplitude.
Generally, patients with cold agglutinins with higher
thermal amplitudes have a greater risk for cold agglutinin
C D
disease. For example, active hemolytic anemia has been
observed in patients with cold agglutinins of modest titer
Figs 1A to D IgG anti-red cell autoantibodies. (A) Structure of an (e.g. 1:256) and high thermal amplitudes. More than 90
IgG molecule demonstrating its variable and constant regions percent of cold-active antibodies have the I antigen as their
and the heavy and light chains; (B) Agglutination of red cells by target on the RBC, and the I antigen is the binding site for a
pentameric IgM antibodies, which can join the cells into a lattice; significant portion of the remaining 10 percent. The closely
(C) Coating of red cells by IgG antibodies. The antibodies are related I/i antigens are high-frequency carbohydrates
unable to agglutinate the cells; (D) Agglutination of IgG-coated similar to the ABO antigens. Neonatal RBCs exclusively
red cells by an anti-IgG antibody expressing large amounts of i antigen, converting to
exclusively I antigen by 18 months of age. Other uncommon
but reported antigen targets include Pr. The fact that M.
antibody AHA. Cytotoxic activities of macrophages and pneumoniae induces anti-I antibodies in the majority of
lymphocytes also may play a role in the destruction of patients is potentially related to the finding that sialylated
RBCs in warm-antibody AHA. I/i antigens serve as specific Mycoplasma receptors. Minor
Warm autoantibodies are panagglutinins, i.e. they modification of this antigen may incite autoantibodies.
react with all the RBCs in the diagnostic panel. Of the The pathogenicity of a cold agglutinin depends upon
reported specificities, Rh is by far the most common (70%), its ability to bind host RBCs and to activate comple
including all but the Rhnull erythrocytes. ment. IgM sensitized RBCs generally are associated
The degree of anemia in AIHA depends not only on the with a combination of intravascular and extravascular
rate of red cell destruction but also on the ability of marrow hemolysis. The pentameric structure of IgM enables
to increase erythrocyte production. With an adequate efficient complement activation. Destruction of erythro
supply of nutrients and growth factors, bone marrow can cytes sensitized with IgM antibodies is mediated by
overcome a hemolytic rate of about three times normal; the complement system. Complement mediates RBC
anemia does not appear until the half-life of the red destruction either directly by cytolysis or indirectly via
cell population drops to about 10 days (the half-life of a interaction of RBC-bound activation and degrad ation
population of normal red cells is about 30 days as measured fragments of C3 with specific receptors on reticuloendo
with 51Cr-labeled red cells). A half-life of 5 or 6 days is not thelial cells, principally liver macrophages (Kupffer cells).
unusual in autoimmune hemolytic anemia. The marrow Due to the presence of regulatory RBC proteins such as
can compensate for accelerated red cell destruction by decay accelerating factor (DAF, CD55) and membrane
increasing the number of red cell precursors by upto inhibitor of reactive lysis (MIRL, CD59), overwhelming
10 times the normal number (erythroid hyperplasia), complement activation usually is required to produce
accelerating the release of reticulocytes, and in some cases, clinically evident intravascular hemolysis. However, in
allowing nucleated red cells to enter the blood. most clinical situations, IgM antierythrocyte antibodies
are present in sublytic quantities. Under these conditions,
Cold Agglutinins and Hemolysins— DAF (CD55) and MIRL (CD59) are able to prevent direct
RBC lysis. More commonly, IgM sensitized RBCs undergo
Pathogenic Effects extravascular hemolysis. While RE cells do not have
Cold-active antibodies exhibit increasing titer and RBC- receptors for the Fc fragment of IgM antibodies, they do
binding activity as the temperature decreases toward 0°C have receptors for the abundant RBC-bound C3b and iC3b
resulting from complement activation. The principal site paroxysmal cold hemoglobinuria, a proposed term for this
of IgM mediated extravascular hemolysis is liver and not latter entity is Donath-Landsteiner hemolytic anemia.21, 22
the spleen.
Cold agglutinins may bind to RBCs in superficial
DRUG-INDUCED IMMUNE
vessels of the extremities, where the temperature generally
ranges between 28 and 31°C, depending upon ambient HEMOLYTIC ANEMIA
temperature.18 Cold agglutinins of high thermal amplitude The most common drugs associated with DIIHA and the
may cause RBCs to aggregate at this temperature, thereby hypotheses for the mechanisms thought to be involved
impeding RBC flow and producing acrocyanosis. In have changed during the last few decades. There are
addition, the RBC-bound cold agglutinin may activate two types of drug-related antibodies. Drug-independent
complement via the classic pathway. Once activated antibodies are those antibodies that can be detected in
complement proteins are deposited onto the RBC surface, vitro without adding any drug; thus, in vitro and in vivo
the cold agglutinin need not remain bound to the RBCs characteristics are identical to cell red blood cell (RBC)
for hemolysis to occur. Instead, the cold agglutinin may autoantibodies. Drug-dependent antibodies are those
dissociate from the RBCs at the higher temperatures antibodies that will only react in vitro in the presence
in the body core and again be capable of binding other of drug (e.g. bound to RBCs or added to the patient’s
RBCs at the lower temperatures in the superficial vessels. serum in test systems to detect drug antibodies); these
As a result, patients with cold agglutinins of high thermal are antibodies directed at epitopes on the drug and/or its
ampli tude tend toward a sustained hemolytic process metabolites, or a combination of drug plus RBC membrane
and acrocyanosis.19 In contrast, patients with antibodies protein. The mechanisms involved in the serological and
of lower thermal amplitude require significant chilling clinical findings are controversial. It is still unknown why
to initiate complement-mediated injury of RBCs. or how some drugs can affect the immune system to cause
This sequence may result in a burst of hemolysis with RBC autoantibody formation, with or without HA.23
hemoglobinuria.19 Combinations of these clinical patterns
also occur.
In contrast to cold agglutinins, cold hemolysins the so
Clinical Features
called Donath and Landsteiner (D-L) antibodies, are IgG Preschool children have the peak incidence of AIHA in
antibodies and are polyclonal. They were first described pediatric age group. It is 2.5 times more common in boys
by Donath and Landsteiner in 1903 in patients with a as compared to girls. The clinical findings in WAIHA are
characteristic syndrome-paroxysmal cold hemoglobinuria variable. They are determined by the rate of hemolysis and
(PCH).20 The Donath and Landsteiner (D-L) antibody is the ability of the body to process breakdown products and
a hemolysin that binds to RBCs at low temperatures and mount a reticulocytosis. Two general clinical patterns are
fixes complement. When the RBCs are warmed, they are seen. In 70 to 80 percent patients mostly in the age group
destroyed by complement lysis. The D-L IgG antibody is of 2 to 12 years the disease has an acute transient pattern
a potent hemolysin, causing significant RBC destruction lasting for 3 to 6 months. It is frequently preceded by an
even in low titers. The D-L antibody is classically described infection, usually respiratory.
as a “biphasic” hemolysin. The antibody requires the cooler Onset may be acute, with prostration, pallor, jaundice,
temperatures (0–4o C) to bind to the RBC, but complement- pyrexia, and hemoglobinuria, or more gradual, with
mediated lysis does not proceed until the temperature primarily fatigue, dyspnea on exertion and pallor. The
is raised (37o C). The antibody in PCH is directed against spleen is usually enlarged. Hepatomegaly, and lympha
the P antigen, found on the RBCs of most individuals. The denopathy may also accompany the anemia.
P antigen is similar to the Forssman glycolipids present Mild, chronic hemolytic anemia with exacerbations in
in many micro-organisms. This similarity suggests that the winter is the general rule for cold agglutinin disease.
infectious agents, may elicit D-L antibodies as a result of Rarely, does the hemoglobin drop below 7 g/dL. Pallor
crossreactivity. and jaundice may occur, if the rate of hemolysis is greater
The D-L antibody occurs in three clinical syndromes: than the endogenous capability to metabolize bilirubin.
(a) chronic PCH associated with late-stage or congenital Some patients have intermittent bursts of hemolysis
syphilis, (b) acute transient PCH occurring after an infectious associated with hemoglobinemia and hemoglobinuria on
illness, and (c) chronic idiopathic PCH. An increasing exposure to cold and may be forced to move to warmer
proportion of Donath-Landsteiner autoantibody-mediated climates to prevent attacks. Acrocyanosis can occur from
hemolytic anemias occurs as a single postviral episode agglutination of RBCs in the cooler vessels of the hands,
in children, without recurrent attacks (paroxysms). The ears, nose, and feet. Digits may become cold, stiff, painful,
prognosis for such cases is excellent. Thus, rather than or numb and may turn purplish. Limbs may manifest
livedo reticularis, a mottled appearance that is readily 5 days, the anemia is severe, and erythropoiesis increases
reversible upon warming of the affected area. Only rarely 8-10 fold. The reticulocyte count may rise, sometimes
does actual gangrene of digits develop. If hemolysis does upto 40 percent. Reticulocytes may be depressed early
occur after Mycoplasma infections, it typically begins in the course. Reticulocytopenia may be because of
when the patient is recovering from the pneumonia and marrow shutdown from intervening infection, malignancy
titers for cold autoantibodies are at their peak. Hemolytic myelophthisis, parvovirus B19 infection, or the possibility
anemia in infectious mononucleosis develops either at the of the autoimmune antibody being directed at antigens in
onset of symptoms or within the first 3 weeks of illness. great concentration on the reticulocytes themselves.
In children <5 years, paroxysmal cold hemoglobinuria On PBF examination, polychromasia indicates reticu
accounts for almost 40 percent of the patients. The onset locytosis. Spherocytes are seen in patients with moderate-
of the disease is sudden with fever (even up to 40oC), to-severe hemolytic anemia. RBC fragments, nucleated
back or leg pain, and hemoglobinuria after exposure RBCs, and occasionally erythrophagocytosis by monocytes
to the cold even of a few minutes. Symptoms may and rarely neutrophils may be seen in severe cases.
follow shortly or several hours later. Abdomen cramps, Most patients have mild leukocytosis and neutrophilia.
headache, nausea, vomiting, and diarrhea may also Leukopenia and neutropenia may also occur sometimes.
occur. Dark red-to-black color urine is voided after the Patients with severe hemolytic anemia and markedly
onset and typically clears in a few hours. Rarely, it persists increased erythropoiesis occasionally develop folate
for a few days. The spleen may be palpable during an deficiency and frank megaloblastosis with raised MCV
attack and shortly thereafter, and mild jaundice may levels.
appear. Systemic symptoms may appear without the Platelet counts typically are normal but may be low in
hemoglobinuria and vice versa. Reports of Vasomotor systemic lupus erythematosus or in Evans’ syndrome.
phenomena manifest as cold urticaria, tingling of hands The combination of a high MCV (because of reticulo
and feet, cyanosis, and Raynaud phenomenon are there cytosis), a high RDW (because of the dimorphic population
in the literature. of reticulocytes and spherocytes), and a high reticulocyte
An antecedent upper respiratory infection in children is count points to hemolytic anemia.
usually identified in PCH. Measles, measles vaccinations, Clumping from the cold agglutinins complicates both
mumps, Mycoplasma pneumoniae, influenza A, adenovirus, the peripheral blood smear and the calculation of the red
varicella, cytomegalovirus, Haemophilus influenzae, and
cell counts and red cell indices in cold agglutinin disease.
infectious mononucleosis have been identified as ante
Disolution of the clumping upon warming indicates
cedent illnesses. The original chronic PCH associated with
the presence of a cold agglutinin rather than Rouleaux
syphilis has all but disappeared.
formation or fibrin clumping.
A careful history of drug exposure should be obtained
from all patients with hemolytic anemia and/or a positive Bone Marrow Examination
DAT. In drug, induced AIHA, clinical manifestations are
the same as above except there is history (taken carefully) Not recommended routinely. Indicated if uncommon
of use of the offending drug and absence of hepatomegaly findings or in cases where lymphoma is suspected.
and significant lymphadenopathy. Cefotetan or quinidine The characteristic finding in bone marrow is erythroid
(by ternary complex mechanism) has been implicated in hyperplasia.
many severe hemolytic reactions. Fatal reactions may
occasionally occur. Cefotetan and ceftriaxone have been Biochemical Tests Suggesting Increased
associated with fatalities. Patients with hapten/drug Destruction of Erythrocytes
adsorption (e.g. penicillin) and autoimmune (e.g. alpha-
Raised serum bilirubin levels, rarely >5 mg/dL with
methyldopa) types of drug-induced hemolytic anemia
conjugated (direct) fraction constituting less than 15
exhibit mild-to-moderate hemolysis, with insidious
percent of the total. Increased urinary urobilinogen,
onset of symptoms developing over a period of days to
hemoglobinemia and depressed or absent haptoglobin
weeks.
can be seen in rapid hemolysis rarely in warm AIHA but
more commonly in patients with cold agglutinin disease,
LABORATORY FINDINGS24-27 and characteristically in patients with paroxysmal cold
hemoglobinuria and with drug-immune hemolytic anemia
Anemia
mediated by the ternary complex mechanism, even if
By definition, patients with AHA present with anemia, the extravascular. Hemoglobinuria and hemosiderinuria may
severity of which ranges from life-threatening to very mild. be seen after severe hemolysis. Serum haptoglobin levels
In fulminant cases, in which the RBC lifespan is less than are low, and lactate dehydrogenase levels are elevated.
Antiglobulin (Coombs) DAT Test Table 3 Results of DAT in 100 pediatric patients with different
and Indirect Antibody Test (IAT) serologic type of AIHA
Diagnosis of AIHA or drug-immune hemolytic anemia AIHA
requires demonstration of immunoglobulin and/or DAT Warm Cold Mixed PCH
complement bound to the patient’s RBCs outer membrane (n = 64) (n = 26) (n = 4) (n = 6)
by DAT, also known as the Coombs test (first described in DAT neg 5 11 5
1945 by Robin Coombs) which is pathognomonic for this
disease. It is a screening procedure. If agglutination is DAT pos 59 15 4 1
noted with this broad-spectrum reagent, antisera reacting Compl (C) 5 15 1
selectively with IgG (the “gamma” Coombs) or with C3 IgG 19
(the “nongamma” Coombs) are used to define the specific IgG + C 31 3
pattern of RBC sensitization. Monospecific antisera to IgM
IgG + IgA 1
or IgA also have been used in selected cases.
Nevertheless, a positive antiglobulin test requires IgG + IgA + C 1 1
cautious interpretation when there are no other features IgG + IgA 1
of autoimmune hemolytic anemia. False-positive test + IgD + C 1
results are not unusual. The reported incidence of positive IgA
antiglobulin tests in normal blood donors and general Vaglio et al. 2007
populations of hospitalized patients varies widely—from
1 in 100 to 1 in 15000. Differences in the technique used
in performing the test account for this variation. The most Patients with cold agglutinin disease have a more
common reason for a false-positive direct antiglobulin test homogeneous DAT results than patients with warm AIHA.
is low-avidity adherence of nonspecific IgG to red cells. In Since IgM antibodies are involved in this disease DAT is
rare cases, however, the result is not a false-positive but a positive almost exclusively with anti-C3 and polyspecific
harbinger of the development of autoimmune hemolytic reagents and negative with anti-IgG.
anemia. False-negative tests are usually due to low-affinity PCH is caused by Donath-Landsteiner antibody—
autoantibodies that spontaneously elute from the red cell a biphasic IgG antibody. It fixes complement at low
in vitro or amounts of erythrocyte-coating antibodies temperature and ultimately dissociate at higher tempera
that are below the limit of detection by the antiglobulin tures. As a result, DAT is positive with anti-C3, but is
test. The distinction between a true-positive and a false- generally negative with anti-IgG unless performed at colder
positive direct antiglobulin test can be made by eluting temperature. Biphasic IgG autoantibodies bind RBCs
the antibody from the red cells and testing its ability to efficiently at 0–4oC and subsequently, fix complement C1
bind to normal red cells. In a false-positive reaction, the at that temperature.
eluted antibody does not bind to normal red cells, whereas D-L antibodies are potent, so even a small titers can
binding occurs in a true-positive test. produce hemolysis.
In >95 percent of warm AIHA cases, the DAT is positive:
Series vary in their DAT results. Between 20 and 66 percent THERAPY17,22, 24,28
have only IgG on the surface, 24 to 63 percent have IgG General principles of treatment are guided by the severity
and C3, 7 to 14 percent have only C3, and 1 to 4 percent of hemolysis. Severe cases with very low hemoglobin
are DAT-negative. Patients with SLE are particularly may warrant immediate blood transfusion while mild-to-
prone to positive tests for complement on their RBCs. IgG1 moderate cases may either need only observation or else
predominates, either alone or in combination with other need to modulate the immune system’s production of
subclasses (Table 3). autoantibody and destruction of antibody-coated RBCs.
“Free” autoantibody may be detected in the plasma
or serum of these patients by the IAT. In general, patients Blood Transfusion in Autoimmune
whose RBCs are heavily coated with IgG more likely exhibit
plasma autoantibody. Patients with a positive IAT as a
Hemolytic Anemia
result of a warm-reactive autoantibody should also have In AIHA, the decision to transfuse does not depend on
a positive DAT. A patient with a serum anti-RBC antibody compatibility test results and, instead depends on an
(positive IAT) and a negative DAT probably does not evaluation of the patient’s need for transfusion. The
have an autoimmune process but rather an alloantibody indications for transfusion in patients with AIHA are not
stimulated by prior transfusion. significantly different than for similarly anemic patients
without AIHA. Patients with autoimmune hemolytic of RBCs, as little as 0.5 to 1 units. Transfusion should be
anemia (AIHA) frequently have anemia of sufficient given when indicated even before all serological tests are
severity as to require a blood transfusion. It is impossible completed.
to find compatible blood when, as is frequently the case, Compatibility testing in cold antibody AIHAs is
the autoantibody in the patient’s serum reacts with all less labor intensive. In cold agglutinin syndrome, the
normal red blood cells. autoantibody does not often react upto a temperature of
Because the antibody in this disease is usually a 37°C, whereas clinically significant RBC alloantibodies will
“panagglutinin,” reacting with nearly all normal donor react at this temperature. Accordingly, the compatibility
cells, compatible cross-matching is impossible. The goal test can be performed strictly at 37°C (Petz & Garratty,
in selecting blood for transfusion is to avoid administering 2004). If the transfusion service is not able to perform
RBC with antigens to which the patient may have testing strictly at 37°C, one or two cold autoadsorptions
alloantibodies. should be done, which will not remove a high titer cold
A common procedure is to adsorb the panagglutinin agglutinin completely, but are likely to eliminate reactions
present in the patient’s serum with the patient’s own that occur at 37°C.
RBC from which antibody has been previously eluted.
Serum cleared of autoantibody can then be tested for the GLUCOCORTICOIDS
presence of alloantibody to donor blood groups. ABO-
compatible RBC matched in this fashion are administered Glucocorticoids are the main stay of treatment in
slowly, with watchfulness for signs of an immediate-type warm AIHA. If the disease is mild and compensated, no
hemolytic transfusion reaction. treatment is needed. However, if the hemolysis is severe
Physicians should provide as many RBCs as may be with significant anemia with its antecedent signs and
reasonable because the autoadsorption procedure is symptoms, glucocorticoid treatment is started.
the most effective method for detecting alloantibodies Glucocorticoids decrease the rate of hemolysis by
in patients with warm autoantibodies. The warm blocking macrophage function by downregulating Fcγ
autoadsorption test is not useful in patients who have receptor expression and thus may suppress RBC seques
been transfused recently (within about the last 3 months) tration by splenic macrophages, decreasing the production
because even a small percentage of transfused cells may of the autoantibody, and perhaps by enhancing the elution
adsorb the alloantibody during the in vitro adsorption of antibody from the RBCs. The standard of practice is
procedure, thus invalidating the results. An alternative administration of prednisone in a dose of 1.0 to 2.0 mg/kg/
approach, which may be about as effective in avoiding day. In some patients with severe hemolysis, doses upto 6
the effects of alloantibodies, but which is not widely mg/kg/day may be required to reduce the rate of hemolysis.
implemented in transfusion services, is to perform A response, manifested by a rise in the hematocrit and a fall
extensive RBC phenotyping of the patient and the donor in the reticulocyte count usually within 3 to 4 weeks. The
units. Other simple tests that provide safety include routine duration of treatment at this dose is an unsettled question.
testing of the patient’s serum against a red cell panel and A patient who fails to improve within this time is unlikely to
diluting the patient’s serum before doing compatibility respond to further treatment with prednisone. In a patient
testing. who responds, slow reduction of the dose of prednisone
However, one need to remember that only a few percent is essential to avoid a relapse. The dose is tapered only
of all hospitalized patients have RBC alloantibodies so when the rate of hemolysis decreases significantly and
that if transfusion is extremely urgent, the lesser risk then reduced to 5 to 15 mg/day. Thereafter, slow, cautious
may be to transfuse rather than waiting for completion tapering over a period of at least 4 months is the rule. A
of the compatibility testing. In very urgent situations, rise in the reticulocyte count or a fall in the hematocrit
the quickest, but least reliable, techniques for detection should prompt an increase in the dose, usually to the
of alloantibodies are the dilution technique and partial previous level. The disease tends to remit spontaneously
RBC phenotyping. Warm autoadsorption test should within a few week or month. The Coombs test result may
be performed if there is adequate time, since it is highly remain positive, even after hemolysis has subsided. About
effective for detection and identification of alloantibodies 25 percent of patients treated with corticosteroids enter
and only requires one to three adsorptions of the patient’s a stable, complete remission; half the patients require
serum with the patient’s (ZZAP-treated) RBCs. Allogeneic continuous, low-dose prednisone; and the remaining 25
adsorption, which is the most time consuming, is indicated percent respond only transiently or not at all or are unable
if the patient has been transfused recently or if the patient’s to tolerate continuous corticosteroid treatment. There
RBCs are not available for autoadsorption. is no reliable evidence that alternate-day maintenance
Alleviation of signs and symptoms of anemia usually treatment is superior to daily treatment, but some patients
can be accomplished with relatively small quantities tolerate this schedule better than daily prednisone.
Very high doses of intravenous methylprednisolone A rise in the platelet count occurs after splenectomy in
may be tried in cases who do not respond to oral almost all patients. The increase rarely exceeds 500000/
prednisolone before other treatments are initiated or are μL and usually subsides within 3 to 5 months. Routine
critically ill. antithrombotic prophylaxis is not indicated for post-
splenectomy thrombocytosis as there is a low risk for
SPLENECTOMY thromboembolism.
Because the spleen is the major site of red cell destruction
in autoimmune hemolytic anemia, splenectomy should
Immunosuppressive Therapy
be considered for patients who have not responded to Patients who have failed to respond to splenectomy or
corticosteroids or who have maintained a stable, but have relapsed after splenectomy, when splenectomy poses
corticosteroid-dependent remission. A complete, durable an unacceptable risk, and for patients who cannot tolerate
remission follows splenectomy in half to two-thirds of steroid therapy, are the candidates for immunosuppressive
cases. Attempts to predict responsiveness to splenectomy therapy.
with measurement of splenic sequestration of 51Cr-labeled Cytotoxic and immunosuppressant drugs, such as
erythrocytes are not reliable. However, the relapse rate cyclophosphamide, azathioprine and cyclosporine A, give
following splenectomy is disappointingly high. Many a 40 to 60 percent response rate. However, these treatments
patients require further glucocorticoid therapy to maintain may be associated with serious side effects, such as bone
acceptable hemoglobin levels, although often at a lower marrow suppression, nephrotoxicity and secondary
dose than required prior to splenectomy. The only way of malignancies, while the effectiveness of other options, such
knowing the effectiveness of splenectomy in a given patient as IVIG, plasmapheresis and, danazol, is controversial.
is to perform the procedure. Laparoscopic splenectomy, a A reasonable immunosuppressive regimen might
safe method of removing the organ, is now the preferred include azathioprine (80 mg/m2/day) or cyclophosphamide
surgical technique. In most cases, a reasonable approach is (60 mg/m2/day), concomitantly with prednisone (40 mg/
to continue glucocorticoids for 1 to 2 months while waiting m2/day). Prednisone may be tapered over 3 months or
for a maximal response. However, if no response is noted so, and the cytotoxic agent continued for 6 months before
within 3 weeks, the patient’s condition deteriorates, or the reducing the dose gradually. Bone marrow suppression
anemia is very severe, splenectomy should be performed may dictate minor dose adjustments. Rapid withdrawal has
sooner. led to rebound immune response. Alternatively, high-dose
Splenectomy removes the primary site of RBC cyclophosphamide (50 mg/kg/day × 4 days) has produced
trapping. The beneficial effect of splenectomy may be a complete remission in 66 percent of patients who were
related to several factors interacting in complex fashion. refractory to other therapies. Severe myelotoxicity and
The spleen is also believed to be a major producer its attendant potential for complications are expected.
of IgG antibodies. Continuation of hemolysis after Hemorrhagic cystitis, bladder fibrosis, secondary malig
splenectomy is partly related to persisting high levels nancies, sterility, and alopecia are some of the major side
of autoantibody, favoring RBC destruction in the liver effects.
by hepatic Kupffer cells. Splenectomy has little effect on
the clearance of IgM-coated RBCs and therefore would
not be indicated in the unusual patient with a warm-
RITUXIMAB29,30
active IgM antibody. Rituximab is a monoclonal antibody directed against the
Splenectomy is complicated by a heightened risk of CD20 antigen expressed on B-lymphocytes and is used for
infection with encapsulated organisms, particularly in treatment of B-cell lymphoma. Its use for treatment of AHA is
patients younger than 2 year. Prophylaxis is indicated with based on the antibody’s ability to eliminate B lymphocytes,
appropriate vaccines (pneumococcal, meningococcal, and including presumably those making autoantibodies to
Haemophilus influenzae type B given atleast 2 weeks before RBCs. However, the mechanism of action is more complex
splenectomy) and with oral penicillin/amoxicillin after than that, as the effect of rituximab can occur very early,
splenectomy. The usual dose of penicillin for prophylaxis before the autoantibodies can recede. Rituximab (375 mg/
is 250 mg twice daily for 2 to 3 years after splenectomy m2 weekly for a median of 4 weeks) is effective in treating
(or at least until the age of 5). Education of the patient both warm AIHA and CAD, with an overall response rate
concerning the risk for serious infection after splenectomy ranging from 40 to 100 percent (median with 60%), and with
is also important. Subsequent to splenectomy, patients patients of all ages responding. Moreover, many of these
should be given antibiotics promptly with any febrile responses were durable, lasting >3 years in some patients.
illness preferably after sending a blood culture. In a large prospective series,30 13 of 15 (87%) children with
warm-antibody AHA responded to rituximab 375 mg/ 15. De Angelis, De Matteis MC, Cozzi MR, et al. Abnormalities
m2 weekly for 2 to 4 weeks. Twenty-three percent of the of membrane protein composition in patients with auto-
responders relapsed, but subsequent courses of rituximab immune hemolytic anemia. Br J Haematol. 1996;95:273-7.
induced additional remissions in this series. Thus far, few 16. Shlomchik MJ. Mechanisms of immune self-tolerance
and how they fail in autoimmune disease. In: Silberstein
side effects have been reported, but rare reactions to the
L (Ed). Autoimmune disorders of blood. Bethesda, MD:
infusion have been documented. B-cell counts remain low
American Association of Blood Banks. 1996.pp.1-34.
for months after treatment, raising the risk of infections due 17. Schwartz RS. Autoimmune and intravascular hemolytic
to poor immune response. If remissions remain durable anemias. Chapter 164 Goldman: Cecil medicine, 23rd edn.
and potential side effects are less harmful than other Saunders: An imprint of Elsevier. 2007.
treatments for warm AIHA, such as prolonged steroid use 18. Logue GL, Rosse WF, Gockerman JP. Measurement of
or splenectomy, its use may become more common. the third component of complement bound to red blood
cells in patients with the cold agglutinin syndrome. J Clin
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1984;72:245. associated with Donath-Landsteiner antibodies. Acta
4. Vaglio S, Arista MC, Perrone MP, et al. Autoimmune Haematol. 1982;68:268.
hemolytic anemia in childhood: serologic features in 100 22. Gottsche B, Salama A, Mueller-Eckhardt C. Donath-
cases. Transfusion. 2007;47:50. Landsteiner autoimmune hemolytic anemia in children: A
5. Carapella de Luca E, Casadei AM, di Pietro G, et al. Auto- study of 22 cases. Vox Sang. 1990;58:281.
immune haemolytic anemia in childhood. Vox Sang. 23. Petz LD, Garratty G. Acquired immune hemolytic anemias.
1979;36:13-20. New York: Churchill Livingstone. 1980.
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autoimmune hemolytic anemia: an observational study. autoimmune cytopenias in childhood. Pediatr Clin North
J Pediatr (Rio J). 2006;82(1):58-62. Am. 2013;60:1489.
7. Naithani R, Agrawal N, Mahapatra M, et al. Autoimmune 25. Powers A, Silberstein LE. Autoimmune hemolytic anemia,
hemolytic anemia in India: clinico-hematological Hematology Basic Principles and Practice. Hoffman R
spectrum of 79 cases. Hematology. 2006;11(1):73-6. et al. (Eds) Churchill Livingstone. 2009.
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anemia. Indian Journal of Pediatrics. 2008;75(5):451-4. hemolytic anemia in children. Pediatric Hematology-
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2009. Edinburgh: Churchill Livingstone. 1992.
10. Garratty G, Arndt PA. An update on drug-induced immune 28. Aladjidi N, Leverger G, Leblanc T, et al. New insights
hemolytic anemia. Immunohematology. 2007;23:105-19. into childhood autoimmune hemolytic anemia: a
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Ceftriaxone induced hemolysis complicated by acute Haematologica. 2011;96:655.
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C H A P T E R 23
Paroxysmal Nocturnal
Hemoglobinuria
Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired clonal disorder of hematopoetic stem cells. The molecular defect in
PNH is a somatic mutation in the PIG-A gene causing defect in glycosyl phosphatidyl inositol (GPI) anchored proteins. Deficiency of
these GPI-anchored proteins (GPI-AP) on the membrane of hematopoietic cells lead to the various manifestations of PNH.
The clinical manifestations of PNH are characterized by the hematopoetic stem cell, resulting in a deficiency in
a triad of cytopenias, intravascular hemolysis and venous the surface expression of all GPI anchored proteins due to
thrombosis at unusual sites. PNH can be broadly classified defective synthesis of GPI in the endoplasmic reticulum.
into hypoplastic and classical type. The predominant The defect in PIG-A gene is always in the somatic cells and
manifestation of hypoplastic type is bone marrow failure is never identified in the germ line; thus it is always acquired.
with a small clone of PNH cells.
The classical type is characterized by: Effect of the Defect
• Intravascular hemolysis
The mutation in PIG-A gene leads to little or no GPI anchor
• Thrombosis
being made. This leads to lot of proteins missing on the
• With or without cytopenias. membrane of these cells which are GPI anchored.
The disease is insidious in onset and causes significant
morbidity due its prolonged natural course. Management
of PNH depends upon proper classification, as the
INCIDENCE AND NATURAL HISTORY
treatment differs in different subgroups. PNH is a rare disease whose incidence is reported to be
15/million population in United States. As a result of
MOLECULAR GENETICS OF PAROXYMAL improved diagnostic tools, more and more patients are
NOCTURNAL HEMOGLOBINURIA suspected and screened for PNH.
Glycosyl Phosphatidyl Indian Perspective

Inositol Anchor (GPI-anchor) Koduri PR, Gowrishankar S et al. from Apollo Hospital
Although most membrane proteins traverse the lipid Hyderabad, in their series of 11 patients of PNH in
bilayer one or more times, certain membrane proteins 1992 reported that the Indian patients were younger
(i.e. GPI-anchored proteins) adhere to the cell surface by and showed a marked male preponderance. Severe
means of a glycolipid moiety. In PNH cases, the defect is thrombocytopenia with its hemorrhagic manifestations
attributed to mutations in the PIG-A gene. and infectious complications were not seen. However,
thrombotic complications were common. Parab RB et
al. in 1990 presented a retrospective study of 17 patients
PIG-A Gene and its Significance in PNH of PNH diagnosed on positive sucrose lysis. Anemia was
PIG-A is an endoplasmic reticulum membrane protein. present in all of them, whereas about 50 percent of the
In PNH, there is a somatic mutation in the PIG-A gene in patients had jaundice, fever and bleeding tendency.
Chapter-23 Paroxysmal Nocturnal Hemoglobinuria 239
Agarwal MB, Mehta BC et al. also reported a study of Table 1 Clinical manifestations of PNH
20 PNH patients in 1981. Neelam Verma et al. from PGI
Chandigarh found that 9.6 percent and 30.7 percent of the Due to intravascular hemolysis
Aplastic anemia patients were positive for PNH on flow • Anemia, hemoglobinuria, fatigue
cytometry on diagnosis and on follow up respectively. The • Acute/chronic renal failure, recurrent urinary tract infection
conventional tests, however, diagnosed only 4.6 percent • Back pain, headache
patients on follow up. None of the MDS patients tested • Abdominal pain, bloating
positive by any of these means, on diagnosis or at follow up. • Esophagospasms
• Cholelithiasis
CLINICAL MANIFESTATIONS (TABLE 1) • Erectile dysfunction
Rare
The clinical presentation of PNH is variably overlapped by • Choledocho dyskinesia
three features: • Acute pancreatitis
I. Intravascular hemolysis • Ischemia and ulceration of duodenum or colon
II. Thrombosis
Due to thrombosis
III. Hematopoiesis deficient.
Venous thrombosis
Intravascular Hemolysis • Abdominal vein thrombosis
– Budd-Chiari
It is one of the most important manifestations of PNH and – Splenic vein
the disease derives its name from this symptom. – Mesenteric veins
– Portal hypertension, esophageal varices, caput medusae
Pathophysiology of Intravascular Hemolysis (dilated abdominal veins)
– Renal vein thrombosis
The cause of the hemolysis is the deficiency of 2 GPI-AP
• Cerebral vein thrombosis
in the RBC membrane, that is CD55 (Decay accelerating
– Headache
factor-DAF) and CD59 (Membrane Inhibitor of Reactive
– Hemorrhagic infarct
Lysis-MIRL). CD55 and CD59 protect the RBCs from
– Retinal vein thrombosis
complement mediated hemolysis, especially CD 59. PNH
– Loss of vision
cells are divided into three types (Table 2).
– Deep vein thrombosis
Aggravating factors for hemolysis: – Pulmonary emboli
1. Nocturnal hemolysis: The nocturnal pattern of hemoly- Rare
sis, initially thought to be a consequence of systemic • Cutaneous vein thrombosis
acidosis due to retention of CO2 during sleep is now • Pyoderma gangrenosum
attributed to the nocturnal absorption of lipopolysac- • Arterial thrombosis (less common)
chrides (LPS), a byproduct of bacterial cell wall from • Stroke
the gut. LPS markedly activates complement system. • Myocardial infarction
LPS is normally bound by monocytes through a GPI Due to bone marrow failure
linked protein, CD14, which is missing in PNH.
• Anemia, infections, bleeding
2. Concurrent inflammation or immune reactions to
• Myelodysplastic syndrome
infections: Complement is also activated by con
• Bone pain
current inflammation or the immune reaction to
Rare: Transformation to acute myeoloid leukemia (AML)
infections. Most viral disorders will result in a burst
Table 2 Types of PNH cells

Type CD55/CD59 Complement Erythrocyte survival (t1/2) Cell of origin
sensitivity
I Normal Normal 120 days Normal stem cell
II 10–50% of normal 3–15 times 30–60 days Defective clone with limited ability to
make GPI anchor
III Absent 15–25 times 4–6 days Clone unable to make GPI anchor
of hemolysis. The most serious hemolysis results 3. Other manifestations: The free hemoglobin in
from gastrointestinal inflammation, usually due to plasma diffuses in the tissues and binds to nitric
viral gastroenteritis. This may be due to increased oxide (endothelial derived relaxing factor) causing
absorption of LPS and the effect of the immune a local deficiency of nitric oxide in the tissues. This
reaction against the virus. leads to contraction of smooth muscles leading to
3. Immune reactions, which otherwise cause minimal various clinical manifestations such as early morning
hemolysis in normal persons, can cause severe hemolysis substernal tightness, dysphagia (esophageal contrac
in PNH patients’, e.g. infectious mononucleosis. tion), penile erectile dysfunction in males, sense of
– Immunizations and vaccinations: Special precau fatigue and weakness, abdominal pain, Jaundice due to
tion should be taken while giving repeated doses of indirect hyperbilirubinemia. The patients of PNH may
polysaccharide containing vaccine against Strepto be chronically jaundiced due to the ongoing hemolysis
coccus pneumoniae. in the circulation. This is sometimes aggravated in
– Blood transfusion in a PNH patient can lead to a acute hemolysis.
burst of hemolysis, due to the activation of comple
ment by immune reactions involving leukocytes or Thrombosis
plasma proteins. This can be avoided by washing of
Thrombosis has been reported to be a leading cause of
RBCs prior to transfusion, in a susceptible patient.
morbidity in PNH patients.
– Few reports suggest that intravenous hematinics
and injection erythropoietin may aggravate hemo
lysis. Pathophysiology of Thrombosis
The pathophysiology of thrombosis in PNH is not clearly
Clinical Effects of Intravascular Hemolysis understood. Various mechanisms are postulated:
1. Hemoglobinuria: It is due to the excretion of αβ globin • Absence of CD59 on the platelets may play a role in the
dimers in urine as the reabsorption capacity of proximal hypercoagulable state of PNH.
tubules is exceeded. The incidence of hemoglobinuria • The PNH platelets are more sensitive to aggregation by
presenting anytime during the course of the disease thrombin than normal platelets.
ranges from 27 to 84 percent. • The abnormal monocytes of PNH lack the receptor
2. Renal dysfunction: PNH patients can develop renal for plasminogen activator and thus are less efficient in
complications in the form of either acute renal failure fibrinolysis.
or chronic kidney damage. Thrombosis in PNH is mostly venous, and at unusual
a. Acute renal failure: This occurs when the sites. Preference for these sites may be due to the locally
concentration of hemoglobin in the tubular filtrate retarded blood flow, sufficient for the formation of platelet
becomes sufficiently high to impair renal function, aggregates. This also permits activation of complement on
and acute renal failure sets in. This situation is the surface of RBC that can be transferred to the neighboring
usually seen with gastrointestinal illnesses and is endothelial cells resulting in the expression of tissue factor
complicated by the inability of the patient to take and initiating clotting. The common sites for thrombosis are:
sufficient amount of water orally. Although there Hepatic veins and veins of portal system: These are
is usually good recovery if the condition is treated particularly affected. Hepatic and portal vein thrombosis
with hydration and careful monitoring of blood in PNH can present in either of the forms:
pressure, there often is residual renal impairment. • Acute onset of thrombosis: This results in the classic
b. Chronic renal damage: Hemoglobinuria can lead to Budd-Chiari syndrome, often seen in the setting of
chronic damage to the kidney. It manifests in two severe hemolysis, leading to
forms. – Tender hepatomegaly
c. Proximal renal tubular acidosis: This happens – Ascites
when the concentration of globin dimers in the – Jaundice
glomerular filtrate becomes so great that the – Elevation of serum enzymes indicative of liver
resorption capacity of the proximal tubule for other damage.
molecules normally resorbed by this epithelium is • Gradual hepatic vein thrombosis
impaired. – Pain in the right upper quadrant
d. Chronic renal failure: This is usually slowly – Hepatomegaly
progressive and may result in death. It is the cause – Gradual onset of ascites
of death in 8 percent of the PNH patients. – Signs of portal hypertension
Radiological diagnosis can be made in both instances. Classification

• Inferior vena cava thrombosis:
– Can lead to lower body anasarca PNH is a disease of varied clinical presentations. It is
classified into subtypes, based on predominant clinical
– Renal vein thrombosis can cause renal dysfunction
manifestations and laboratory features.
in the form of proteinuria and renal failure.
There are various proposed systems of classification of
• Splenic vein thrombosis:
PNH.
– Causing massive splenic enlargement and even
rupture. Clinically PNH can be divided into two types:
• Splanchnic vein thrombosis: 1. Classical PNH: Patients have predominant hemolytic
– Results in a syndrome of recurrent abdominal pain and thrombotic manifestations and some degree of
sometimes bowel necrosis hematopoietic deficiency.
• Cerebral venous thrombosis: 2. Hypoplastic PNH: Patients have features of bone
– Common in PNH patients marrow failure with a PNH clone which may or may
– Sagittal sinus is most frequently involved not have hemolytic or thrombotic manifestations.
– Patients have headache, signs of raised intracranial Wendell P et al. divided PNH into five groups:
tension and focal neurological deficits 1. Aplastic anemia with detectable PNH cells [AA-PNH]:
• Retinal vein thrombosis: This group is characterized by bone marrow failure with
– Manifests as diminution of vision. detection of fewer than 5 percent PNH granulocytes in
These are the common sites of venous involvement in the peripheral blood by flow cytometry. These patients
PNH. In addition to this rarely there can be may not develop overt PNH symptoms in future.
• Thrombosis of dermal and epididymal veins: 2. Aplastic anemia—PNH [AA-PNH]: In this group the
– In the former, there is necrosis of the skin predominant syndrome is bone marrow failure, but
– The later results in a syndrome that is confused with presence of >5 percent PNH cells which can lead
with epididymitis, orchitis or torsion of the testis to some clinical features.
– Thrombosis of the veins of the uterus can cause a 3. PNH-aplastic anemia [PNH-AA]: In this group the
syndrome resembling pelvic inflammatory disease. predominant clinical syndrome is PNH with significant
It is postulated that the PNH granulocyte clone size is evidence of bone marrow hypoplasia including
predictive of the risk of thrombosis. The overall incidence granulocytopenia and thrombocytopenia.
of thrombosis in patients with granulocytic clone size 4. Classic PNH [PNH]: Syndrome of PNH without bone
marrow hypoplasia.
larger than 50 percent is 53.5 percent, as compared to an
5. MDS-PNH or PNH-MDS: In this group, PNH cells are
incidence of only 5.8 percent in patients with a small PNH
present in a patient with predominant myelodysplastic
granulocytic clone. Thrombosis as a complication predicts
hematopoiesis in the former or when the clinical
poor survival.
syndrome is predominantly due to the abnormal
Factors associated with high-risk of thrombosis during PNH cells with some elements of MDS as in the later
the disease course: (Table 3).
• Thrombosis at diagnosis
• Age over 54 years WHO SHOULD BE SCREENED FOR PNH?
• Presence of infection at diagnosis.
(TABLE 4)
Thrombosis in PNH mostly involves the venous system.
However, there are few case reports of arterial thrombosis PNH is a rare disease with varied clinical manifestations.
also. The International PNH interest group has suggested
screening for PNH in the following patients (Table 3):
• Patients with hemoglobinuria
Hematopoiesis Deficient • Patients with Coombs-negative intravascular hemo
Many patients of PNH have a history of aplastic anemia lysis (based on abnormally high serum LDH), especi
and diminished hematopoiesis to a greater or smaller ally patients with concurrent iron deficiency.
extent. Nearly 50 percent of patients of aplastic anemia • Patients with venous thrombosis involving unusual
have a readily detectable PNH clone, particularly during sites:
or after recovery with antithymocyte globulin therapy. – Budd-Chiari syndrome
Many patients of PNH develop aplastic anemia in – Other intra-abdominal sites (e.g. mesenteric or
the final stage. PNH like cells are found in 20 percent portal veins)
of patients of MDS. Anemia and hemorrhage are more – Cerebral veins
common in pediatric than in adult PNH patients. – Dermal veins.
Table 3 The International PNH interest group (Parker et al. Dec. 2005) classification
Types Hemolysis/Thrombosis Bone marrow Cytogenetic Flow cytometry Hams/Sucrose
abnormality lysis test
Classical Yes Erythroid hyperplasia Absent Positive Positive
PNH/SAA*-MDS** Yes Associated disorder May be seen Positive Positive
(SAA, MDS, MF)
Subclinical No Associated disorder May be seen Positive Negative
(SAA*, MDS**, MF***
*SAA: Severe aplastic anemia; **MDS: Myelodysplastic syndrome; ***MF: Myelofibrosis
Table 4 Who should be tested for PNH and how often?

Once Repeatedly #
• All patients with hemoglobinuria • All patients with PNH
• All patients with unexplained hemolysis (increased LDH) • All patients who have aplastic anemia
• All patients with abdominal and cerebral vein thrombosis • All patients who have had aplastic anemia (except after bone
• All patients with thrombocytopenia and macrocytosis or signs marrow transplantation)
of hemolysis • All patients with myelodysplastic syndrome (MDS)
Initially once every 6 months; then annually
#
• Patients with aplastic anemia (screen at diagnosis This discrepancy reflects underlying marrow dysfunc
and once yearly even in the absence of evidence of tion that is invariably a component of the disease.
intravascular hemolysis).
• Patients with refractory anemia—MDS. Urine Hemosiderin
• Patients with episodic dysphagia or abdominal pain
with evidence of intravascular hemolysis. Due to intravascular hemolysis, there is continuous
presence of hemoglobin in the glomerular filtrate in the
LABORATORY DIAGNOSIS (TABLE 5) kidney. This excess of hemoglobin is deposited in cells
of the proximal convoluted tubule as hemosiderin and
Laboratory workup of a patient suspected of PNH includes can be detected in urinary sediments. At least 3 samples
the essential tests to diagnose and classify PNH as well of urine should be tested for hemosiderinuria which
as some ancillary tests to determine the prognosis and indicates chronic intravascular hemolysis.
treatment.
The various laboratory tests include: Bone Marrow Examination
Morphologic analysis of the bone marrow aspirate and
CBC
biopsy and cytogenetics are needed for proper classi
The blood picture varies. There may be: fication, as PNH is often observed in association with
• Severe pancytopenia marrow failure syndromes.
• Bicytopenia • Bone marrow cellularity may vary from hypo, normo to
• Normal counts hypercellular.
• Virtually all patients are anemic with mild macrocytosis • In classic PNH it is normo to hypercellular with
• Occasionally, when urinary iron loss is considerable, erythroid hyperplasia.
the red cells may appear microcytic and hypochromic • It is usually hypocellular in hypoplastic PNH.
• In addition polychromasia, normoblasts and fragmen • The presence of dysplasia other than in erythroid
ted red cells may be seen on peripheral smear series and cytogenetic abnormalities (present in 20%)
• Relative reticulocytosis. suggest associated hematological disorder.
This may be marked but the absolute reticulocyte count • The bone marrow iron stain shows decreased iron in
is often lower than that found in association with other case of iron loss due to hemolysis and increased iron in
hemolytic disorders at comparable degrees of anemia. case of transfusion dependency.
Table 5 Laboratory tests for the diagnosis of PNH

Diagnostic tests
Traditionally
• Ham test (acidified serum lysis) The lysis of PNH red blood cells exposed to activated complement
• Sucrose lysis test tests for the deficiency of CD59 and CD55 on red blood cells. The
• Thrombin lysis test tests vary in the pathways activating complement.
Advantage: Cheap and simple to perform.
Disadvantage: Labor intensive, decreased sensitivity due to the
short half-life of circulating PNH red blood cells.
Today
• Flow cytometric analysis Advantage: Useful to determine the degree of GPI anchor
– CD59 and/or CD55 peripheral blood red cells deficiency (PNH type I, type II, type III).
Disadvantage: Decreased sensitivity due to the short half-life of
circulating PNH red blood cells.
CD59, CD24, CD16, or any other GPI-linked proteins expressed on Advantage: The deficiency of at least 2 linked proteins is sensitive
peripheral blood granulocytes and specific for the diagnosis of PNH.
Disadvantage: Might be difficult to perform in severe aplastic
anemia when the number of circulating granulocytes is very low.
FLAER (fluorescently labeled inactive toxin aerolysin) binding of FLAER binds the GPI anchor.
peripheral blood granulocytes Advantage: The lack of FLAER binding on granulocyte is sufficient
for the diagnosis of PNH.
Disadvantage: Cannot be used for the analysis of red blood cells or
platelets. Might be difficult to perform in severe aplastic anemia
when the number of circulating granulocytes is very low.
PIGA gene mutation analysis Although very specific is NOT used for diagnosing PNH
Serum Iron Studies and Ferritin lysis in the patient’s own acidified serum, sucrose lysis test
is negative and the expression of GPI-AP is normal.
Due to chronic urinary iron loss in patients with hemolytic The standard Ham test can be negative when there
PNH, they may have iron deficiency. In contrast, patients are less than 5 percent PNH Type III cells or less than 20
of aplastic anemia with a clone of PNH may have normal or percent PNH Type II cells.
sometimes even increased serum iron due to transfusion
dependency. Sucrose Lysis Test
The sucrose lysis test is based on the fact that red cells absorb
Complement Based Assays complement from serum at low ionic concentrations. PNH
Principle cells, because of their greater sensitivity will undergo lysis
but normal red cells do not. In PNH, lysis usually varies
PNH cells are unusually susceptible to lysis by complement. from 10 percent to 80 percent, but exceptionally may be
This can be demonstrated in vitro by a variety of tests. as little as 5 percent. Sucrose lysis test is more sensitive
and less specific than the Ham test, as red cells from some
Acidified Serum Lysis Test (Ham Test) cases of leukemia or myelofibrosis may undergo a small
amount of lysis, almost always less than 10 percent. In
The principle of this test is that patient’s red cells are these cases the Ham test is usually negative.
exposed at 37°C to the action of normal or patients own
serum, suitably acidified to the optimum pH for lysis (pH GPI—Anchor-based Assays
6.5–7.0). In PNH, 10 to 50 percent lysis is usually obtained.
The Ham test is relatively specific but less sensitive. The Today these tests are the mainstay of the diagnosis of PNH.
only disorder, other than PNH that may appear to give a
Principle
clear cut positive test is a rare congenital dyserythropoietic
anemia CDA Type II or HEMPAS. In contrast to PNH, It involves the use of monoclonal antibodies against
however HEMPAS red cells undergo lysis in only a specific GPI-anchored proteins in conjunction with flow
proportion (about 30%) of normal sera, do not undergo cytometry to diagnose PNH.
Advantages Effects
Flow cytometry offers several advantages over complement • Stabilization of hemoglobin levels, reduction or
based assay for diagnosing PNH: cessation of transfusion requirement
• It is more sensitive and specific • Reduction of the intravascular hemolysis, cessation of
• Measures the size of the PNH clone hemoglobinuria
• It is less affected by blood transfusions. • Clinically significant improvement in the quality of life
RBCs are the easiest to deal with but are not the most • Significant reduction in the rate of thrombosis.
sensitive cell type to test because hemolysis or transfusion
may decrease or even eliminate the PNH clone. Assaying Corticosteroids
granulocytes is better, assuming there is not severe granu The use of corticosteroids in treating chronic hemolysis
locytopenia, but not all GPI-anchored surface antigens is debated because of the empiric nature of therapy
expressed granulocytes will give identical results. The and no experimental data to support the explanation in
recommendation is that more than one GPI-AP must ameliorating complement mediated hemolysis. It may
be demonstrated to be abnormal and depending upon have a role in attenuating acute hemolytic exacerbation
circumstances, more than one cell line should be evaluated. given in a dose of 0.25 to 1 mg/kg of prednisolone. A
Flow cytometry can be useful to know the extent rapid response (within 24 hours) suggests complement
of involvement and monitoring therapy in PNH. The inhibition either by directly inhibiting alternate pathway
standard flowcytometer can detect a PNH clone of or dampening the inflammation.
>3 percent. The best estimate of clone size is given by
CD55, CD59 and CD66b in granulocytes and CD55, CD59
Androgens
and CD14 in monocytes.
The mechanism of the amelioration of anemia in PNH
Aerolysin Based Assays is thought to be complement inhibition and stimulating
erythropoiesis. Danazol is usually used in these cases. A
A fluorescently labeled aerolysin, FLAER, is now the gold starting dose of 400 mg twice a day is recommended, but
standard for diagnosis of PNH. These flow cytometry test a lower dose (200–400 mg/d) may be adequate to control
are now done in many centers. chronic hemolysis. Benefit of danazol was attributed to
reduced hemolysis rather than enhanced erythropoesis.
TREATMENT OF PNH Lack of venous thrombotic complications is an advantage
of danazol over other androgens. Danazol has been shown
Treatment of PNH is under evolution. It aims at: to increase fibrinolytic activity thus offering protection
• Supportive treatment against thromboembolism.
• Definite treatment
Supportive treatment is as follows:
Folate
Anemia: It is invariably present in PNH patients. It may be
either due to hemolysis or bone marrow failure. Supplemental folate (5 mg/d) is recommended in all
patients to compensate for increased utilization associated
Anemia due to Hemolysis with heightened erythropoiesis that is a consequence of
hemolysis.
If anemia is predominantly due to ongoing hemolysis, it
needs to be treated. The various treatment options are:
Chronic Transfusion Therapy
Eculizumab Transfused red cells are CD55 and CD59 positive and thus
have normal survival. It also suppresses erythropoesis
This is a recombinant humanized monoclonal antibody in addition to increasing hemoglobin. Iatrogenic hemo
against the terminal complement components and is an chromatosis is delayed in PNH patients due to iron loss.
effective therapy in PNH. It is a remarkably safe drug and
has been approved for the treatment of PNH. However,
there is a slightly increased risk of developing infections Iron Replacement Therapy
with encapsulated organisms’ especially meningococcal Patients of PNH often become iron deficient as a result of
infections. Thus, all patients should receive appropriate hemoglobinuria and hemosiderinuria. Iron replacement,
vaccinations and early therapy as required. both oral and parenteral causes exacerbation of hemolysis.
However, iron therapy should not be withheld because of Stem Cell Transplant for PNH
this and the exacerbation should be treated with steroids,
androgens and blood transfusion. This is the only definite treatment for PNH. The overall
survival for PNH patients who undergo transplantation
using an HLA-matched sibling donor is in the range of 50
Splenectomy percent to 60 percent.
There are only anecdotal reports of amelioration of
Indications for consideration of transplantation: Inter
hemolysis and improvement of cytopenias by splenectomy,
national PNH Interest Group recommendations:
but there is an increased risk of postoperative thrombosis;
• Bone marrow failure
therefore it is not recommended in the treatment of PNH.
Decision on transplantation is based on underlying
marrow abnormality (e.g. aplastic anemia)
Treatment of Nonhemolytic Anemia • Major complications of PNH
Pancytopenia with low reticulocyte count indicates anemia – Recurrent, life-threatening thromboembolic disease
due to bone marrow failure. Thus treatment should aim at – Refractory, transfusion-dependent hemolytic ane-
the underlying disease (Aplastic anemia, myelodysplastic mia. The availability of new treatment options (e.g.
syndrome). Hence, when immunosuppressive therapy is eculizumab) may influence the decision to recom-
used in a patient of PNH, therapy is aimed at the treatment mend transplantation.
of bone marrow failure. Androgens may also be beneficial
in patients with PNH who have a hypoproliferative PEDIATRIC PNH
component to their anemia. PNH can occur in the young (about 10% of patients are
Thrombosis: Thrombosis is the leading cause of mortality younger than 21) but is often misdiagnosed and misman-
in patients of PNH. The occurrence of thrombosis has aged. A retrospective analysis of 26 cases underscored
been related to the size of the granulocytic clone. the many similarities between childhood and adult PNH.
Signs and symptoms of hemolysis, bone marrow failure,
Prophylaxis: Prophylactic anticoagulation in PNH patients and thrombosis dominate the clinical picture, although
is a matter of debate in the International PNH Interest hemoglobinuria may be less common in young patients.
Group and not yet a standard recommendation. A generally good response to immunosuppressive therapy
may be seen. However, based on the lack of spontaneous
Management of Thromboembolic Disease remissions and poor long-term survival (80% at 5 years,
60% at 10 years, and only 28% at 20 years), sibling-matched
Patients of PNH usually present with venous thrombosis.
stem cell transplantation is the recommended treatment
Any acute thromboembolic event requires anticoagulation
of childhood PNH. A recent Dutch study confirmed the
with heparin.
common presentation of bone marrow failure in 11 chil-
Patients presenting with acute Budd-Chiari syndrome
dren with PNH, and reported that 5 patients eventually un-
should be managed with thrombolytic therapy and/or
derwent bone marrow transplantation (BMT; 3 matched
radiological intervention.
unrelated donors and 2 matched family donors), of whom
Thrombocytopenia should not come in way of
4 are alive. Mortality appears high in young patients with
anticoagulant treatment. It is a relative but not an absolute
PNH treated with transplantation using unrelated donors
contraindication for anticoagulation. It can be managed
although surviving cases have been reported.
with repeated platelet transfusions. However this may not
always be possible. BIBLIOGRAPHY
Patients with a thrombotic episode require life-long
anticoagulation. Long-term anticoagulation should be 1. Agarwal MB, Mehta BC. Paroxysmal nocturnal hemo
reassessed in any patient who undergoes a spontaneous globinuria: (a report of 20 cases). J Postgrad Med. 1981;27:
remission or in whom the PNH clone size falls to below 50 231-4.
2. Brodsky RA. New Insights into Paroxysmal Nocturnal
percent.
Hemoglobinuria. In: Berliner N, Linker C, Schiffer CA
Stem cell transplantation is indicated in recurrent life (Eds). Hematology 2006, American Society of Hematology
threatening thrombotic complications. Education Program Book, Orlando, Florida. 2006.pp.24-8.
3. Brodsky RA. Paroxysmal Nocturnal Hemoglobinuria.
Definitive Treatment of PNH In: Hematolgy—Basic Principles and Practice. Hoffman
R, Benz EJ, Shattil SJ, Furie B, Cohen HJ, Siberstein LE,
The definitive treatment of PNH includes stem cell McGlave P. (Eds) 4th Edn. Elsevier Churchill Livingstone.
transplantation and gene therapy. 2005.pp.419-27.
4. Gabrielle Meyers, Charles J Parker. Management Issues 9. Koduri PR, Gowrishankar S. Paroxysmal nocturnal haemo
in Paroxysmal Nocturnal Hemoglobinuria. Int J Hematol. globinuria in Indians. Acta Haematol. 1992;88(2-3):126-8.
2003;77:125-32. 10. Madkaikar M, Gupta M, Jijina F, Ghosh K. Paroxysmal
5. Ghosh K, Madkaikar M, Gupta M, Jijina F. Evaluation of nocturnal haemoglobinuria: diagnostic tests, advantages,
danazol, cyclosporine, and prednisolone as single agent or and limitations. Eur J Haematol. 2009;83:503-11. Review.
in combination for paroxysmal nocturnal hemoglobinuria. 11. Parab RB. Paroxysmal nocturnal hemoglobinuria: a study
Turk J Hematol. 2013;30:66-370. of 17 cases. J Postgrad Med. 1990;36:23-6.
6. Hall SE, Rosse WF. The use of monoclonal antibodies and 12. Parker J Charles. Historical aspects of Paroxysmal
Nocturnal Hemoglobinuria: ‘Defining the disease’. British
flow cytometry in the diagnosis of paroxysmal nocturnal
Journal of Hematology. 2002;117:3-22.
hemoglobinuria. Blood. 1996;87:5332-40.
13. Rosse Wendell F, Nishimura J. Clinical manifestations of
7. Hill A, Stephen J, Hillmen P. Recent developments in
paroxysmal nocturnal hemoglobinuria: present state and
the understanding and management of paroxysmal future problems. International Journal of Hematology.
nocturnal hemoglobinuria. British Journal of Hematology. 2003;77:113-20.
2007;137:181-92. 14. Varma N, Garewal G, Varma Subhash, Vohra H. Flow
8. Hillmen P, Hall C, Marsh JC, et al. Effect of Eculizumab on cytometric detection of PNH defect in Indian Patients with
hemolysis and transfusion requirements in patients with Aplastic anemia and Myelodysplastic Syndromes. Letters
paroxysmal nocturnal hemoglobinuria. N Engl J Med. and correspondence. American Journal of Hematology.
2004;350:552-9. 2000;65:263-6.
C H A P T E R 24
Diagnosis and Management of Acquired
Aplastic Anemia in Children
Nitin K Shah
Aplastic anemia (AA) is defined as pancytopenia caused by bone marrow failure with bone marrow hypocellularity without infiltration
or fibrosis.1,2 It is a difficult proposition to treat in resource crunched set-up lie in developing countries and carries high mortality. It
accounts for 20 to 30 percent cases presenting with pancytopenia2,3 and pediatric AA accounts for nearly 16 percent of all AA.4 In
USA and Europe, incidence of childhood AA is estimated at 2 to 6/million.1 Incidence in India is not known exactly but is perceived as
higher than in the West by Indian pediatric hematologists. A recent study from Canada has reported increased incidence in children
of east and south-east Asian descent (7.3/million/year) compared to white or mixed ethnic groups (1.7/million/year).5 In Western
countries, AA has been reported with equal frequency in boys and girls. However, in Indian studies AA is reported to occur three to
four times more frequently in boys compared to girls.6,7 It can be inherited (Inherited bone marrow failure syndromes like Fanconi’s
anemia) or acquired which may be then primary or idiopathic; or secondary to some insults like drugs, etc. It is also classified as mild
to moderate, severe and very severe depending on the severity of pancytopenia. In large series, nearly 70 percent cases have been
reported to be severe/very severe and almost 70 to 80 percent of acquired aplastic anemia cases to be idiopathic.4,8
DIAGNOSIS OF APLASTIC one of the secondary causes in any case of AA as shown

ANEMIA IN CHILDREN in Table 1.
One should also look for findings suggestive
As in any other disease diagnosis involves detailed clinical of inherited bone marrow failure syndromes like
history, head to toe clinical examination and laboratory skeletal anomalies, mental retardation, short stature,
investigations; and in that order! peri-oral hyperpigmentation, nail dystrophy, renal
anomalies on ultrasound and a strong family history
Clinical Presentations like consanguinity, other sibs affected in family and
cousins and some family members having some of the
Clinical presentation will include effects of depressions anomalies without frank AA.
of all three peripheral blood cell lines, viz anemia, • One should also look for findings like presence of
neutropenia and thrombocytopenia; and effects of the lymphadenopathy, hepatosplenomegaly, bone pains
cause of AA. and weight loss which may suggest pancytopenia
• It will include effects due to anemia like pallor, easy due to other causes like leukemia, lymphomas,
fatigability, tiredness, headache, breathlessness, myelodysplastic syndromes, myelofibrosis, megalo-
puffiness of face and edema of the feet, tachycardia, blastic anemia, osteopetrosis, etc.
tachypnea, frank cardiac failure.
• Effects due to neutropenia like fever, sepsis, oral ulcers.
• Effects due to thrombocytopenia like petichea, pur
Laboratory Investigations
pura, ecchymosis, mucosal bleeds like epistaxis, gum Basic tests done in a case of suspected AA include
bleeds, gastrointestinal (GI) hemorrrhage, hematuria, complete blood count (CBC), PS examination and
intracranial bleeds, etc. One should carefully look for corrected reticulocyte count. CC will show different
Table 1 Classification of aplastic anemia in children test is required after 2 to 3 weeks to see for evolution
of changes of hypocellular MDS. When hypocellular
• Acquired: MDS is suspected, heparinized bone marrow should
– Viruses be also sent for cytogenetic study.
i. EBV • Lastly, one should also do stress cytogenetic study to
ii. Hepatitis rule out Fanconi’s anemia in all cases of pediatric AA
iii. HIV and irrespective of presence of physical anomalies as
– Immune diseases some cases of Fanconi’s anemia may be phenotypi
– Eosinophilic fascitis cally absolutely normal. It is important to rule out
– Hypoimmunoglobulinemia Fanconi’s anemia in all cases of pediatric AA as the
– Thymoma treatment for Fanconi’s anemia is totally different
– Pregnancy and immunotherapy has no role in these patients.
– Paroxysmal nocturnal hemoglobinuria (PNH) Stress cytogenetic study can be done on peripheral
• Inherited: blood lymphoctyes. Recently, even genetic study for
– Fanconi anemia Fanconi’s anemia is available in some centers which is
– Dyskeratosis congenital useful to confirm Fanconi’s anemia in rare cases where
– Shwachman-Diamond syndrome there is strong suspicion clinically but repeated stress
– Reticular dysgenesis cytogenetic studies are inconclusive.
– Amegakaryocytic thrombocytopenia • Other tests that are required include:
– Familial aplastic anemia – Liver function tests as a baseline before starting
– Nonhematological syndromes androgenic steroids.
– Down syndrome – Renal function tests before starting cyclosporine.
– Dubowitz syndrome – Serum lactate dehydrogenase (LDH) and uric acid
– Seckel syndrome which, if high may suggest leukemia as diagnosis.
– HbF levels which, if high may suggest Fanconi’s
anemia.
severity of pancytopenia with anemia, neutropenia and – One should also look for secondary causes like
thrombocytopenia. HIV/HCV/HBsAg/Parvovirus as aplastic anemia
• Corrected retic count will be lower than 1 percent. can follow one of these viral infections.
Mean corpuscular volume (MCV) is usually high with – PNH study by flow cytometry for CD 55 and
normal differentiating it from megaloblastic anemia CD 59 cells or HAMs test or sucrose lysis test, if flow
where MCV is high but red cell distribution width cytrometry is not available.
(RDW) is also high.
The PS will confirm macrocytosis without aniso RISK STRATIFICATION OF APLASTIC
cytosis, neutropenia and thrombocytopenia. Presence ANEMIA IN CHILDREN
of some changes would suggest other diagnoses
like blasts (leukemia), leukcoerythroblastic changes International Aplastic Anemia Study Group takes in to
(osteopetrosis), etc. consideration three peripheral blood criteria, i.e. absolute
• Bone marrow aspiration and bone marrow trephine neutrophil count (ANC) below 0.5 × 109/L, platelet count
biopsy: Diagnosis is confirmed by bone marrow below 20 × 109/L and corrected retic count below 1 percent
aspiration as well as bone marrow trephine. On aspirate (or absolute retic count below 20 × 109 /L); and two bone
smears, one will see hypocellularity with increased fat marrow criteria, i.e. bone marrow biopsy showing severe
spaces, however, this could also be due to technically hypocellularity < 25 percent; or moderate hypocellularity
dilute marrow or a hypocellular myelodysplastic (25–50%) with hematopoietic cells representing less than 30
syndrome (MDS). On the other hand, a hypercellular percent of residual cells.9 Based on these AA is classified as:
marrow does not necessarily rule out aplasia on bone • Severe aplastic anemia (SAA) when any 2 of the
marrow aspirate alone as it could have hit one of the 3 peripheral blood criteria and either marrow criterion
few persisting cellular foci. Hence, a trephine biopsy, are present.
which gives a wider area to look at, is mandatory for • If ANC is below 0.2 × 109/L, then it is labeled as very
diagnosis of aplastic anemia. A hypocellular MDS severe aplastic anemia (vSAA).
should be kept in mind, even if hypocellular marrow • All other cases are classified as mild-moderate AA.
is found on trephine biopsy, if there are dysplastic It is important to classify AA as inherited vs. acquired
changes present. In such cases a repeat bone marrow and acquired AA as severe, very severe or mild-moderate
Chapter-24 Diagnosis and Management of Acquired Aplastic Anemia in Children 249
as the outcome and treatment needed are different for Table 2 Indications of using prophylactic platelets in AA
different severity. Most inherited causes need stem cell
transplant (except TAR which normally resolves by 1 year Prophylactic platelets (without bleeding)
and Diamond-Blackfan syndrome which has 25 percent • < 500–10000/cumm in a non-sick child
chances of spontaneous regression) whereas many • < 20000/cumm in a sick child with:
mild-moderate and some severe AA may resolve with – Severe mucositis
immunosuppressive therapy or sometimes even with – DIC
supportive care alone; and many severe and all very severe – Platelet likely to fall < 10,000/cumm before next evaluation
AA will need stem cell transplant.10,11 – Associated coagulopathy/anticoagulation
The number of days taken for ANC to improve with • Before surgery
G-CSF and non-response to any specific therapy at – Bone marrow aspiration/biopsy can be without platelet
6 months have also been proven to be risk factors in one support
study.12 Certain cytogenetic changes at diagnosis like – Lumbar puncture < 30,000/cumm
presence of telomerase gene mutations leading to short – Other surgeries < 50,000/cumm
telomeres make the prognosis unfavorable.14 Presence – Surgery at critical sites like CNS, eyes < 100,000/cumm
of cytogenetic abnormalities after diagnosis also alter • < 50,000/cumm with acute bleeding, massive hemorrhage,
prognosis like trisomy 8, chromosome 13 abnormalities, head trauma, multiple trauma
chromosome Y deletion, etc. suggest favorable outcome,
whereas monosomy 7 and complex abnormalities depict
not required prophylactically unless platelets counts
an unfavorable outcome.13
are less than 500 to 10,000/cumm in a non-sick child,
<20,000/cumm in a sick febrile child or patient is
Supportive Care undergoing some surgical procedure as shown in
Supportive care is the main stay besides definitive Table 2. Platelets may be liberally used in presence of
therapy, and for many ill affording patients it is the only sepsis and DIC with platelets < 20,000/cumm as there is
treatment available. It includes use of blood components a lot of consumption of platelets in these conditions. For
to control anemia and thrombocytopenia, management of minor mucosal bleeds (except hematuria) one can use
infections, psychosocial support and financial help. platelets sparing drugs like tranexamic acid in the dose
of 75 mg/kg/day in 3 divided doses orally. Similarly,
Transfusion Support menorrhagia may be a problem in adolescent girl who
can benefit with hormonal replacement therapy.14,15
• Packed red blood cell: Target is to keep hemoglobin
in near physiological range of about > 9 gm% so as Care of Infections
to improve quality of life. One should do complete
antigen phenotype before first transfusion so as to Infections are the most common problems and cause of
use a particular rare blood group donor should there death in untreated severe and very severe AA patients, and
be sensitization after multiple transfusions. It is are difficult to eradicate in spite of effective antimicrobials.
mandatory to use leukodepletion by using white blood Infections are also related to use of immune suppressive
cell (WBC) filters with each transfusion so as to avoid therapy as well as in post-transplant period. Bacterial
HLA sensitization as well as prevent febrile transfusion infections are the most common infections in AA patients
reactions. It is preferable to use irradiated blood followed by fungal, parasitic and of course viral infections
products to prevent transfusion associated graft versus which are otherwise also so common in children in general.
host disease, especially in post-transplant period. Gram negative sepsis is more common than gram positive
One should also always avoid relative donors for any sepsis in India. Recently several hospitals are facing
transfusion, especially if the patient is for a potential extended spectrum beta lactamase (ESBL) producing
transplant candidate. Cytomegalovirus (CMV) negative gram-negative organisms as well as methicillin resistant
donor is desirable but not practicable as most of the Staphylococcus aureus sepsis in neutropenic patients.
donors in India are CME positive. In presence of Partly, this is because of haphazard use of antimicrobials
bleeding and infection, packed red blood cells (PRBCs) in general. Initial choice of antimicrobials in a patient with
are used more liberally. suspected sepsis in AA patients will depend on the recent
• Platelets: Platelet transfusion is reserved for patient local experience with the type of microorganisms grown
with mucosal bleeds. It is not required for only skin and treatment failure with a particular antimicrobial.
bleeds, howsoever grotesque they may look. It is also Initially, broadspectrum antimicrobials that will cover
gram negative as well as gram positive organisms will be avoided even in siblings and inactivated poliovirus
be 1st line choice like third generation cephalosporins vaccine (IPV) could be used in to stead. Live vaccines
like ceftriaxone plus aminoglycoside like amikacin. In are especially contraindicated in transplant patients.
case, ESBL producing organisms are common in a local • Intramuscular injections are contraindicated in
set-up, one may have to start cefaperzone-sulbactum patients with severe thrombocytopenia due to fear of
or piperacillin-tazobactum plus amikacin as the 1st line bleeding.
therapy. Drugs will be the changes based on cultures • IV access may be difficult in patients due repeated
and antimicrobial sensitivity. However if there is no infections and use of peripheral lines, hence central
growth and patient is not responding, one will have to lines or a port may be a better option especially for
add vancomycine to cover for MRSA on day 3 to 5. If patients on immune suppressive therapy or transplant
patient still fails to respond or deteriorates, one may patients as they will need prolonged supportive care.
have to switch to carbapenums like meropenum with
vancomycine. Lastly, in desperate ELBS producers which SPECIFIC THERAPY IN APLASTIC ANEMIA
are carbapenum resistant one will have to add IV colistin.
Anti-fungals will be added either empirically on day 5 to 7 Options available: There are several options available to
when patient does not respond to second line antibiotics offer a cure to patients with AA. Several mild-moderately
or if culture grows a specific fungus.16 Recently several severe AA patients may need supportive care alone or with
hospitals are faced with problems of fluconazole resistant immune suppressive therapy (IST). Severe and very severe
candida species sepsis or increasing trends of infection AA patients will need either immune suppressive therapy
with Aspergillus, especially if some construction work is or stem cell transplant (SCT). Many patients in India
going on in near vicinity.17 There is no role of prophylactic cannot afford IST or SCT and would receive only supportive
antimicrobials or antifungals as it will only add to the care that too haphazardly leading to high mortality. Such
problem of drug resistance. Lastly unlike in patients with patients are often treated with corticosteroids, androgenic
leukemia, there is no role of giving Pneumocystis carinii steroids alone. There are also anecdotal reports of use of
pneumonia (PCP) prophylaxis to AA patients, and sulfa danazol, cyclophosphamide, splenectomy, etc. which are
drugs will be contraindicated in patients with AA. sort of given up now with the advent effective IST and SCT.
Immunosuppressive Therapy for

Prevention of Infections
Aplastic Anemia
Various measures are required to prevent infections
Immunosuppressive therapy (IST) currently comprises
which include chlorhexidine mouth washes after every
use of anti-thymocyte globulin (ATG) and cyclosporine
major meals, chlorhexidine bath, betadine application
(CsA) as standard first line therapy along with short course
to groins and axillae after bath, sterile or well-cooked
low dose steroids. Clinical experience and laboratory data
diet, avoidance of contaminated, uncooked and open or
suggests that the mechanism leading to bone marrow
overnight left over food, hand sanitization by care taker
failure is probably secondary to activated cytotoxic
before handling patients, cleaning well fruits and eating
lymphocytes, which produce T-helper type 1 (Th1)
only well preserved or fully skin covered fruits after
cytokines including interleukin-2, interferon gamma and
peeling, avoiding going to crowds, etc.
tumor necrosis factor. These cytokines in turn induce
apoptosis of the hematopoietic stem cells. Immuno
General Measures suppressive agents exert their action by inhibiting T cell
• No drugs should be administered per rectally due to activation and dose dependent lympholytic activity as well
fear of infection and bleeding. Avoid contact games as stimulation of hematopoietic growth factors.
and injuries
• Avoid altogether brushing or brush with soft tooth Indications
brush, especially if patient is thrombocytopenic.
• Psychological support to the patient and family Children tolerate hematopoietic stem cell transplantation
members is of utmost importance like in any other (HSCT) exceedingly well with excellent outcome making
chronic life-threatening illnesses stressing the chronic it as the modality of choice for all severe and very severe
nature of disease and slow response to treatment.14,15 aplastic anemia cases. However, a large chunk of children
• All vaccinations should be avoided during the active with SAA/vSAA in developing countries cannot afford
disease as live vaccines can lead to vaccine induced HSCT or do not have a matched donor ; for them IST is the
infection and killed may not be efficacious. OPV should only alternative. For non-severe aplastic anemia cases IST
is the first modality of choice as it is associated with less day in 3 divided doses should be given along with ATG
risk than HSCT. infusion and continued for 2 weeks and then tapered
over next one week to prevent serum sickness. One has
Eligibility to use IV hydrocortisone or dexamethasone to treat
serum sickness should it occur and then switched to
All patients should have a stress cytogenetics test done oral course thereafter. Requirements of PRBc, platelets
to rule out Fanconi anemia as IST is of no use in patients may go up after IST course and even infections may
with Fanconi anemia. Patient should be relatively well increase post-IST.
and free of serious infections. Central venous access is
required as ATG can cause peripheral venous sclerosis. Cyclosporine (CsA)
Platelet and packed red cell transfusion should be given
to keep platelets above 20,000/cumm and Hb above 7 g/ Oral cyclosporine is given in the dose of 5 to 10 mg/kg
dL before and during ATG course. Post-ATG transfuse per day in 2 divided doses and the dose is adjusted by
packed red cells to maintain Hb >7 g/dL. It is desirable to keeping trough levels at 100 to 150 ng/mL by testing the
use leukodepleted blood products. Even use of irradiated levels 15 days after starting the dose and any increments
blood products is desirable post IST as there is profound done thereafter. CsA is usually started on day 21 after
immune suppression following IST. Immunosuppressive stopping prednisolone, as combined administration
therapy should be instituted with facilities for resuscitation often leads to hypertension. Common side effects of
and intensive life support under care of a qualified medical CsA include hirsuitism and gum hypertrophy. Toxicities
team familiar with this treatment. include hypertension, liver function abnormality, and
renal toxicity; hence monitor liver function tests and renal
ATG/ALG functions, and blood pressure weekly. CsA is continued
for a minimum 3 to 6 months before any response can be
• Horse and Rabbit ATG is available in India containing seen. The oral liquid is highly concentrated and comes as
100 to 250 mg in 5 mL vial. ALG is used in the dose of 100 mg/mL. Hence a small child may need fraction of an
40 mg/kg/day for 5 to 10 days, and ATG is used in the milliliter as the dose and patient should be taught how to
dose of 15 mg/kg/day for 5 to 10 days, however one measure tiny doses using 1 mL syringe.
should follow manufacturer’s instructions. Chances
of serum sickness go up when given for more
Response to IST (ATG Plus CsA)
than 7 to 10 days. Lyophilized powder is reconstituted
in normal saline shaking it vigorously, avoiding plastic It takes 3 to 6 months for response to be seen, hence,
bottles as ATG tends to stick to the sides, looking for one should not stop therapy before 6 months. Patient is
visible contamination, discoloration or precipitation, assessed after 3 to 4 months and periodically thereafter for
and given over 8 hours under close monitoring after response as well as toxicities.
obtaining informed written consent. Reconstituted Response is defined as complete when patient main
solution should be used as early as possible. Pre- tains hemoglobin levels normal for age, ANC above 1500/
medication in form of paracetamol, chlorpheniramine cumm and platelets > 150,000/cumm on two occasions
and hydrocortisone or dexamethasone is given prior 1 month apart without any transfusion support. In case
to starting ATG/ALG infusion daily to prevent allergic patient where the counts improve but is still transfusion
reactions. First vial should be administered slowly. dependent to maintain normal counts it is taken as partial
Look for toxicities like allergic reactions, anaphylaxis, response.
urticaria, etc. If patient satisfies all criteria of AA and is completely
• Abandon ATG/ALG infusion if patient develops transfusion dependent for PRBCs and platelets it is taken
anaphylaxis as suggested by development of reactions as failure of first course of IST in which case either HSCT
like hypotension, dyspnea, poor peripheral circulation, should be considered or if that is not feasible, a second
etc. and immediately start standard measures of course of IST can be given using a different brand of ATG/
resuscitation.1,18 ALG than what was used in the first course. When giving
• Patient may develop serum sickness by 10 to 14 second course patient should be closely monitored for
days after ATG infusion presenting with fever, rash, anaphylaxis as there might have occurred sensitization
arthralgia and arthritis. Hence observe the patient in with first course. Chances of response to first course
hospital for 2 weeks after ATG course for the same. are 40 to 50 percent and that with a second course is
Steroids in form of prednisolone in dose of 1 mg/kg/ around 60 percent in moderate cases.
Other Drugs price in public health set-up. As such very few centers
have invested for HSCT even in private set-up and hence
Various drugs like corticosteroids including high dose the patient has to travel long distance to get HSCT done
methylprednisolone, androgenic steroids, cyclopho outside major metro cities. In elderly patients HSCT has
sphamide, danazol, stanozolol, etc. have been in past with its own complications rates especially due to GVHD and
anecdotal response. success rates are less with higher mortality making IST as
Nandrolone enanthate is used in a dose of 2 to 5 mg/ the first choice especially for > 40 years of age.21
kg/day of injectable form once in 10 days and continued Ideal is HLA matched sibling or family donor. Partial
till response is evident. Efficacy is doubtful and the matched family donor or unrelated matched donor are
outcome may be in fact adverse due to side effects like less desirable as complications like GVHD and graft
masculanization, stunted growth, hepatotoxicity, Ca liver, rejection are higher in this settings. Donor need not be
etc. Though these are currently not the ideal choice of ABO compatible, but should be healthy. Best experience
treatment, they can be tried in those children with AA who is with bone marrow or peripheral blood stem cells as the
cannot afford HSCT or IST. source of HSCT. Umbilical cord blood stem cells are nor
Corticosteroid stimulates erythropoiesis. It also preferred. For peripheral blood stem cells donor is given
stabilizes capillary membrane and decreases bleeding. G-CSF for 4 to 5 days and then subjected to apheresis to
They are useful to counteract side effects of androgenic collect enough stem cells from peripheral blood.
steroids on growing epiphysis and the serum sickness of Most protocols would use non-myeloablative condi
immunotherapy. Oral prednisolone is used in the dose of tioning based on combination of ATG, cyclophosphamide
0.5 to 1 mg/kg/day and tapered to a minimum effective and fludarabine.22-25 Engraftment would occur normally
dose. High dose IV methylprednisolone was popular in in 10 to 15 days and till then patient will need effective
Europe in past and probably effective in patients treated supportive care. All aseptic precautions should be
within few weeks of diagnosis, this therapy is reserved taken to avoid infections during the critical neutropenic
for occasional patients due to tremendous toxicity. It is period that includes chlorhexidine bath, chlorhexidine
used in the dose of 15 to 20 mg/kg/day for 5 days followed mouth washes, sterile drinking water, sterile diet or well
by tapering doses over next 15 days. Side effects include cooked food, avoiding of crowds, sterile cloths or at least
hypertension, fluid electrolyte imbalance, infections, clean ironed cloths. And hand sanitizer for the contacts.
suppression of neuroendocrinal axis, psychosis, avascular Only irradiated blood products should be used to avoid
necrosis of head of femur, etc.19 transfusion associated GVHD which has very high fatality
Danazol 100 mg BD/TDS for girls, stanozolol 10 mg BD should it occur. Once engraftment is well established and
for boys have shown some response in children with non- patient has no infection, patient can go home and follow-
severe AA. up on outdoor basis for further management and usually
is kept under follow-up for 3 to 6 months.
HEMATOPOIETIC STEM CELL
TRANSPLANTATION IN APLASTIC ANEMIA COMPLICATIONS AFTER SUCCESSFUL
ENGRAFTMENT
In young patients hematopoietic stem cell transplantation
(HCST) is the best modality of therapy as it is quite safe • Graft-versus-host disease (GVHD) and rarely graft
at that age, has almost 80 to 90 percent cure rates and rejection are two main complications of HSCT.22
except for initial period of profound neutropenia related Acute GVHD can occur up to 100 days post-transplant
infections, and graft-versus-host disease (GVHD), there and will characterize with skin rash, diarrhea and
are not many long term complications.7 Most of the patients liver disease with jaundice and sometimes fever.
are free from transfusion support and medications within Treatment includes immune suppression with steroids
1 year or so. This is in contrast to IST which has lesser cure and cyclosporine. Chronic GVHD develops after
rates, more toxicities, slower response, need for prolonged 100 days and is characterized by scleroderma with
support with blood products and quite a cost.20 However skin rash, sicca complex, hepatic dysfunction, and
HSCT needs a HLA matched donor and less than 40 percent sclerosing bronchiolitis. Management includes again
of the patients will be lucky to find such a matched sibling immunosuppressive agents. Most patients develop
donor from the family. This is a big problem now with tolerance after 1 year or two and are able to stop
smaller family size. Unrelated matched donor is difficult to immune suppression thereafter. Other complications
get in India and is prohibitively expensive to get from the include viral infections like CMV, EBV, HSV; fungal
Western registry. Besides, HSCT itself is quite expensive infections, bacterial infections with encapsulated
and not many centers offer such a therapy at an affordable organisms, etc.
• Immunization: Revaccination with routine childhood 2. International Agranulocytosis and Aplastic Anaemia
vaccines against polio, diphtheria, tetanus, pertussis, Study. Incidence of aplastic anemia: relevance of
Hib, and pneumococcal vaccines is carried out at 1 year diagnostic criteria. Blood. 1987;70:1718-21. PubMed.
after successful HSCT and when the patient is off 3. Kumar R, Kalra SP, Kumar H, Anand AC, Madan H.
Pancytopenia-a six year study. J Assoc Physicians India.
all immune suppression. Typically 2 doses of Tdap,
2001;49:1078-81.
IPV, Hib, hepatitis B, and PCV13 are given at one 4. Bhatnagar SK, Chandra J, Narayan S, Sharma S, Singh V,
month interval followed by 3rd dose after 6 months. Dutta AK. Pancytopenia in children: etiological profile. J
These children also receive age appropriate doses of Trop Pediatr. 2005;51:236-9.
inactivated influenza vaccine which is then continued 5. Mary JY, Baumelou MG and the French cooperative
annually. In India they will also receive hepatitis A 2 group for epidemiological study of aplastic anemia.
doses at 6 months interval and Vi typhoid vaccine one Epidemiology of aplastic anemia in France: A prospective
dose to be repeated every 3 to 5 years. Live vaccines like multicentric study. Blood. 1990;75:1646-53.
MMR and Varicella are given as 2 doses 8 to 12 weeks 6. McCohan E, Tang K, Rogers PC, McBride ML, Schultz KR.
The impact of Asian descent on the incidence of acquired
apart starting at 2 years after HSCT.
severe aplastic anemia in children. Br J Haematol.
2003;121:170-2.
ROLE OF PEDIATRICIAN IN SHARED CARE 7. Marwaha RK, Bansal D, Trehan A, Varma N. Androgens in
childhood acquired aplastic anemia in Chandigarh, India.
Before HSCT primary pediatrician should encourage Trop Doctor. 2004;34:149-52.
the patient to take proper treatment, treat infections 8. Chandra J, Naithani R, Narayan S, Sharma S, Ravi R,
vigorously, discuss options of IST and HSCT with parents, Singh V, Pemde H, Dutta AK. Antithymocyte globulin plus
refer the patient to appropriate referral center for further ciclosporin in children less than 12 years with acquired
management, avoid unnecessary transfusions, and avoid aplastic anemia. Br J Haematol. 2006;133(suppl 1):118.
any transfusion from family members. After discharge 9. Shimamura A, Guinan EC. Acquired Aplastic Anemia.
primary pediatrician can continue to follow-up patient for In Nathan and Oski’s “Hematology of Infancy and
minor ailments and carry out immunization as advised. Childhood” Vol I. Nathan DG, Orkin SH, Ginsburg D, Look
AT eds; WB Saunders Co 6th Edn. 2003.pp.256-79.
10. Camitta B, O’Reilly RJ, Sensenbrenner L, Rappeport J,
FOLLOW-UP AND PROGNOSIS Champlin R, Doney K, et al. Antithoracic duct lymphocyte
globulin therapy of severe aplastic anemia. Blood. 1983;62:
Patient, if followed clinically for regression or reappearance 883-8.
of symptoms that may suggest response or graft failure like 11. Führer M, Rampf U, Baumann I, Faldum A, Neimeyer C,
pallor, bleeding, fever. They are followed for complication Janka-Schaub G, et al. Immunosuppressive therapy for
like skin rash, diarrhea, jaundice, skin changes, dryness aplastic anemia in children: a more severe disease predicts
of mucosal surfaces which may suggest acute or chronic better survival. Blood. 2005;106:2102-4.
GVHD. If patient is on immune suppression like steroids 12. Alter BP. Bone marrow failure: A child is not just a small adult
or cyclosporine, patient is monitored for side effects like (But an adult can have a childhood disease). Hematology
hypertension, renal toxicities, hyperglycemia, etc. In a sick Am Soc Hematol. Educ Program. 2005.pp.96-103.
13. Kojima S, Ohara A, Tsuchida M, Kudoh T, Hanada R,
child, follow-up is on a daily basis and in a well child, it
Okimoto Y, et al. Risk factors for evolution of acquired
could be once in 2 to 3 weeks. Laboratory tests are also aplastic anemia into myelodysplastic syndrome and acute
done periodically including CBC, LFT, RFT depending myeloid leukemia after immunosuppressive therapy in
presence of complications. CBC are usually done weekly children. Blood. 2002;100:786-90.
initially and then monthly once patient shows successful 14. Maciejeweski J, Risitano A, Sloand E, Nunez O, Young N.
engraftment.26,27 Distinct clinical outcomes for cytogenetic abnormalities
evolving from aplastic anemia. Blood. 2002;99:3129-35.
15. BCSH General Haematology Task Force. Guidelines for the
LONG-TERM COMPLICATIONS diagnosis and management of acquired aplastic anaemia.
Br J Haematol. 2003;123:782-801.
Patient may develop long-term complications like relapse
16. BCSH Transfusion Task Force. Guidelines for the use of
of disease, leukemia, PNH, etc. Check bone marrow platelet transfusions. Br J Haematol. 2003;122:10-23.
aspiration and bone marrow biopsy are done after 6 to 12 17. Weinberger M, Elatta L, Marshall D, Steinberg SM, Redner
months. RL, Young NS, Pizzo PA. Patterns of infection in patients
with aplastic anemia and the emergence of Aspergillus as
a major cause of death. Medicine. 1992;71:24-43.
REFERENCES
18. Ascioglu S, Rex JH, de Pauw, et al. Defining opportunistic
1. Davies JK, Guinan EC. An update on the management invasive fungal infections with cancer and hematopoietic
of severe idiopathic aplastic anemia in children. Br J stem cell transplants: an international concensus. Clin
Haematol. 2007;136:549-64. infectious Dis. 2002;34:7-14.
19. Aplastic Anaemia in childhood management guidelines. conditioning regimen for allogeneic peripheral blood
UK Childhood Leukaemia working party. 2005. stem cell transplantation performed in non-HEPA
20. Young NS, Calado RT, Scheinberg P. Current concepts in filter rooms for multiply transfused patients with
the pathophysiology and treatment of aplastic anemia. severe aplastic anemia. Bone Marrow Transplant.
Blood. 2006;108(8):2509-19. 2006;37(8):745-9.
21. Frickhofen N, Heimpel H, Kaltwasser JP, Schrezenmeier H. 25. George B, Mathews V, Viswabandya A, Kavitha ML,
Antithymocyte globulin with or without cyclosporin A: 11- Srivastava A, Chandy M. Fludarabine and cyclopho
year follow-up of a randomized trial comparing treatments sphamide based reduced intensity conditioning (RIC)
of aplastic anemia. Blood. 2003;101(4):1236-42. regimens reduce rejection and improve outcome in Indian
22. Marsh J. Making therapeutic decisions in adults with patients undergoing allogeneic stem cell transplantation
aplastic anemia. Hematology Am Soc Hematol Educ for severe aplastic anemia. Bone Marrow Transplant.
Program. 2006.pp.78-85. 2007;40(1):13-8.
23. Copelan EA. Hematopoietic stem-cell transplantation. N 26. Guidelines for the diagnosis and management of aplastic
Engl J Med. 2006;354(17):1813-26. anemia. British J of Hematology. 2003;123:782-801.
24. Kumar R, Prem S, Mahapatra M, et al. Fludarabine, 27. Neal S. Young: Acquired Aplastic Anemia: Annals Int.
cyclophosphamide and horse antithymocyte globulin Medicine. 2002;136:534-46.
C H A P T E R 25
Inherited Bone Marrow
Failure Syndromes
Revathi Raj
Inherited bone marrow failure syndromes (IBMFSs) are rare genetic disorders characterized by defective production of red cells, white
cells and platelets. This results in a single cell line failure or pancytopenia depending on the gene mutation inherited.
Based on the cell lines affected IBMFS can be classified as Table 1.

Table 1 Classification of inherited bone marrow failure syndrome
Disorder Cell line Gene mutation Mode of inheritance Chromosome affected
affected
Fanconi anemia Pancytopenia FANC A to G Autosomal recessive Several 16, 9,13,3,6,11
Dyskeratosis congenita Pancytopenia DKC1/TERC X linked or autosomal Xq28, 3 q26
recessive
Pearson syndrome Pancytopenia Mitochondrial DNA Maternal Maternal
Reticular dysgenesis Pancytopenia Unknown Unknown Unknown
Congenital amegakaryocytic Pancytopenia cmpl Autosomal recessive 1p34
thrombocytopenia
Diamond-Blackfan anemia Anemia RPS19 Autosomal dominant 19q
Congenital dyserythropoietic anemia Anemia CDAN1 Autosomal recessive 15q, 20q
Congenital sideroblastic anemia Anemia ALAS2 X-linked recessive Xp11
Kostmann syndrome Neutropenia ELA2 Autosomal dominant 19p
Shwachman-Diamond syndrome Neutropenia SBDS Autosomal recessive 7q11
Thrombocytopenia absent radii Thrombocytopenia Unknown Autosomal recessive Unknown
Fanconi Anemia hyperpigmentation, microphthalmia, café-au-lait spots,

Children with Fanconi anemia (FA) present with distinct renal anomalies like horse shoe kidney, cardiac anomalies
dysmorphic features and can be diagnosed even at like atrial or ventricular septal defects, cryptorchidism and
birth before the onset of cytopenias. REFAIN—Registry radial ray defect with hypoplastic thumb. Hypopigmented
for Fanconi anemia in India has followed up over 150 spots appeared in the palms with the onset of pancytopenia.
children with FA over 15 years. The main somatic features Fanconi anemia results from multiple defects in FANC
noticed in the Indian FA registry are growth retardation, proteins and the incidence is high in South India due
to high consanguinity. FANC proteins help in monou Congenital Amegakaryocytic

biquitination that helps DNA repair. Mutations in FANC Thrombocytopenia (CAMT)
proteins thus result in defective DNA repair and increased
chromosomal fragility which is the hallmark of FA. Gene Defects in thrombopoiesis ultimately result in pancy
complementation studies have helped in antenatal topenia due to marrow failure. Children can also present
diagnosis and prevention of new births and this is possible with developmental delay and cardiac defects such as
in our country through the FA registry. ASD/VSD. Treatment is with hematopoietic stem cell
The children present with cytopenia from the age of transplantation although rejection rates are high. Cancer
3 years. Peripheral blood testing to assess chromosome predisposition is also noted in CAMT.
breakage with mitomycin C or diepoxybutane helps confirm
the diagnosis. Bone marrow aspiration and karyotyping Diamond-Blackfan Anemia
are required as the presence of an abnormal clone such as Pure red cell aplasia can present at birth or later in life. The
monosomy 7 heralds the onset of acute myeloid leukemia. children show dysmorphic features such as craniofacial
The children initially respond to androgenic steroids such anomalies and thumb anomalies often in association
as stanzolol or oxymetholone. Careful monitoring of liver with deafness and growth retardation. The majority of
enzymes is required during drug therapy. children respond to prednisolone starting at 2 mg/kg/
Hematopoietic stem cell transplantation offers the day and then tapered over 8 to 12 weeks. Steroid dose is
sole chance of cure. Outcomes are better when children kept at a minimum required to sustain hemoglobin levels.
are referred early as multiple transfusions increase the Prednisolone dependent or refractory children need to
chance of graft rejection and graft-versus-host disease. be treated as per guidelines for thalassemia major with
Transplantation is done using low dose conditioning as transfusion and oral chelation. Transplantation helps
the children are extremely sensitive to chemotherapy. achieve cure and children need long-term follow-up for
Overall survival after transplantation for FA approaches screening for malignancies.
90 percent survival with sibling donors and 60 percent
with matched unrelated cord blood transplantation. All Kostmann Syndrome
children need long-term surveillance for malignancies,
especially head and neck cancers. Severe mutations in the ELA2 gene causes Kostmann
syndrome whilst milder mutations result in cyclical
neutropenia. Management consists of aggressive treat
Dyskeratosis Congenita ment of infections and GCSF at a dose between 5 and
Dyskeratosis congenita can present at any age from 20 mcg/kg/day or more is needed to keep the neutrophil
preschool to late thirties with pancytopenia. There are count above 500. Clonal evolution and transformation to
characteristic nail changes with dystrophy and a bald myelodysplasia or acute myeloid leukemia is known to
tongue with skin hyperpigmentation especially around the occur after the second decade.
neck. These children are particularly prone to pulmonary
fibrosis and transplantation carries a higher risk of mortality Thrombocytopenia Absent Radii (TAR)
due to lung complications. Mutations in the DKC gene or
TERC genes are pathognomic of this condition. Cancer Most children show spontaneous regression of throm
predisposition is high as in other marrow failure syndromes. bocytopenia following the first birthday. Orthopedic
procedures to correct hand anomalies are best postponed
till there is platelet recovery above 75,000. Children with
Pearson Syndrome TAR have the best outcome of all the inherited marrow
Pearson syndrome is a mitochondrial cytopathy that failure syndromes.
causes failure to thrive, pancreatic insufficiency and pan
cytopenia. Bone marrow aspiration shows characteristic Key Points in Management of Children with
changes with vacuolation in the marrow precursor cells. Cytopenias
Death in infancy results from infections and the role of
transplantation is not clearly defined. • Always suspect and screen for inherited marrow failure
syndrome even when single cell line is involved as the
disease may progress to pancytopenia
Reticular Dysgenesis • Avoid marrow suppressive drugs for treatment of
This is a severe defect in the lymphohematopoietic system infections
with features of severe combined immune deficiency and • Preserve DNA for future counseling and guiding
marrow failure. families through subsequent pregnancy
Chapter-25 Inherited Bone Marrow Failure Syndromes 257
• Early referral to a transplant center results in improved 4. Butturini A, Gale RP, Verlander PC, Adler-Brecher B, Gillio
outcomes AP, Auerbach AD. Hematologic abnormalities in Fanconi
• Careful screening of sibling donors prior to anemia: an International Fanconi Anemia Registry study.
transplantation is important as they may be affected Blood. 1994;84:1650-5.
asymptomatic individuals 5. Dokal I. Dyskeratosis congenita in all its forms. Br J
• There is an increased risk of malignancies in all forms Haematol. 2000;110:768-79.
6. Dror Y. Shwachman-Diamond syndrome. Pediatr Blood
of IBMFS and cancer surveillance is an important
Can. 2005;45(7):892-901.
aspect of follow-up
7. Freedman MH, Bonilla MA, Fier C, et al. Myelodysplasia
• Test for Fanconi anemia before ATG is given as part of syndrome and acute myeloid leukemia in patients with
immunosuppressive therapy for aplastic anemia. congenital neutropenia receiving G-CSF therapy. Blood.
2000;96:429-36.
BIBLIOGRAPHY 8. Gluckman E, Auerbach AD, Horowitz MM, et al. Bone
marrow transplantation for Fanconi anemia. Blood.
1. Alter BP. Cancer in Fanconi anemia, 1927–2001. Cancer.
1995;86:2856-62.
2003;97:425-40.
2. Alter BP. Inherited bone marrow failure syndromes. 9. Gluckman E, Vanderson R, Ionescu I, et al. Results of
In: Nathan DG, Orkin SH, Ginsberg D, Look AT (Eds). unrelated cord blood transplants in Fanconi anemia.
Hematology of Infancy and Childhood. Philadelphia: WB Blood. 2004;104:2145.
Saunders. 2003.pp.280-365. 10. King S, Germeshausen M, Strauss G, Welte K, Ballmaier M.
3. Ball SE, McGuckin CP, Jenkins G, et al. Diamond–Blackfan Congenital amegakaryocytic thrombocytopenia (CAMT):
anaemia in the UK: analysis of 80 cases from a 20-year A detailed clinical analysis of 21 cases reveals different
birth cohort. Br J Haematol. 1996;94:645-53. types of CAMT. Blood. 2004;104:740A.
C H A P T E R 26
Benign Disorders of Neutrophils
Bharat R Agarwal
This chapter describes benign disorders of neutrophils discrimination, i.e. they were reasonably common in,
(Malignant disorders are described elsewhere). Disorders and reasonably specific for, appendicitis. The most useful
of neutrophils can be quantitative (Neutropenia and and easily obtained is the neutrophil count – a value of
neutrophilia) or qualitative (Cytoplasmic or nuclear 15 × 109/L is a useful cut-off. The difference in frequency
abnormalities). All these various conditions are considered of atypical (‘viral’) lymphocytes is an indication of the high
in the following sections as follows: frequency of viral infection in non-surgical abdominal
• Neutrophilia pain.
• Neutropenia Substantial neutrophilia (< 50 × 109/L) is not infrequent
• Neutrophils: cytoplasmic anomalies in bacterial infection, especially bacterial pneumonia,
• Neutrophils: nuclear anomalies. empyema, bacterial meningitis, septicemia, urinary tract
infection and bacterial endocarditis, and is characteristic of
NEUTROPHILIA the rare genetic deficiency of integrin adhesion molecules
on neutrophils and lymphocytes (delayed separation of
Except for the first weeks of life, the upper limit of normal umbilical cord, severe bacterial infections, periodontitis,
is approximately 7.5 × 109/L. Rarely, artifactually high gingivitis, poor pus formation).
counts may be obtained by automated particle counters:
• Precipitation of cryoprotein on cooling. Error revealed Sweet’s Syndrome (Acute Febrile
by examination of stained film. Examine fluid
Neutrophilic Dermatosis)
preparation by subdued light or phase contrast for
crystals. A rare syndrome of unknown cause, characterized by
• Incomplete lysis of erythrocytes. Examination of high neutrophilia, raised, tender, erythematous plaques
stained film will reveal error. and bullae in the skin, spiking fevers and often arthralgia,
Neutrophilia (Table 1) has little value in specific myalgia and headache. It may occur without underlying
diagnosis, with some exceptions considered below. disease or in association with leukemia, usually myeloid
(in remission and not in a neutropenic phase). Diagnosis
Infection requires skin biopsy (dermal infiltration with mature
neutrophils, edema, vesiculation). Without treatment,
As a general rule, neutrophilia is more likely in bacterial resolves spontaneously in weeks; no response to anti
than in viral infection. However, neutrophilia is not infre biotics, but prompt relief from steroids.
quent in the early stages of viral infection and neutropenia
may occur in severe bacterial infection. The neutrophil
Familial Neutrophilia
count (and other ‘routine’ characteristic) is of value
in assessing the likelihood of appendicitis in children A familial, lifelong, substantial (to 62 × 109/L) neutrophilia
admitted to surgical wards with abdominal pain (Table 2). of segmented and, to a lesser degree, stab forms. Neutro
All characteristics examined in this study were useful in phils are normal in morphology and function. NAP may
Chapter-26 Benign Disorders of Neutrophils 259
Table 1 Benign neutrophilia Toxic Change in Neutrophils

• Artifact Toxic change is characterized by various combinations of
• Infection1 the abnormalities listed in Table 3. When an apparently
• Inflammation toxic change occurs in isolation, e.g. shift to left or heavy
– Kawasaki granulation, suspicion should be raised of mimicry
– Inflammatory bowel disease
(Table 4).
i. Crohn’s
ii. Ulcerative colitis Toxic change in neutrophils, in combination with other
– Rheumatoid arthritis abnormalities in the film, is a useful guide in distinction
– SLE of bacterial from viral infection (Table 5). However,
– Histiocytosis toxic change per se is not specific for infection (Table
• Tissue necrosis (e.g. hepatic)1 8). A quantification of toxic change has been devised
• Leukemoid reactions1,2 (Table 6); this may be combined with other leukocyte
– Viral infection (may mimic e.g. AML M2, M3, M4) changes and the platelet count to produce a score for risk
– TAR syndrome of sepsis in the neonate, in whom (more so than in older
– Randall’s syndrome children) infection is likely to produce rapid deterioration
– Disseminated tuberculosis (Table 7). However, C reactive protein levels on 2 succes
• Nonhemopoietic malignancy
sive days appear to be more useful than morphologic
• Postsplenectomy
• Drugs assessment (if readings can be obtained rapidly); sepsis
– Steroid is likely if values are raised on day 1 and/or day 2, and
– Ranitidine can be confidently excluded if values are normal on both
– Leukocyte stimulating factors1 days.
i. Colony stimulating factors Each feature has a score of 1. A total score of > 3 is
ii. Interleukins strong evidence for, and < 2 is strong evidence against
– Lithium sepsis. If no mature neutrophils in film, score 2 rather than
• Stress 1 for abnormal total count.
– Vomiting
– Convulsions
– T Hypoxia, near-drowning Phagocytosis by Neutrophils
– Acidosis–diabetic, other • Of cells (leukocytes, erythroblasts, erythrocytes): Phago
– Postoperative cytic neutrophils are less conspicuous than phagocytic
• Sweet’s syndrome monocyte, which accompany them (see below).
• Familial1
• Of organisms: Careful and systematic examination
1
Count often exceeds 50 x 109/L of the film with a low to medium power objective
2
Nonleukemic shifts to left, including myeloblasts (×10–×25) for 5-10 minutes will detect organisms
Note: Placement of some disorders, e.g. Kawasaki, histiocytosis in a significant proportion of cases of septicemia,
is tentative. especially those with severe effects such as circulatory
shut-down. On the other hand, blood from an
be increased. Associated features include increased indwelling venous line may contain a profusion of
incidence of chromatid breaks in blood chromosomes, organisms (from colonization) with surprisingly mild
Gaucher-like cells in marrow and spleen, thickening of accompanying symptoms.
skull bones (widening of diploë) and hepatosplenomegaly. Optimal parts of the film for search are edges and tails.
Regarded as inherited, autosomal dominant. Infected leukocytes are more numerous in films of the first
drop of blood from the previously unmanipulated ear-
lobe (because of trapping in capillary bed), and in films
QUALITATIVE CHANGES IN LEUKOCYTES IN of buffy coat. Densely-staining organisms (e.g. cocci) are
INFECTION more readily visible than the pale-staining organisms
Appraisal of leukocyte numbers and morphology usually (Gram-negative rods).
gives some indication of the likelihood of infection, its broad Criteria for acceptability of organisms in films as
nature (bacterial vs viral) and occasionally the precise cause. genuine evidence of septicemia are summarized in
Changes in morphology (Table 3) are considered here. Table 9A; descriptions are given in Table 9B.
Table 2 Hematology of appendicitis (n=50)1 vs nonsurgical abdominal pain (n=50)2

Sensitivity3 Specificity4
Total leukocytes, x 10 /L
9
>15.0 29/50 43/50
> 20.0 15/50 48/50
> 25.0 7/50 50/50
> 30.0 1/50 50/50
Neutrophils, x 109/L >15.0 25/50 48/50
> 20.0 7/50 49/50
> 25.0 3/50 50/50
> 30.0 1/50 50/50
Neutrophils, shift to left, % >10.0 17/49 37/50
> 20.0 11/49 50/50
> 30.0 4/49 50/50
Atypical lymphocytes: 0 in 200 leukocytes 13/50 48/50
ESR, mm in 1 h > 30.0 5/31 38/38
1
Histologically proven
2
Admitted to surgical wards with abdominal pain – 46 with various diagnoses discharged without laparotomy. 4 with histologically
normal appendix removed
3
Sensitivity = proportion of patients with positive test
4
Specificity = proportion of controls with negative test
Table 3 Qualitative changes in leukocytes in infection Cell Death

• Neutrophils Death (apoptosis) of leukocytes is common in infection.
– Shift to left Because anticoagulation and storage also cause damage,
– Toxic granulation cell death is most reliably assessed in films made directly
– Degranulation from vein, finger or ear.
– Döhle bodies
The process affects leukocytes in general. Whole
– Vacuolation
– Pelgeroid change cells as well as fragments may be observed. Though
– Chromomeres phenomenon may be noted in non-infective conditions
– Gigantism such as malignancy and SLE, in childhood it almost
– Phagocytosis of: always is a result of infection and may be striking in viral
i. Leukocytes infections such as infectious mononucleosis, measles and
ii. Erythroblasts/cytes neonatal herpes in viral infection death may affect atypical
iii. Organisms lymphocytes as well as normal leukocytes.
– Cell death, fragmentation Possible mechanisms include immune effect, inter
– Agglutination leukin-2 starvation as a result of cell hyperactivity and
– NAP usually ↓ in bacterial infection, ↑ in viral invasion by virus. Dead cells are a stimulus to phagocyto
• Lymphocytes sis by monocytes/macrophages and neutrophils.
– Cell death, fragmentation
• Monocytes/macrophages TRANSIENT NEUTROPENIA
– Vacuolation, enlargement, activation, Döhle-like bodies
Neutropenia is occasionally spurious.
– Phagocytosis of:
i. Leukocytes • Clotting in sample (likely to depress counts of all cell
ii. Erythroblasts/cytes types rather than neutrophils only)
iii. Organisms • MPO deficiency (neutropenia wrongly identified by
– Chromomeres counters, e.g. Hemalog D, which recognize neutrophils
– Cell death, fragmentation cytochemically)
– Transformation to ‘histiocytes’ • Aggregation, in some cases EDTA-induced
– Infection-associated hemophagocytosis (marrow, blood) • Cell fragility (smudging) in chylomicronemia.
• Giant cells with intranuclear inclusions (marrow, CMW Neutropenia in childhood (Table 10) is most commonly
infection) due to infection, especially viral. Neutropenia is usually
Table 4 Differential diagnosis of toxic changes in neutrophils Table 7 Hematologic scoring system for sepsis in the neonate
Changes Differential diagnosis Abnormality
Toxic granulation Alder granulation Total neutrophil count ↑ or ↓
Degranulation Genetic disorder Immature/total neutrophil ↑
Myeloid leukemias ratio
Immature/mature neutrophil ≥ 0.3
Döhle bodies Döhle-like bodies in May-
ratio
Hegglin, Fechtner and
Sebastian syndromes Immature neutrophil count ↑
Total leukocyte count ↓ or ↑
Vacuolation Anticoagulant/storage artifact
Jordans anomaly (≤ 5.0 x 109/L or ≥ 25.0,
30.0 or 21.0 at birth,
Giant neutrophils Hereditary giant neutrophils 12-24 h and day 2 onward
Pseudo-Pelgerization Anticoagulant/storage artifact respectively
Cell death
Toxic change in neutrophils ≥ 3 + for vacuolation, toxic
granulation or Döhle bodies
Platelets ≤ 150 x 109/L
Table 5 Common changes in leukocytes in bacterial and viral

infection Table 8 Toxic changes in neutrophils. Some causes other than
Bacterial infection Viral infection infection
Neutrophilia Common Uncommon • Collagen disease
Neutropenia In severe infection Common – Rheumatoid arthritis
Toxic change in Common, often Slight; marked in – SLE
neutrophils severe some infections, • Other vasculitides
e.g. measles – Kawasaki
– Stevens-Johnson
Atypical Small numbers or Increased
• Inflammatory bowel disease
lymphocytes absent
– Crohn’s
Organisms in Sometimes 0 (zero) – Ulcerative colitis
leukocytes • Near-drowning1
• Heat stroke
• Tissue necrosis
– Hepatic
Table 6 Quantitation of toxic change in neutrophils1 • Necrotizing enterocolitis2
% cells affected Score – Burns1
• Drugs
Vacuolation 0 0
– Colony-stimulating factors
Döhle bodies <25 +
– Cytotoxics
25–50 2+ 1
Added infection common
51–75 3+ 2
Septicemia from normal gut flora a significant component
>75 4+
Toxic granulation Normal granulation 0 evidence in blood films of antibody/opsonic effect

Slight toxic granulation + includes agglutination and phagocytosis of neutrophils
Approx 50% cells affected 2+ by monocytes or rarely neutrophils. The marrow appears
Toxic granules most cells 3+ normal or shows ‘maturation arrest’ at the myelocyte/
gross; nucleus obscured by 4+ stab stage or, rarely, destruction. In a high proportion the
toxic granules organism is not identified (or sought). Recovery of count
1
For use with (Table 7) after treatment with antiviral agents, e.g. ganciclovir,
suggests a viral cause.
Mechanisms for neutropenia in sepsis include
not severe (>0.2 x 109/L) and usually begins to recover destruction by endotoxin or phagocytosed organisms
within 5 days, rarely not for 2-3 weeks. Destruction may be and agglutination by activated complement components,
due to direct effect or immune mechanism. Morphologic e.g. 5a. The marrow may be depleted of granulocytes,
Table 9A Organisms in blood films: acceptability as genuine Table 10 Neutropenia transient
evidence of septicemia • Infection
At least a proportion intracellular; appearances must be typical. – Viral (including HIV)
Intracellular organisms are unequivocal evidence of septicemia – Bacterial
only in films of capillary blood or films of venous blood made i. Pyogenic infection Staphylococcus, Streptococcus,
directly from needle (skin organisms may rarely be phagocytosed Coliforms, Meningococcus, Haemophilus, if severe
by leukocytes in venous samples in the interval before films are ii. Brucella, typhoid, paratyphoid1
made) – Tularemia1
Malaria, occasional cases1
Disregard
• Burns
• Inclusions in lymphocytes
• Hemodialysis
• Infected skin squames, epidermal cells
– Drugs
• Granules in basophils: More variable in size and shape than
cocci, often hollow, no characteristic grouping, no capsule,
1
Neutropenia may be chronic
paler than most cocci, no or minimal staining with Gram toxic
granules.
Differential diagnosis
especially in neonate. Transient neutropenia associated
• Granules in basophils: More variable in size and shape than with burns and hemodialysis is attributed to agglutination
cocci, often hollow, no characteristic grouping, no capsule, by activated C5.
paler than most cocci, no or minimal staining with Gram toxic
granules NEUTROPENIA, CHRONIC (MORE THAN 3
• Coarse azurophil granules MONTHS)
• Nuclear appendages
• Chromomeres (detached, pyknotic nuclear fragments) Less frequent than transient neutropenia. An empiric
• Phagocytosed nuclear fragments classification has been given (Tables 11 to 14), as in many
cases the mechanism is unknown. As suggested schema
for initial investigation is given in Table 11A.
Table 9B Characteristics of bacteria in Romanowsky-stained

Cyclic Neutropenia
blood films1
N. meningitides Plump cocci, a proportion in pairs, with some Periodicity of infections, especially upper respiratory and
flattening of opposed faces, intense blue- oral, should suggest the diagnosis. The cycling period is
black. N. gonorrhoeae indistinguishable in usually 19–21 days; neutropenia lasts for 3–6 days, with
morphology—rare in childhood and clinical complete absence for 1–3 of these. In neutropenic phases,
features different. Gram negative patients feel unwell from fever and infection, especially
S. pneumoniae Fine cocci, some slightly elongated, most staphylococcal with streptococcal. Oral infection may be
in pairs, with clear space (capsule) around; associated with cervical lymphadenopathy. Monocytes
intense, almost black staining. Infection may are usually increased in neutropenic phases, but infection
be heavy postsplenectomy is nevertheless a problem, as monocytes are less efficient
S. aureus Coarse cocci, usually in small groups, but than neutrophils in tracking and destroying bacteria.
may be single, in Pairs or short chains (to Monocytes, lymphocytes, eosinophils and platelets
about 4), redbrown to violet or almost black, also show some cycling (usually from normal to above
no capsule normal), while reticulocytes cycle above to below normal.
b-hemolytic Fine cocci in groups or chains, intense violet An increase in large granular lymphocytes is often noted
streptococci to almost black in cases of adult onset.
Coliforms Small to large rods, gray to gray-pink, In neutropenic phases the marrow shows ‘maturation
Klebsiella often encapsulated. Organisms arrest’ at the myelocyte stage. Increase in mature forms
usually not profuse in individual cells and and paucity of precursors preceded by some days increase
infected cells usually sparse in neutrophils in blood.
H. influenzae Small rods or coccobacilli, often encapsulated, In some cases oscillations tend to dampen over the
usually not profuse years and evolve to chronic neutropenia. The condition
does not appear to predispose to leukemia, though
Clostridia Large rods, often square ended; gray to gray-
pink, no capsule cyclic neutropenia may rarely be a harbinger of ALL or
myelodysplasia.
1
Gram and PAS stains may be useful on spare films if infected
In most cases, especially those of adult onset, inheri
cells plentiful.
tance cannot be discerned. In familial cases (about one
Table 11A Chronic neutropenia: initial assessment Table 12 Genetic syndromes with visible somatic anomalies
• Clinical history, age at onset, physical examination which may be associated with neutropenia
• Blood values
Shwachman Exocrine pancreatic insufficiency, metaphyseal
• Blood film
dyschondroplasia, growth retardation.
• Neutrophil count, once or twice weekly for 6 weeks
• Bone marrow Fanconi Abnormal pigmentation short stature, thumb
– Aspirate and radius anomalies, abnormal head/face,
- Morphology hypogonadism.
- Karyotype Dyskeratosis Reticular hyperpigmentation, depigmen
– Trephine biopsy congenita skin tation, atrophy; hair loss, nail dystrophy,
– Electron microscopy leukoplakia, dental dystrophy.
• Serum Chediak-Higashi Partial oculocutaneous albinism, bacterial
– Immunoglobulins infections, esp. S. aureus, gingivitis and perio
– Complement components dontitis, cranial and peripheral neuropathies,
– Autoimmune screen hepatosplenomegaly.
– B12, folate Cartilage-hair Short-limbed dwarfism, fine hair, infections,
– Viral serology hypoplasia esp. varicella
– Antigranulocyte antibodies Cohen Nonprogressive psychomotor retardation,
• Lymphocyte subsets microcephaly, short stature, delayed puberty,
• As appropriate: Tests for disorders in Tables 12–14 hypotonia, joint hypermobility, peculiar faces
and teeth, myopia, narrow hands and feet, not
infection-prone
Table 11B Chronic isolated neutropenia in childhood –
Hernandez Dystrophy of nails and hair, mild mental
inherited1
retardation
• No other anomalies
- Microcephaly, psychomotor retardation, reti
– Cyclic
nitis pigmentosa, marrow normal by light
– Kostmann
microscopy
– Lazy leukocyte syndrome
– With abnormal marrow neutrophils: Myelokathexis and
others
• As part of genetic syndrome usual cause of death. There is often a ‘compensatory’
– With visible somatic anomalies (Table 12) monocytosis which, even if striking (in a personal case
– Without visible somatic anomalies (Table 13) to 14.6 × 109/L), assists little in ameliorating infection
1
Neutropenia usually congenital because of the inefficiency of monocytes in handling
infection. The marrow is cellular usually with maturation
third), transmission is autosomal dominant with variable arrest at the promyelocyte/myelocyte stage, or in some
penetrance. cases normal maturation. Electron microscopy shows
The defect appears to reside in the stem cell/committed dysgranulopoiesis in some cases.
progenitor cell, as it is transmissible by transplantation in A deficiency in responsiveness to granulocyte colony
humans and can be cured in affected animals (Collie dogs) stimulating factor (GCSF) is likely, which can however be
by transplantation of normal marrow. overridden in vivo by administration of GCSF. Evolution to
(myeloid) leukemia may occur. Inheritance is autosomal
Differential Diagnosis recessive.
A degree of cycling may be noted in neutropenias such as Lazy Leukocyte Syndrome

Shwachman’s syndrome and monosomy 7 MPD. Cycling,
however, does not have the predictability of true cyclic A rare, chronic neutropenia (<0.2 × 109/L) with mor
neutropenia. phologically normal marrow and severe curtailment of
chemotaxis and random mobility of neutrophils. Neutro
Kostmann’s Syndrome (Infantile Genetic penia shows no response to intravenous adrenaline and
subnormal response to Pneumococcus polysaccharide. An
Agranulocytosis)
abnormality of surface configuration may make the cell
Neutrophils are persistently lower than 0.3 × 109/L; bac rigid. A similar disorder may occur as a temporary (< 12
terial infections are frequent and severe and are the months) phenomenon.
Table 13 Genetic syndromes without visible somatic anomalies Table 14 Chronic isolated neutropenia in childhood–acquired
which may be associated with neutropenia • Alloimmune
Neutropenia with O lgG, IgA, n to ↑ IgM • Autoimmune1
immunodeficiency1 • Infection
X-linked – Virus
agammaglobulinemia – Other2
dysgammaglobulinemia – Brucellosis
type – Typhoid, paratyphoid
- Hyper-IgA, eosinophilia, defective – Malaria
neutrophil chemotaxis, T, B cell – Leishmaniasis
function • Marrow pathology2
– Leukemia
Reticular dysgenesis Hypogammaglobulinemia,
- Monosomy 7 MPD
lymphopenia (imperceptible
- Pre-ALL
tonsils, impalpable lymph nodes,
– Neuroblastoma
no thymus on X-ray). Marrow
– Hypoplasia
depleted of granulocytes.
– Myelosclerosis
Lymphocytes. AR
– Osteopetrosis
mild variant described
• Deficiency of hematinics2
- Lymphopenia, T cell deficit, partial – Folate, B12
Pelgerization, benign course, – Copper
X-linked recessive • Drugs
Organic acidemias: Recurrent metabolic acidosis, – Immune
– Isovaleric often with vomiting, variable – Nonimmune
– Propionic mental retardation, odor of • Sequestration
– Methylmalonic ‘sweaty feet’ in isovaleric acidemia; – Spleen2
thrombocytopenia/pancytopenia – Marrow macrophages
in acidotic episodes of isovaleric 1
In neonates passive transfer may occur from a mother with
acidemia autoimmune disease
Glycogen storage 1b Hypoglycemia, convulsions, failure 2
Usually with other cytopenias
to thrive, infections, bleeding,
hepatomegaly
Abbreviations: AR: Autosomal recessive; n: Normal. Neutropenia with Gigantism and
1
Neutropenia may be a result rather than a cause of infection, Multinuclearity of Marrow Neutrophils
as neutropenia appears to be infrequent in those treated with
immunoglobulin Marrow promyelocytes contain up to 4 nuclei, and seg
mented neutrophils up to 16. Some cells are hypogranular
and others hypergranular. Cells are severely deficient in
lactoferrin (specific granules). The abnormalities impair
Neutropenias with Abnormal Marrow survival in marrow and are attributed to aberration of
Neutrophils centrioles. Karyotype is normal.
Blood neutrophils by contrast are normal. Increase is
Myelokathexis (Kathexis = Retention) inconstant in infection and minimal after adrenaline and
A rare neutropenia (chronic, non-cycling) associated with, dexamethasone.
and attributed to, abnormality of segmented neutrophils in
marrow which are abundant but show signs of degeneracy Neutropenia with Large, Binucleate,
(nuclear pyknosis, hypersegmentation with slender con Tetraploid Neutrophils and Monocytes in
necting filaments, cytoplasmic vacuolation). Neutrophils
Marrow
show variable impairment of function (decreased NAP,
impaired dye exclusion, mobility and phagocytosis). MPO The proportion of binucleate cells increases with maturity
is normal. Count increases in infection and after injection (metamyelocytes 42%, segmented 100%). Other leukocytes
of GCSF. Possible variants include familial occurrence are normal. Neutropenia is attributed to impaired egress
(father, daughter) with hypogammaglobulinemia, and as of abnormal cells from marrow. Binucleate cells are rare
sociation with growth retardation and dysmorphism. (< 1%) in blood.
In a personally observed case, neutropenia was Table 15 Alloimmune neonatal neutropenia : diagnosis
associated with abnormal granule structure and pancreatic Combination Result
fibrosis. Maternal serum + paternal granulocytes pos
Maternal serum + maternal granulocytes1 neg
Neutropenia as Part of a Genetic Syndrome Baby serum2 + paternal granulocytes pos
(Tables 12 and 13) Baby serum2 + baby neutrophils3 pos
Maternal serum + baby neutrophils3 pos
Neutropenia in these disorders is due to a variety of 1
To exclude maternal autoimmune neutropenia, e.g. SLE
mechanisms. 2
Collected while neutropenia
• Precursor cell deficiency, e.g. Fanconi, Shwachman, 3
Collected when numbers become normal
cartilage-hair hypoplasia
• Intramedullary destruction, e.g. Chediak-Higashi
(abnormal myeloid precursors in marrow, increased Table 16 Autoimmune neutropenias of childhood
serum muramidase in absence of monocytosis or Combination
decreased survival of blood neutrophils) • Autoimmune neutropenia of infancy (‘chronic benign’)
• Suppression of myeloid maturation, e.g. by organic • Viral infection
acids and glycine in organic acidemias. • Autoimmune disease
– SLE
Antibody-induced Neutropenias – Feity
• Passive transfer from mother with autoimmune disease, e.g.
Antibodies to neutrophil-specific antigens are an impor SLE
tant cause of neutropenia (Minchinton & Waters 1984). • With autoimmune hemolysis and thrombocytopenia (Evans
Antigens shared with other cells types (e.g. HLA) do not syndrome)
appear to be significant in neutropenia. Serum may be • Bone marrow transplantation
tested against a panel of neutrophils of known phenotype • Drugs (some)
or against films of marrow aspirate. Serum should be tested • T lymphocytosis with neutropenia
fresh, but if delay in transport to a reference lab is likely,
blood should be taken into citrate-phosphate-dextrose-
EDTA solution. Serum or plasma should be heated before fetal neutrophils. Immunization is usual with the first
application; to remove complement with interferes with child.
antibody binding. Marrow films should be fresh, or stored Because of paucity of cells, realistic testing can
at -30°C till and fixed with paraformaldehyde at time be done only for antibody in the mother’s serum
of testing. Antibody is demonstrable on more mature which reacts with the father’s but not with her own
stages (metamyelocyte onward) in mild to moderate neutrophils (Table 15).
neutropenias, and on myelocytes and promyelocytes as Marrow examination excludes the unlikely occur
well, in severe neutropenias. rence of leukemia as the cause of isolated neutrope
nia. Cellularity is variable. Stages from metamyelocyte
Alloimmune Neutropenias onward may be normally represented, deficient or
absent. ‘Maturation arrest’ is due to destruction of
It is two important types in childhood: mature forms and not to suppression of maturation.
1. Alloimmune neutropenia of infancy: Estimates of Neutropenia persists for 3 weeks to 3 months
incidence vary from 1/200 to 3 percent of neonates. depending on rate of catabolism of antibody. Mortality
Neutropenia is severe (0-0.5 × 109/L, often with a (bacterial septicemia) is about 5 percent.
‘compensatory’ monocytosis. In infected infants anti 2. Autoimmune neutropenias (Table 16): The most
body should be sought if neutropenia is excessive for common in childhood is autoimmune neutropenia of
the infection (neutropenia is unlikely unless bacterial infancy.
infection is severe).
Antibodies (IgG) are directed against one of the Autoimmune Neutropenia of Infancy
normal cell-specific antigens (well represented on
(Chronic Benign)
cord neutrophils), most commonly NA1, NA2, NB1,
NC1 and 9a. Rarely, the mother has no NA specific Neutropenia is severe (0-0.5 × 109/L); however there is no
neutrophil antigens (NA null, CD16 negative) and as a excess of infections compared with normal children of the
consequence reacts to any NA (NA1, NA2) antigens on same age. The count increases with infection and urticaria,
especially in the recovery period; there is little or no response always is permanent. Some infants have, singly or
to adrenalin (draws cells from marginating pool), variable in combination, neutropenia, thrombocytopenia or
response to steroid (enhances release from marrow), and autoimmune hemolysis.
good response to intravenous immunoglobulin (usually • Felty’s syndrome (rheumatoid arthritis, splenomegaly
temporary occasionally permanent, effect attributed to Fc and neutropenia): Rare in childhood. The neutropenia
receptor blockade and decreased synthesis of antibody). is of complex causation:
‘Compensatory’ monocytosis is common, so that usually – Neutrophil antibodies demonstrable in most cases
total leukocyte count is within normal limits. – Hypersplenism, most patients showing sustained
Marrow examination is recommended to exclude improvement in count after splenectomy
the rare possibility of leukemia as a cause of isolated – Inadequate compensatory marrow production.
neutropenia. Myeloid hyperplasia is usual; mature forms
(bands and segmented) are normally represented or Evan’s Syndrome
deficient (‘maturation arrest’). Autoimmune thrombocytopenia and hemolysis is without
Median age at detection is approximately 8 months detectable underlying cause such as viral infection or
(range 3-30). Blood counts shortly after birth have been SLE. Autoimmune neutropenia also occurs in some cases.
normal. Girls are more often affected than boys (1.5/L). Antibodies to the various cell lines are different.
The antibody is IgG, with some IgM in occasional cases.
Specificity is usually for NA1 or NA2; in some cases the Marrow Transplantation
target antigen cannot be identified. Evidence of parvovirus
infection (PCR on marrow cells, serology) was obtained in Antibodies to neutrophils (and platelets) occur frequently
a majority of cases; the occurrence of neutropenia rather after marrow transplant (allogeneic or autologous).
than the usual erythroblastopenia may be due to altered Antibodies post-allogeneic transplant can be shown by
immune response, the antibody recognizing myeloid cells immunoglobulin allotyping to be of donor origin, i.e.
as well as virus. antibody against engrafted cells is autoimmune, whether
Neutropenia is self-limiting with a median duration of allogeneic or autologous.
30 months (range 6-60), 95% recovering by 4 years. There
are no known long-term or other effects. Drug-immune Neutropenia (Table 17)
Rare in childhood.
Viral Infection • In most cases the condition is drug-specific; in contrast
to drug-specific hemolysis, antibody will not simply
Antineutrophil antibodies have been detected in some
react with pretreated granulocytes, but only when
viral infections, e.g. infectious mononucleosis, HIV and
serum, drug and neutrophils are incubated together.
parvovirus infection. Neutropenia, however, occurs in
• True autoantibody, comparable to methyl dopa immune
only a minority of those with antibody. The target antigen
hemolysis; though autoimmune, antibody cannot be
is not clear—it does not appear to be one of the known
detected in serum after withdrawal of the drug.
polymorphous, neutrophil-specific antigens, NA1, NA2,
Usually antibody is active against both mature and
etc. Neutropenia is only occasionally (< 1%) severe and
immature cells, in a minority against only precursor or
prolonged enough in itself to predispose to bacterial
only mature cells. The nature of the target antigen/s is
infection.
for the most part unknown; the specific neutrophil series
(NA1, NA2, etc.) is not involved. For quinine, at least it is a
Autoimmune Disease membrane glycoprotein. Antibodies from different drugs
• SLE: Antineutrophil antibodies are detectable in about occasionally cross-react (e.g. quinine, quinidine).
50% of patients. The antibody does not have specificity Neutropenia affects only a minuscule proportion of
for known polymorphous neutrophil-specific antigens those exposed, and is unpredictable in occurrence and
and is distinct from the anti-DNA present in most course. Usually onset is abrupt, 1-5 weeks after start of
cases. treatment, sooner (or immediately) after re-exposure.
Neonatal lupus syndrome is a risk if the mother has Neutropenia lasts usually for 1-4 weeks after withdrawal of
SLE (not necessarily symptomatic in the pregnancy). the drug, occasionally as briefly as 1 day. It may be severe
Major manifestations are cutaneous lupus (not enough to cause serious infection.
manifest at birth but becoming so within 2 months) Marrow may be grossly depleted of neutrophils in
and complete heart block (at birth), usually one or the general or show ‘maturation arrest’ at the myelocyte/
other, occasionally (< 10%) both. Skin lesions resolve metamyelocyte stage, with or without hyperplasia of
usually within 6 months, but the heart block almost precursor cells.
Table 17 Pediatric drugs which may be associated with T-Lymphocytosis with Neutropenia
immune neutropenia
Rare in childhood. Neutropenia (antibody demonstrable
• Antiarrhythmic in some cases) with substantial is increase in normal
– Aprinidine HCl mature lymphocytes (CD8 suppressor/cytotoxic; large
– Flecainide acetate granular lymphocytes in some cases). Lymphocytosis
– Procainamide may first manifest or become more obvious after sple
– Quinidine
nectomy (for other reasons) and may affect marrow.
• Antibiotics
– Penicillin and derivatives
Karyotype normal. It may be associated with polyclonal
- Ampicillin hyperimmunoglobulinemia. Blood is otherwise normal,
- Amoxycillin no anemia or thrombocytopenia. Chronic (years) is with
- Dicloxacillin little effect on health. Spleen may be moderately enlarged.
- Nafcillin Evidence of EBV infection is found in some cases.
- Oxacillin
– Cephalosporins Marrow Infiltration/Replacement (Table 14)
- Cephradine
- Cefotaxime Neutropenia is rarely the only finding. In monosomy 7
- Ceftazidime MPD, however, neutropenia, with or without macrocytosis,
- Cefuroxime may be a prodrome over many years to overt disease.
• Sulphonamides Rarely, neutropenia is a prodrome to ALL.
– Sulphamethoxazole
– Sulphathiazole Deficiency of Hematinics
– Sulphafurazole
– Sulphapyridine • Folate, B12 deficiency
• Antimalarial • Copper deficiency.
– Amodiaquine A rare cause. Neutropenia is an important and early
– Chloroquine characteristic, usually severe (<0.5 × 109/L); marrow usually
– Quinine shows vacuolation of precursor cells and ‘maturation
• Analgesic/anti-inflammatory arrest’ at myelocyte/metamyelocyte stage. Anemia is
– Amidopyrine usually severe (to about 4.5 g/dL) and macrocytic, with
– Aminosalicylic acid megaloblastosis, vacuolated erythroblasts and ringed
– Diclofenac sideroblasts (10-15% of cells) in marrow. The genesis of
– Ibuprofen
these changes obscure; they do not occur in the best-known
– Propyphenazone
• Antithyroid
copper deficiency in man (Menkes kinky hair syndrome).
– Propylthiouracil1,2 Possible mechanisms include defective synthesis of
– Carbimazole cytochrome oxidase and ascorbic acid oxidase; which
– Methimazole keep copper in the reduced state. Treatment with copper
• Other produces rapid and striking response.
– Phenytoin Deficiency is most likely to occur in infants with
– Chloral hydrate prolonged diarrhea and malnutrition, and in those on
– Gold thiomalate prolonged total parenteral nutrition without copper
– Levamisole supplementation. Deficiency may, however, occur without
1
Neutropenia may not occur till months or years after exposure obvious cause in infants who are thriving.
2
True autoimmune in some cases
Drugs and Neutropenia
Selective granulocytopenia as an idiosyncratic, unpre
In some cases antibody is generated against platelets dictable effect of drugs is rare in childhood, though it is
as well as neutrophils (different antibodies), and rarely possible with a large variety of drugs. Mechanisms include
against other cells, e.g. erythrocytes and T lymphocytes in personal idiosyncrasies in pharmacokinetics, sensitivity
a hemolytic-uremic-like syndrome attributed to comple of myeloid precursors and immune response. Drugs with
ment-mediated activation and adhesion of neutrophils to potential for immune destruction are listed in Table 17. The
endothelium. same drug may produce granulocytopenia by different
mechanisms in different patients and possibly even at green or gray-black, may resemble basophil granules),
different times in one patient. and unusually coarse or densely staining granules in
basophils, monocytes and mast cells. Precursor cells in
Neutropenia due to Sequestration marrow are also affected. To be distinguished from toxic
granulation (finer, often associated with other signs of
• Hypersplenism (e.g. biliary atresia, liver cirrhosis,
toxicity, temporary).
cavernous transformation of portal vein).
• Hyperphagocytosis of band and segmented forms by
marrow macrophages is a rare cause of isolated neutro Sparse, Coarse Azurophil Granules
penia; e.g. hemophagocytosis, in which macrophages A minority of neutrophils contains a light sprinkling of
contain a variety of inclusions. There is no evidence coarse granules. Rare granules may be metachromatic.
of neutrophil antibody, no or slight increase in serum Other granulocytes are normal.
muramidase, no response to adrenalin (marginating Characteristic of MPS IV (Morquio) type A (early
pool) but good response to hydrocortisone (marrow onset). Main features: Normal intelligence, gross and
reserve). distinctive skeletal change, mild facial coarsening and
corneal clouding, risk of spinal cord compression from
GRANULOCYTES: CYTOPLASMIC ANOMALIES odontoid hypoplasia; valvular heart disease. Onset
(TABLE 18) 1-3½ year’s survival beyond 30 years is unusual auto
somal recessive. Definitive diagnosis is by galactose-
Qualitative changes in leukocytes in infection are des 6-sulphatase (arylsulphatase A) assay in leukocytes or
cribed in Tables 3 to 9. cultured fibroblasts. Milder forms occur, with no excess of
mucopolysaccharide in urine and longer survival.
Alder Anomaly (Table 19)
Coarse, densely-staining granulation in neutrophils, Vacuolation
anomalous staining of eosinophil granules (violet, Vacuolation is uncommon as a genetic anomaly. More
common causes are toxic states (infection, inflammation
Table 18 Anomalies of granulocyte cytoplasm
and cytotoxics) and artifact of anticoagulation.
• Alder anomaly
• Sparse coarse azurophilic granules
Vacuoles of Neutral Lipid (Jordan’s Anomaly)
• Vacuolation Vacuoles are ORO and Sudan III positive, stain red with
• Vacuolation of granulocyte precursors and erythroblasts Nile blue sulphate and occur in most to all neutrophils,
• Döhle-like bodies eosinophils, basophils and monocytes and in a proportion
• Neutrophil specific granule deficiency
of plasma cells, but not in other hemopoietic cells. Absent
• Eosinophil specific granule deficiency
• Peroxidase deficiency in neutrophils
from myeloblasts and increase with cell maturity from
• Giant granulation in granulocytes and monocytes promyelocyte onward.
• Amorphous rounded ‘gray’ bodies
• Hemosiderin Ichthyosis and Neutral Lipid Storage Disease
• Bilirubin
Triglyceride droplets also in other tissues, e.g. muscle, liver.
Main features: Ichthyosis, myopathy, ataxia, sensorineural
deafness, cataracts, liver dysfunction, variable mental
Table 19 Alder granulation retardation, evident at birth, autosomal recessive.
• MPS VI (Maroteaux-Lamy)1
• MPS VII (Sly) Carnitine Deficiency
• Multiple sulphatase deficiency A heterogeneous and incompletely characterized group of
• Infantile free sialic acid storage disease2
disorders is in which carnitine deficiency may be genetic
• Asymptomatic3
or acquired (e.g. renal Fanconi syndrome, hemodialysis,
1
Granules metachromatic and birefringent total; parenteral nutrition). The defect/s in some of the
2
Partially developed Alder granulation
genetic deficiencies is unidentified and the traditional
3
Existence doubted
distinction between ‘muscle’ and ‘systemic’ deficiency
may be artificial. In some there is a defect in carnitine May-Hegglin Anomaly

transport across mitochondrial membranes, in others
a defect in carnitine synthesis. Jordan’s anomaly is Inclusions usually easily seen, but may be inconspicuous
associated with muscle carnitine deficiency (skeletal and in some cases; occur in granulocytes and monocytes but
cardiac), less so with systemic deficiency. not lymphocytes; pyroninophilic (reaction abolished by
ribonuclease), with distinctive ultrastructure of 7-10 nm
filaments oriented in parallel in long axis. It is associated
Wolman’s Disease with enlarged platelets and, in about one quarter, mild
Lipid vacuoles are inconsistent and infrequent. thrombocytopenia autosomal dominant.
Neonatal Hemochromatosis Fechtner’s Syndrome (Alport’s Syndrome

Neutrophils with ORO positive vacuoles may occur Variant)
in neonatal hemochromatosis. In infants vacuolation Dohle-like inclusions, giant platelets, nephritis (micros
may be associated with nuclear pyknosis/cell death, copic hematuria to renal failure), sensorineural deafness
and vacuolated cells contained (between the vacuoles) and congenital blue-spotted (‘cerulean’) cataracts. The
fine Perls positive granulation (finer than in adults with Döhle-like bodies occur in most neutrophils and some
hemochromatosis). eosinophils, are smaller and less intensely staining than in
May–Hegglin and consist of segments of rough endoplas
Other tic reticulum and ribosome cluster’s but no filaments.
ORO positive neutrophils may occur transiently in biliary Platelets are large, moderate to severe thrombocytope
atresia and following GCSF treatment. nia common autosomal dominant.
Pearson’s Marrow-Pancreas Syndrome Sebastian Platelet Syndrome

Vacuolation of granulocyte and erythroid precursors, It is similar to Fechtner’s syndrome but without the clinical
ringed sideroblastosis, increased storage hemosiderin, abnormalities.
transfusion-dependent macrocytic anemia, reticulocy
topenia; variable neutropenia, thrombocytopenia and Neutrophil Specific Granule Deficiency
splenic atrophy. It may evolve to marrow aplasia or leuke
mia. A deletion of mitochondrial DNA has been identified Specific granule deficiency is usually acquired (myeloid
with possible maternal inheritance. leukemias, burns, infection, normal neonate). The genetic
It presents in infancy with growth failure and refractory deficiency is never severe and is rare.
steatorrhea (pancreatic fibrosis). About half die before the Main features:
age of 3 years, others improve with age. • In stained films cells appear poorly granulated and
Differential diagnosis is from other marrow-pancreas washed out (MPO-deficient cells appear normal).
syndromes and from hereditary sideroblastic anemia. • NAP decreased or absent. NAP is not a constituent
• Shwachman syndrome: No vacuolation or sidero of specific granules but plasma membrane linked,
blastosis; marrow hypoplasia earlier and more pro deficiency being attributed to an anomaly common to
minent than in Pearson’s. both plasma membrane and specific granules.
• Hereditary sideroblastic anemia: A proportion of eryth • Granules ultrastructure consists only of the enveloping
rocytes are microcytic. No vacuolation or pancreatic vesicle.
insufficiency. • Deficiency of specific granule components, e.g. lacto
• Atypical cystic fibrosis with marrow hypoplasia: No ferrin, TCII, can be shown by biochemical or immuno
vacuolation or sideroblastosis. logic methods. Deficiency of TCII in serum may be
associated.
• Deficiency of defensins (normal component of sub
Döhle-like Bodies
population of azurophil granules) accompanies the
These are usually more sharply defined and larger than specific granules defect.
Döhle bodies, are not accompanied by toxic changes and • Pelgeroid changes—bilobed nuclei of uneven size;
are permanent. micronuclei in some cells.
• Cells are defective in chemotaxis (specific granules CAE (constituents of azurophil granules) and contain
produce chemotactic receptors) and in bactericidal lactoferrin (specific granules). Normal specific granules
capacity (deficiency of lactoferrin, defensins). are sparse and azurophil granules absent. Defects in
• Neutropenia due to intramedullary destruction may chemotaxis, mobilization and degranulation are attributed
occur. to mechanical impediment imposed by granule size. These
• Manifests as pyogenic infections, which may be indolent. defects, together with neutropenia and deficient natural
• Probably autosomal recessive. killer cell activity, contribute to susceptibility to infection.
Neutrophil precursors (marrow) contain large, MPO
Peroxidase Deficiency and Monocytes positive, pink to purple staining inclusions, often within
vacuoles. Neutropenia and increase in serum muramidase
The peroxidase in neutrophils and monocytes (MPO)
are attributed to intramedullary destruction of precursor
differs from that in eosinophils and is under different
cells. Eosinophil precursors may contain giant, densely
genetic control. In neutrophils MPO is localized to azuro
staining azurophil granules.
phil (primary) granules. A minority of platelets may contain large granules.
• Acquired deficiency occurs in myeloid leukemias (M2, Bleeding is due to deficiency of dense bodies and, in the
M3 and M4 especially) and myelodysplasias. Usually accelerated phase, thrombocytopenia.
a proportion of neutrophils is completely lacking in The main clinical features are partial oculocutaneous
enzyme. The gene for MPO lies in 17q in the vicinity of albinism, cranial and peripheral neuropathy (muscle
the breakpoint for the t(15;17) of AML M3. weakness, ataxia, sensory loss, nystagmus), infection
• Activity is diminished by some drugs—sulphonamides, (especially S. aureus) and bleeding. In the accelerated
antithyroid drugs, phenothiazines, ascorbic acid. phase (usually preterminal, first or second decade),
• MPO deficiency is the most common inherited disorder organ enlargement and pancytopenia are due to
of neutrophils (complete deficiency about 1/4000, lymphohistiocytic proliferation and hemophagocytosis.
partial about 1/2000. The partial deficiency affects all Autosomal recessive. Heterozygotes may show giant
or almost all neutrophils, though not necessarily to the granulation in occasional leukocytes, but this is an
same degree. Identification is by standard cytochemical unreliable test for the carrier state.
methods (MPO, SBB) or, for partial deficiency especi
ally, automated flow cytometry using 4-chloro-1- Pseudo-Chediak-Higashi Granulation
naphthol as substrate (e.g. Hemalog D counter).
Giant granulation in granulocytes, but not lymphocytes,
MPO-deficient bloods will be wrongly identified as
may occur in myeloid leukemias. Abnormal granules are
neutropenic by automated counters which identify formed by fusion of azurophil granules and may contain
neutrophils cytochemically. EPO is normal. Auer-like microcrystals, which differ from true Auer rods
Morphology of neutrophils and monocytes in stained in periodicity of ultrastructure.
films is normal.
Surprisingly, bactericidal capacity of MPO—deficient
granulocytes is only mildly diminished; killing of fungi
Gray-staining Bodies
(candida, aspergillus) however is severely affected. Amorphous, rounded, gray-staining bodies have been
Patients have little susceptibility to bacterial infection, noted in:
but there is a risk of disseminated candidiasis if MPO • Granulocytes of the three types, monocytes and mast
deficiency is associated with diabetes mellitus. Mode of cells in an infant with livedo reticularis of the skin and
inheritance is uncertain. Simple Mendelian genetics are extrahepatic biliary atresia. Leukocyte morphology
unlikely and polygenic inheritance has been proposed. in both parents was normal. Inclusions negative by
routine cytochemical procedures. The anomaly is not
Giant Granulation in Granulocytes and a result of the biliary atresia.
• Eosinophils and basophils only, as a dominantly inheri
Monocytes ted, apparently asymptomatic anomaly. Inclusions
Chediak-Higashi Syndrome absent from other hemopoietic cells, including mast
cells; mildly positive with some cytochemical procedures
An unidentified membrane abnormality results in (e.g. PAS), distinctive in ultrastructure. (Charcot-Leyden
fusion of azurophil and specific granules to form giant crystals, which may also occur in eosinophils, are not
granules. These are positive for MPO, SBB AchPh and latticed).
• Granulocytes (all types), monocytes, lymphocytes intermediate degree of pelgerization in one parent
and plasma cells, from birth in an infant with hepato available for study
spleno megaly, spherocytic hemolysis, thrombocy iii. A syndrome of leukopenia and infections, probably
topenia, neutropenia and infections. These features X-linked.
disappeared and the child is apparently well at the age The homozygous state appears to be usually lethal
of 2 years. Inclusions negative with routine cytochemis in utero. Pelgerization is conspicuous in inherited
tries. Electron microscopy showed ribosomal type neutrophil specific granule deficiency.
composition without identifiable organelles.
• In a (possibly) similar case, inclusions identified Excessive Tags
as collections of actin microfilaments occurred in
all hemopoietic cells, but mainly granulocytes in a Short projections, with head as wide as or slightly wider
13-month-old boy with transfusion-dependent ane than the attachment stalk, to be distinguished from
mia, splenomegaly, gray skin discoloration and inter drumsticks (larger mass of dense chromatin, attached by
mittent neutropenia and thrombocytopenia. Clinical short filament) and clubs (larger than drumstick, longer
abnormalities resolved spontaneously at about 18 filament). The chromatin may be coarse and lumpy and
months but inclusions have persisted. separation of lobes indistinct.
Occurrence of 2 or more tags in > 15% of neutrophils
Other Inclusions is characteristic of trisomy 13, whether isolated or as part
of triploidy. Hereditary persistence of nuclear appendages
Neutrophils and/or monocytes may contain hemosiderin is descried as an autosomal dominant, asymptomatic
(brown-yellow staining, often refractile, and bilirubin defect, but its status as a genuine, independent anomaly is
(yellow-green to green black). uncertain.
GRANULOCYTES: NUCLEAR ANOMALIES Hypersegmentation of Neutrophil Nuclei

(TABLE 20) (Table 21)
Pelger-Huet Anomaly The normal mean lobe count, after the neonatal period
• Pelgerization is most commonly acquired, occurring is 2.8 (2.5–3.1). Only the last 2 in Table 21 are considered
especially in myeloid leukemias, myelodysplasias, here. Others are discussed elsewhere.
bilineage ALL, toxic states and colchicine poisoning. • Hereditary constitutional hypersegmentation—mean
• The inherited anomaly may be heterozygous or lobe count approximately 4 but cells normal in size.
homozygous and affect all or only some (5-20%) cells. Asymptomatic, autosomal dominant.
In the common, heterozygous state, neutrophil nuclei • Hereditary giant neutrophils (macropolysytes)—5-15%
have rod, dumb-bell, peanut or pince-nez shapes, and of neutrophils abnormally large, with 6-10 lobes (prob
eosinophil nuclei > 2 lobes; abnormality is not readily ably tetraploid. Normally up to 2 cells per 1000, slight
recognizable in basophils. In the rare homozygote, ly more in a variety of illnesses, including cytotoxic
most cells have rounded nuclei. exposure. Asymptomatic, autosomal dominant, but
Inheritance is autosomal dominant, with some rare only the heterozygous state is known.
exceptions (following). The common, heterozygous state is
not convincingly linked to any clinical illness. Associations
Table 21 Hypersegmentation of neutrophil nuclei
have however been suggested with:
i. Muscular dystrophy • Toxic states
• Megaloblastosis1
ii. A syndrome of episodic fever and abdominal pain
• Triploidy
(thought to be autosomal recessive because of an • Myelokathexis
• Neutropenia and hypogammaglobulinemia with abundant
• Abnormal neutrophils in marrow
Table 20 Abnormalities of granulocyte nuclei • Lightsey anomaly2
• Pelger-Huet and like anomalies • Iron deficiency (uncommon)
• Excessive tags • Hereditary constitutional hypersegmentation
• Hypersegmentation of neutrophil nuclei • Hereditary giant neutrophils (macropolycytes)
• Hypersegmentation of eosinophil nuclei 1
Benign or as a part of myeloid leukemia/myeloidysplasia
• Chromomeres 2
Neutropenia with gigantism and multinuclearity of marrow
• Toxic states neutrophils
Nuclear Changes in Toxic States 5. Lyall EGH, Lucas GF, Eden OB. Autoimmune neutropenia
of infancy. Journal of Clinical Pathology. 1992;45:431-4.
• Infection, treatment with cytotoxics 6. McClain K, Estrov Z, Chen H, Mahoney DH. Chronic neu
• Heat stroke, hyperpyrexia ‘Botryoid’ nuclei (shrinkage, tropenia of childhood: frequent association with parvovi
pyknosis, clustering of lobes), often with fragmentation rus infection and correlations with bone marrow culture
and chromomere formation, may occur in heat stroke studies. British Journal of Haematology. 1993;85: 57-62.
and hyperpyrexia. Changes disappear within 24 hours 7. Minchinton RM, McGrath KM. Alloimmune neonatal
after removal from injury. neutropenia–a neglected diagnosis? Medical Journal of
Australia. 1987;147:139-41.
• Colchicine poisoning 8. Pui CH, Williams J, Wang W. Evans syndrome in childhood.
Extrusions, chromatin damage and Pelgerization may Journal of Pediatrics. 1980;97:754-8.
be noted. 9. Rodwell RL, Leslie AL, Tudehope DI. Early diagnosis
of neonatal sepsis using a hematologic scoring system.
Journal of Pediatrics. 1988;112:761-7.
BIBLIOGRAPHY 10. Schooley RT, Dolin R. Epstein-Barr virus (infectious
mononucleosis). In: Mandell GL, Douglas RG, Bennett JE
1. Dacie JW, Lewis SM. Practical hematology, 7th edn.
(Eds) Principles and practice of infectious diseases, 3rd
Churchill Livingstone, Edinburgh. 1991.
edn. Churchill Livingstone, New York. 1990.
2. Dale DC, Hammond WP. Cyclic neutropenia: a clinical
11. Shastri KA, Logue GL. Autoimmune neutropenia. Blood.
review. Blood reviews. 1988;2:178-85. 1993;81:1984-95.
3. Harmon DC, Weitzman SA, Stossel TP. The severity of 12. Stroncek DF. Drug-induced immune neutropenia.
immune neutropenia correlates with the maturational Transfusion Medicine Reviews. 1993;7:268-74.
specificity of antineutrophil antibodies. British Journal of 13. Young GAR, Vincent PC. Drug-induced agranulocytosis.
Haematology. 1984;58:209-15. Clinics in Haematology. 1980;9:485-504.
4. Kozlowski C, Evans DIK. Neutropenia associated with 14. Zipursky A, Palko J, Milner R, Akenzua GI. The haematology
X-linked agammaglobulinaemia. Journal of Clinical of bacterial infections in premature infants. Pediatrics.
Pathology. 1991;44:388-90. 1976;57:839-53.
S E C T I O N 4
Bleeding Disorders
CHAPTERS OUTLINE
27. Approach to a Bleeding Child
28. Diagnosis and Management of Hemophilia Patients
Farah Jijina
29. von Willebrand Disease and Other Rare Coagulation Disorders
30. Acquired Inhibitors of Coagulation
31. Immune Thrombocytopenic Purpura—Diagnosis and Management
32. Platelet Function Disorders
Shanaz Khodaiji
33. Pediatric Thrombosis
Rashmi Dalvi
34. Disseminated Intravascular Coagulation in Neonates
VP Choudhary
C H A P T E R 27
Approach to a Bleeding Child
Hemostasis is maintained by many processes in the Local Versus Systemic

circulating blood and the blood vessels designed to stop
hemorrhage. These functions are delicately balanced Local cause should be suspected when:
so that we will neither clot nor bleed to death. Clotting • Bleeding even if recurrent from the same site, e.g.
reactions are initiated in response to injury to vessels and recurrent epistaxis from one nostril may be due to
leads to formation of a fibrin platelet plug that stops the excoriation of a superficial vessel in the Kisselbach
blood loss but ultimately results in elimination of the clot, triangle.
repair of any damage to the vessels and normal blood flow • Nose picking, polyps, foreign bodies (Fig. 1A) or rarely
through the affected site. vascular anomalies may cause frequent nose bleeds.
Hemostasis involves a complex interplay between the • Profuse prolonged, bilateral nose bleeds that occur
vessel wall, platelets, and coagulation factors. spontaneously without any trauma and are difficult
The main three components of hemostasis are: to stop are suggestive of a coagulation defect. Rarely
1. Vascular and extra vascular factors an angiofibroma or Rendu-Osler-Weber (Hereditary
2. Platelets hemorrhagic telangiectasia) may cause profuse epis
3. Plasma factors—coagulation factors, fibrinolytic factors, taxis (Figs 1A and B).
natural inhibitors of coagulation and fibrinolysis. • Generalized petechiae, purpura, bruising, hematuria
Successful management of an acute bleeding episode in especially if associated with past history of bleeding
a child depends on: from dental extractions or circumcision should arise
• Detailed history and clinical examination leading to suspicion of a bleeding disorder.
appropriate diagnostic tests.
• Ordering an array of available diagnostic tests without IS THE DEFECT INHERITED OR ACQUIRED?
a clinical diagnosis is neither economically viable nor
therapeutically ideal. Inherited Disorders
• Prompt implementation of therapeutic measures.
• Inherited disorders usually present in infancy and
early childhood.
HISTORY • Family history of bleeding disorder.
• Significance of bleeding • Inherited disorers in milder forms may not be seen in
• Nature and site of bleeding early infancy and may present later in life with bleeding
• Local vs generalized causes following injury or during surgery—mild hemophilia,
• Acquired or hereditary disorder von Willebrand disease.
• Vascular, platelet or factor deficiency • The development of bleeding later in childhood usually
• Is it due to a local cause? indicates an acquired disorder.
276 Section-4 Bleeding Disorders
liver dependent factors. Medication can also exacerbate

bleeding in those with existing coagulation defects—use
of aspirin in patient with hemophilia or low platelets.
INDICATIONS FOR EVALUATION

Investigations for bleeding disorders are done when there is:
• Recent bout of bleeding—unusual, spontaneous, pro
longed, or delayed bleeding. Postsurgical and trau
matic bleeding that is unexpected, prolonged or
disproportionate to extent of injury.
• Family history of bleeding
• Abnormal coagulation test results obtained as a part
of preoperative evaluation—preparation for surgery or
invasive procedures
A B
• Systemic diseases known to be associated with bleed
Figs 1A and B (A) Persistent recurrent bleeding from left nostril ing disorders, e.g. liver disorder, renal disorder, DIC
due to foreign body; (B) Bleeding from both nostrils and over the and sepsis, etc.
skin (DIC) (Courtesy: MR Lokeshwar)
Is the Bleeding due to Vascular, Platelet or a
Acquired Disorders Coagulation Abnormality or a Combination of
• Usually present later in life. Immune thrombocyto these?
penic purpura (ITP) may however present during early
Vascular disorder, thrombocytopenia or functional platelet
childhood, i.e. 3 to 5 years of age.
disorders:
• Have a negative family history.
• Usually in the form of subcutaneous and mucus
• Underlying medical disorder that may affect hemo
membrane bleeds like petechiae, purpura (Fig. 2C),
stasis.
ecchymoses, epistaxis and subconjunctival hemorrhage
– Hepatic disorders, malabsorption syndrome, may
(Fig. 2B)
be associated with vit. K dependent coagulation
• Mucous membrane bleeding and menorrhagia can
factors.
occur in von Willebrand disease also.
– Renal disease: Uremia can interfere with platelet
Factor deficiency:
function.
• Hematomas (Fig. 3): Intramuscular, soft tissue bleeding
– Low-molecular-weight coagulation proteins (factors
• Hemarthrosis, retroperitoneal bleeds
IX and XI) are lost through the kidney in children
• Post-traumatic bleeds are often delayed, some times
with nephrotic syndrome.
hours after the injury.
– Cyanotic congenital heart disease with polycy
Mucous membrane bleeding (epistaxis, excessive
themia may have thrombocytopenia and hypofibri
menorrhagia, bleeding from gums) is often the conse
nogenemia with risk of bleeding and or thrombosis.
quence of a problem with primary hemostasis. Namely a
• Infections: Meningococcemia with DIC
platelet disorder or von Willebrand disease (VWD).
• A detailed menstrual history should be obtained when
• Hereditary hemorrhagic telangiectasia may also be
applicable. The prevalence of bleeding disorders
manifested as mucosal bleeding
in women with menorrhagia is as high 20 percent
• Umbilical stump bleeding is typically seen with factor
conversely; menorrhagia is a common initial symptom
XIII deficiency, but it may also occur with deficiencies
in women with VWD and has been reported to occur
of prothrombin, factor X, and fibrinogen
more than 90 percent of patients.
• The immediate history often provides useful clues
• Past surgical procedures, serious injuries, fractures
to diagnosis. A sick child with fever, shock, mucocu
and tooth extractions without any abnormal bleeding
taneous purpura frequently has intravascular coagu
is good evidence against the presence of a congenital
lation (DIC) (Fig. 6) associated with bacterial infection.
hemorrhagic disorder.
• Medications: Aspirin and other nonsteroidal anti- Family History of Bleeding
inflammatory agents affect platelet aggregation. Pro
longed use of antibiotics can lead to decreased levels of History of unusual bleeding in family members is present
vitamin K deficiency leading to decreased production of in hemophilia, von Willebrand and platelet function
Chapter-27 Approach to a Bleeding Child 277
A B C
Figs 2A to C (A) Bleeding in the knee joint; (B) Subconjunctival hemorrhage; (C) Purpura (Courtesy: MR Lokeshwar)
Proper pedigree chart, covering at least 2 to 3 genera

tions also should take note of members who have expired,
especially due to bleeding.
X-linked Recessive Pattern

Maternal brothers, cousins, uncles and maternal grand
father may be affected with X-linked transmission while
the females remain asymptomatic carriers.
Bleeding disorders which have a sex-linked recessive
inheritance are:
• Hemophilia A (factor VIII deficiency)
• Hemophilia B (factor IX deficiency)
Fig. 3 Cephalhematoma in bleeding disorder
• Wiscott-Aldrich’s syndrome.
(Courtesy: MR Lokeshwar)
Autosomal recessive disorders: In autosomal recessive
disorders, the parents of affected person are heterozygote
and hence have a 50 percent plasma concentration of the
relevant clotting factors. History of consanguinity should
be asked for. This genetic pattern is typical of disorders
of factor II, V, VII, X, XI, XII, XIII, prekallikrein and high
molecular weight kininogens.
However, lack of plasma factor XII, prekallikrein or
high molecular weight kininogens do not usually cause
any clinically significant bleeding.
Autosomal Dominant Pattern of Inheritance

• Von Willebrand disease
Fig. 4 Hemorrhagic disease of newborn • Qualitative platelet defects
(Courtesy: MR Lokeshwar) • Dysfibrinogenemia
• Hereditary hemorrhagic telangiectasia.
disorders. Approximately a third of infants and young This type of inheritance may show a variable pene
children with newly diagnosed hemophilia have a negative trance and expressivity. Many members in different gene
family history. rations of the family may be affected.
A B C
Figs 5A to C (A and B) Bleeding in the joints in hemophilia; (C) Glanzmann’s thrombasthenia (Courtesy: MR Lokeshwar)
• A well-looking child covered with petechiae often has

immune thrombocytopenia (Figs 2C and 9)
• Hematuria with bruising localized to the gluteal region,
ankles, and feet—Henoch-Schönlein purpura (Figs
10A and B).
ASSOCIATED UNDERLYING DISORDERS

Certain characteristic hemostatic defects are associated
with specific clinical conditions.
• Liver diseases with factor II, VII, IX and X deficiency and
fibrinolysis due to decreased clearance of activators
and hypercoagulable state because of antithrombin III
and protein C deficiency.
• Malabsorption states may be associated with multiple
factor deficiencies, i.e. Vitamin K dependent factors.
Fig. 6 Intravascular coagulation (DIC) (Courtesy: MR Lokeshwar) • Acute promyelocytic leukemia is known to be associ
ated with DIC due to increased cellular procoagulant
activities.
• Negative family history does not rule out the possibility • Myeloproliferative disorder may have platelet defects,
of inherited bleeding disorders. thrombocytopenia and thrombocythemia.
• Family history might be negative, if the coagulation
• Amyloidosis may be associated with factor X deficiency
defect is mild.
and capillary fragility.
• Spontaneous mutation, as is seen in 20 percent of
• Systemic lupus erythematosis—antibody to acidic
patients with hemophilia A
phospholipase, autoantibodies to coagulation proteins
• Exsanguinating bleeding is uncommon due to bleeding
and glycoproteins may be present, resulting in lupus
disorders and is more likely to be due to injury to major
inhibitors, factor deficiency, thrombocytopenia and
vessels.
thrombocytopathy.
History and physical examination can often help
you make a specific diagnosis and point one to the right
diagnostic tests. CERTAIN SYNDROMES KNOWN TO BE
• A male toddler who has just started crawling exhibits
ASSOCIATED WITH BLEEDING DISORDERS
extensive bruising and or joint bleeding—diagnosis
hemophilia A or B (Figs 2A, 5A and B) • Hereditary hemorrhagic telangiectasia is associated
• A girl who has had severe menorrhagia with frequent with characteristic telangiectatic lesions in the
nose bleeds—possible VWD mucus membrane and skin and may manifest with
Fig. 7 von Willebrand disease (Courtesy: MR Lokeshwar)
B
Figs 10A and B Henoch-Schönlein purpura
epistaxis, melena and bleeding per rectum. Presence

Fig. 8 Qualitative platelet defects (Glanzmann’s thrombasthenia)
of telangiectasia in the mucous membrane of nose,
bulbar conjunctiva, tongue, lips and tips of fingers is
the hallmark of diagnosis.
• Keloids may be seen in children with afibrinogenemia
and factor XIII deficiency.
• Cigarette paper scar, hyperextensible joints suggest
Ehler-Danlos syndrome.
• Presence of syndactyly with history of bleeding episode
is known to be due to factor V deficiency.
• Wiskott-Aldrich syndrome (Fig. 11A) is associated with
thrombo cytopenia, recurrent infection, otitis media,
and eczema.
• Children with albinism may have qualitative functional
defects of platelets.
• Thrombocytopenia with absent radius (TAR syndrome)
is easy to diagnose because of skeletal anomaly.
• Kasabach-Merritt syndrome is characterized by giant
hemangioma associated with evidence of clinical
Fig. 9 Immune thrombocytopenic purpura and subclinical DIC and thrombocytopenia (Figs 11B
(Courtesy: MR Lokeshwar) and C).
A B C
Figs 11A to C (A) Wiskott-Aldrich syndrome; (B and C) Kasabach-Merritt syndrome (Courtesy: MR Lokeshwar)
Hemarthrosis (spontaneous), bleeding in the muscles Table 1 Screening laboratory tests in hemostatic disorders
without significant trauma points towards inherited Platelet BT PT APTT Interpretation
coagulation disorders. ↓ ↑ Normal Normal Thrombocytopenia
↓ ↑ ↑ ↑ DIC
LABORATORY ASSESSMENT (TABLE 1) ↓ ↑ Normal ↑ Type 2A vWD
Sample Collection and Technique Normal ↑ Normal ↑ vWD
Platelet
• A properly drawn blood sample is crucial for Normal ↑ Normal Normal
Dysfunction
interpretation of the results of coagulation test. Normal Normal ↑ Normal Factor VII deficiency
• For coagulation assays blood should be obtained by Normal Normal Normal ↑ XII, XI, IX, VIII
clean venipuncture without air bubbles and without Liver disease, Vit K
contamination by tissue fluids Normal Normal ↑ ↑
def, Combined def
• Drawing of samples from catheter often results in Normal Normal Normal Normal XIII def, vascular
‘sample contamination or intravenous fluids and
spuriously abnormal values
• Screening tests are done after evaluating the nature
• Improper sample collection is one of the most common
and clinical circumstances of bleeding and prior to
reasons for abnormal coagulation test
surgery if indicated
• Samples should be tested within 2 hours of collection
• Specific tests need to be done to confirm the diagnosis
if maintained at room temperature or within 4 hours if
after the history, physical examination and screening
kept cold
test point to a possible diagnosis.
• Plasma samples must be frozen if not tested within
this time frame. When they are to be analyzed, frozen
samples should be rapidly thawed at 37°C and tested
Screening Tests
immediately These are the tests for the initial assessment for bleeding
• A panel of screening tests should be ordered, including tendency and include:
a complete blood count with evaluation of platelet • CBC and PS examination
number, morphology-smear examination, PT, APTT, • Platelet count
PFA (Platelet function analysis). • Bleeding time/PFA
Though a thorough history and clinical evaluation • Clot retraction
helps in suspecting the nature and type of bleeding • Prothrombin time/INR
disorder, laboratory investigations are required to make a • APTT.
specific diagnosis, they can be conveniently divided into CBC can reveal involvement of other cell lines in cases
screening tests and special tests. suspected to have leukemia, aplastic anemia, etc.
Proper Smear Examination also helps in Evaluating The most widely used method is the modified ivy BT
Extent of Thrombocytopenia if Present performed with a template. The BT is performed with a
tourniquet maintained at 40 mm Hg and placement of the
• Presence of clumps of platelets rules out platelet template device on an area of the forearm (Just below the
deficiency and absence of platelets indicates severe elbow), blade makes a linear cut 1 to 2 mm deep.
thrombocytopenia usually less than 10,000–20,000/ With a stop watch and filter paper, the blood coming
cumm from the cut is gently blotted away while taking care to not
• Presence of platelets but not in clumps—indicates touch the filter paper to the cut (which would remove the
absence of aggregation, suggesting platelet functional fragile platelet plug). A normal BT is 3 to 9 minutes with
disorder normally functioning platelets. The BT is an approximate
• Large platelets simulating size of the lymphocytes measure of the relationship between platelet number and
suggest possibility of Bernard-Soulier syndrome function.
• Large platelets also indicate younger platelets as seen Prolongation of bleeding time usually occurs at platelet
in regenerative type of thrombocytopenia where there count of < 50,000/cumm to 100,000/uL. At counts below
is peripheral destruction of platelets 10,000/cumm, bleeding time is usually prolonged and is
• Large platelets are also seen in the hereditary giant often 15 minutes or longer and hence BT should not be
platelet syndromes performed when platelets are low.
• Small platelets are characteristic Wiskott-Aldrich The BT can also be prolonged with congenital and
syndrome. acquired platelet defects.
Platelet Count Prolonged Bleeding Time with Nearly

Normal Platelet Count
• It is a simple first step in evaluating the cellular aspect
of hemostasis Qualitative Platelet Disorders (Table 2)
• However, manual count is not reliable and not
• Glanzmann’s thrombasthenia (Figs 5C and 8)
reproducible and hence platelet count should be
• Bernard-Soulier syndrome
done on particle cell counter or using phase contrast
• Storage pool disorder
microscope
• Wiskott-Aldrich syndrome
• A spuriously low automated platelet count (pseudo
• Acquired defects, uremia and cyanotic congenital
thrombocytopenia) may result from ethylene diamine
heart disease
tetra acetic acid (EDTA) anticoagulant plus an IgG or
• Vasculitis (e.g. Henoch-Schönlein purpura)
1gM platelet antibody, platelet cold agglutinins, or
• Connective tissue disorders such as Ehlers-Danlos
platelet clumping from a partially clotted sample. The
syndrome
normal platelet count (for all ages) ranges from 150,000
• Drugs like aspirin and nonsteroidal anti-inflammatory
to 450,000/uL
agents
• In the setting of thrombocytopenia increased or
• Von Willebrand disease (Fig. 7).
decreased platelet size may suggest platelet turnover
Bleeding time has been replaced by the platelet
or decreased production, respectively
function analyzer (PFA) which is a rapid and automated
• If platelet type of bleeding (petechiae, purpura,
form of platelet function assessment.
mucosal bleeding) is seen with normal platelet count or
Clot retraction: This is not any more a commonly
marginally low platelet count, then platelet functional
ordered test as a screening or diagnostic test any more.
disorders should be kept in mind.
Retraction and exudation of the serum after one hour
• Electronic particle counters also provide a mean
is observed in the clotting tube. Normally, 50 percent
platelet volume and (size) distribution.
exudation at the end of one hour of the original blood
volume is taken as normal retraction.
Bleeding Time
The bleeding time (BT) is a measure of the interaction
Prothrombin Time
of platelets with the blood vessel wall. This test evaluates Normal (reference) range varies depending on the labora
primary hemostatic stage. It has major drawbacks and has tory (its instrumentation and the lot of thromboplastin),
been discarded in children by most hematologists. but it is generally 10 to 11 seconds.
The BT is an approximate measure of the relationship • PT measures extrinsic clotting system and the common
between platelet number and function. pathway, the activities of factors I (fibrinogen), II
Table 2 Platelet aggregation response

Comment/
Condition Platelet Aggregation with
Further tests
Count Size ADP Col Ri AA A23187
IIb/IIa
Thrombasthenia N N 0 0 1 0 0
expression
Bernard-Soulier
Low Large N N O N N Gp1b expression
syndrome
Storage pool
N N 1 R 1 1/0 R ATP: ADP pools
defect (d)
Cyclo-
Responds to
oxygenase N N 1/N R N R R
endoperoxide
deficiency
Thromboxane
synthetase N N 1/N R N R/0 N
deficiency
Aspirin
ingestion NSAID N N 1 R N R/0 N/R Stop aspirin
and retest
Ehlers-Danlos
N N N N N N N
syndrome
von Willebrand Assay vWF:Ag
N N N N 0/R N N
disease and RiCoF
(prothrombin), V, VII, and X. PT is prolonged with to 54 seconds) in term infants (and often even longer in
deficiencies of plasma factor VII, X, V, II and fibrinogen premature infants).
and inhibitors of these factors The APTT measures factors I (fibrinogen), II (pro
• Prolongation of the PT beyond the reference range is thrombin), V, VIII, IX, X, XI, and XII; prekallikrein; and
not generally seen until the functional level of one of high-molecular-weight kininogen.
these factors is less than 30 percent or until fibrinogen
is less than 100 mg/dL Activated Partial Thromboplastin Time is
• A prolongation of PT with normal PTT indicates factor Prolonged
VII deficiency OR early in the course of anticoagulant
therapy, Vitamin K deficiency (Fig. 4) or liver diseases • During deficiency or abnormalities of high molecular
• The PT is also used to monitor the effect of coumarin- weight kininogen, prekallikren, factor XII and XI, IX,
type anticoagulants. VIII, X, V, II and fibrinogen
• By inhibitors of blood coagulation such as lupus inhib
Activated Partial Thromboplastin Time itors, heparin, fibrin/fibrinogen degradation product
• Deficiency of any of the latter three factors (prekal
Activated partial thromboplastin time (APTT) is an likrein; and high-molecular-weight kininogen) can
excellent screening test for determining abnormality of result in a markedly prolonged APTT in the absence of
intrinsic and common pathway. clinically significant bleeding
• It should be noted that the sensitivity and repro • Isolated prolongation of the APTT in a patient with
ducibility of the APTT are highly dependent on the clinical bleeding is likely to result from a deficiency of
specific reagents used (particularly the activator in the factor VIII, IX, or XI.
partial thromboplastin reagent) Mixing study in prolonged PTT: The presumption
• With most APTT reagents, the APTT will not he is that addition of normal plasma to a 50 percent mix
prolonged until the amount of factor VIII is less than should correct the PTT if it is due to factor deficiency.
35 percent (0.35 U/mL). Among hospitalized infants or children, unintentional
The reference range will generally be approximately contamination of patient samples with heparin is a
20 to 35 seconds for children and adults but longer (30 common cause of an unexpected prolongation of the APTT
that does not correct on mixing. Failure to correct after aggregation with ADP, epinephrine and collagen but
a mix of 50 percent of normal plasma is indicative of the absence of platelet aggregation with ristocetin but with
presence of inhibitors or antibody that indiscriminately normal levels of von Willebrand factor.
(not specific against any factor) suppresses fibrin formation In Glanzmann’s thrombasthenia, the patient’s platelets
in patients and normal plasma. Lupus anticoagulant (a will agglutinate normally with ristocetin but not at all with
misnomer as it is not necessarily associated with SLE) is a the addition of adenosine diphosphate, epinephrine,
very common cause of isolated prolonged PTT in children collagen, or arachidonic acid. In Bernard-Soulier synd
with adenotonsillar hypertrophy and infections. rome, platelet agglutination will occur normally on addi
tion of each of these agonists except ristocetin.
Thrombin Clotting Time Bleeding disorders not associated with any abnor-
malities in screening tests are:
The thrombin clotting time (TCT or TT) measures the • Factor XIII deficiency
thrombin-induced conversion of fibrinogen to fibrin and • Alpha 2 antiplasmin deficiency, amyloidosis (may or
is performed by adding bovine thrombin to the patient’s may not be associated with factor X deficiency)
citrated plasma and recording the clotting time. • Vascular disorders like hemorrhagic telangiectasia
An extremely prolonged TT usually indicates a heparin (Rendu-Osler-Weber syndrome)
effect. Reptilase, a snake venom protease, clots fibrinogen • Scurvy—prolonged BT
in the presence of heparin and thus can be used to • Ehlers’-Danlos syndrome—prolonged BT
identify heparin as the cause of a prolonged TT. Thus, in • Henoch-Schönlein’s purpura—prolonged BT
the presence of heparin the TT is prolonged, whereas the • Mild factor deficiencies—factor assay
reptilase time is normal. • Battered baby syndrome (Figs 12A and B).
Thrombin Clotting Time is Abnormal in Patients Special Confirmatory Tests

with • Specific coagulation factors: Each of the coagulation
• Hypofibrinogenemia whether acquired or congenital or factors of the intrinsic pathway (prekallikrein, high-
• Dysfibrinogenemia molecular–weight kininogen, and factors VIII, IX, XL, and
• In presence of inhibitors like heparin, myeloma XII) can be measured by one stage, APTT based method
proteins and fibrin degradation products which block • FDPs are usually measured in serum samples because
either thrombin cleavage of fibrinopeptide or fibrin these degradation products consist predominantly of
monomer polymerization. nonclottable derivatives that remain in solution after clot
• A normal or minimally low platelet count with • The D-dimer assay will identify only cross-linked FDPs
prolonged BT or abnormal PFA and poor clot retraction (indicating that fibrin has formed intravascular, has
indicates the possibility of platelet functional disorders been cross-linked, and has then been cleaved into
• Platelet aggregation studies: Low and high concen D-dimers by plasmin
tration of ADP, epinephrine, collagen and Ristocetin • Euglobulin clot lysis time.
are used for aggregation studies. First phase of aggre The euglobulin clot lysis time (ECLT) is a screening
gation is induced by low concentration of ADP and by test for excessive fibrinolysis. The normal ECLT is 60 to
direct effect of certain agents notably epinephrine and 300 minutes. It is shortened in conditions characterized
thrombin. Second phase is mediated by thromboxane A2 by increased fibrinolysis (e.g. antiplasmin deficiency,
and endogenous ADP released in response to numerous plasminogen activator inhibitor 1 deficiency or systemic
pharmacological and naturally occur ring substance fibrinolysis).
like ADP itself. Absence of first phase of aggregation— Lupus anticoagulant: Abnormal mixing study followed
unresponsiveness to ADP in any concentration is by dilute russel vipor venom test (DRVVT) and platelet
characteristic of Glanzmann’s disease. Deficiency of neutralization procedure (PNP) and for confirmation.
platelet fibrinogen and specific glycoproteins GP-II Solitary thrombocytopenia may be due to either:
and GP-III confirm the diagnosis • Reduced production or increased destruction of the
• von Willebrand disease has characteristic lack of platelets
aggregation with ristocetin alone • Idiopathic thrombocytopenic purpura is characterized
• The classical laboratory findings in Bernard-Souiler by acute onset of isolated thrombocytopenia in other
syndrome are—prolonged bleeding time, throm wise healthy children. Other cell lines are normal and
bocytopenia, very large platelets on peripheral smear, the smear reveals normal RBC, white cells with low
deficient platelet adhesion and normal platelet number of platelets and occasional macrothrombocytes
A B
Figs 12A and B Battered baby syndrome—hematoma over the forehead and punch mark over the thigh (Courtesy: Raj Warrier)
• Thrombocytopenia due to decreased production may BIBLIOGRAPHY

be either due to involvement of only megakaryocytes
1. Blanchette V, Bolton-Maggs P. Childhood immune
as seen in TAR syndrome or in amegakaryocytic
thrombocytopenic purpura: diagnosis and management.
thrombocytopenic purpura and is characterized by Pediastr Clin N Am. 2008;55:393-420.
thrombocytopenia and platelet type of bleeding. 2. James AH, Manco-Johnson MJ, Yawn BP, Dietrich JE,
Nichols WL. von Willebrand disease: key points from the
CONCLUSION 2008 National Heart, Lung, and Blood Institute guidelines.
Obstet Gynecol. 2009;114(3):674-8. Review.
Detailed history, thorough clinical examination and 3. Petrini P, Seuser A. Haemophilia care in adolescents-
screening tests usually give sufficient information to compliance and lifestyle issues. Haemophilia. 2009;15
decide, whether bleeding is due to local causes or a (Suppl 1):15-9. Review.
generalized bleeding disorder. 4. Rodeghiero F, Kadir RA, Tosetto A, James PD. Relevance
Depending upon type and nature of the bleeding of quantitative assessment of bleeding in haemorrhagic
disorder, further tests such as factor assay, aggregation disorders. Haemophilia. 2008;14(Suppl 3):68-75. Review.
5. Rossbach HC. The rule of four: a systematic approach to
tests, etc. have to be carried out to confirm the diagnosis
diagnosis of common pediatric hematologic and oncologic
before planning therapy. disorders. Fetal Pediatr Pathol. 2005;24(6):277-96. Review.
C H A P T E R 28
Diagnosis and Management
of Hemophilia Patients
Farah Jijina
Hemophilia is an X-linked hereditary bleeding disorder. Hemophilia A is the most common of these, with an annual incidence of 1/5000
male births worldwide. Hemarthrosis is the most common, most painful, physically, economically, and psychologically debilitating
manifestation of hemophilia. It occurs in 90 percent of severe hemophiliacs. Prompt replacement therapy with the required factor
remains the mainstay of treatment of a bleed.
The dose and choice of the product are influenced by the CLINICAL FEATURES
severity of the disease, the site and severity of the bleeding,
inhibitor antibody status and the clinical scenario. • haracteristically deep/internal bleeding
C
However, the adjuvant role of antifibrinolytic agents • Usually spontaneous or following minimal trauma
and expert physical therapy in the treatment cannot be • Occurs in frequent unpredictable episodes
undermined in an economically burdened society like ours. • Delayed bleeding due to failure of secondary hemo
It is possible to carry out any procedure on a hemophilic stasis.
patient provided adequate factor is given. Outcome is
directly related to the intensity of treatment and the level of Grades of Severity
compliance. Improvements translate into decrease absence Severe Factor levels < 1% Spontaneous bleeds
from school or work, fewer bleeds and days spent in the
hospital, increased personal and professional productivity,
Moderate Factor levels 2–5% Bleeds following minor
improved overall performance status and a healthier self
trauma or after procedures
image. Carrier testing and prenatal diagnosis can be offered
to women who are interested in children bearing. Mild Factor levels 6–40% Usually do not bleed except
following major trauma or
surgery
INTRODUCTION
The most prevalent of the hereditary disorders of
coagulation are: Diagnosis
Types of hemophilia When to suspect?
Hemophilia A Factor VIII deficiency If any of the following are present:
Hemophilia B Factor IX deficiency • Recurrent spontaneous bleeds
von Willebrand disease von Willebrand factor deficiency • Bleeding following trauma out of proportion to the injury
Hemophilia C Factor XI deficiency • Positive family history
Investigations To Avoid
• A patient who is clinically suspected to have hemophilia • IM injections
should be subjected to coagulation tests. • All contact sports
• In hemophilia, the APTT is prolonged and corrects on • Aspirin and other nonsteroidal anti-inflammatory
addition of normal pooled plasma. The PT and TT are drugs (NSAIDs); all drugs that affect platelet function.
characteristically normal.
• However, in case of combined deficiencies for example, To Do
combined factor V and VIII deficiency, the PT will also
be prolonged. • E arly factor correction; prompt treatment of a bleed.
• Factor assay is then done to confirm the diagnosis and This prevents subsequent damage and deformity.
document the severity of the disease. • Ice application at the site of bleed.
• One of the problems, we face in our country is the lack • All procedures to be done under appropriate factor
cover, including dental procedures.
of adequate laboratory facilities at all places to carry
• Getting all vaccinations is recommended, including
out all the tests. Therefore a clinician suspecting a
hepatitis A and B; these need not be given intramuscular
bleeding disorder should refer the patient to a center
but can be given subcutaneously.
that specializes in carrying out these investigations to
• Maintaining a healthy body weight is important to
ensure an early accurate diagnosis. Now, most of the avoid extra stress on joints. Thus mothers need to be
major cities have these diagnostic facilities. educated on a proper diet for these children.
• Once the patient is accurately diagnosed, further • Regular supervised physical therapy and exercises
treatment can be given by the local treating physician should be taught to children from an early age. This
with some guidance from a specialist center. The helps to develop strong muscles and thereby prevent
patient may then be referred to a higher center only bleeding into the joints. This is one of the most
for complicated problems. A wrong diagnosis at the important preventive measures that the child and his
beginning would result in the patient being given parents can do.
inappropriate treatment and the development of • Very often the local physicians and dentists are not
complications. aware of the problem or the relatives tend to hide the
problem and the patient comes to us with complications
of an avoidable bleed. Thus, it is important to educate
Treatment of Hemophilia the parents and the patient.
In the Western world today, it is possible for a child with • It is also worthwhile to inform the principal and
hemophilia receiving adequate treatment to live a near teachers of the school, regarding the problems faced
normal life. An accurate diagnosis is quickly established, by these children.
the family is educated on the management, and the child is • This ensures immediate institution of measures to
put appropriate factor therapy. With this type of treatment control the bleed when it occurs.
most children with hemophilia (apart from the small • It should be highlighted to the caretakers that no form
number who develop inhibitors) can go to school, enjoy of physical punishment be given to the child. We have
sports, and expect to have minimal or no joint bleeding. had many children coming to us with muscle bleeds
This is however expensive and not feasible for us. Thus following physical punishment in school. This is highly
we need to have our own strategies to treat our patients at unfortunate and all efforts should be made to prevent it.
lower costs.
Definitive Treatment
GENERAL PRINCIPLES Prompt replacement therapy with the required factor
remains the mainstay of treatment of a bleed.
Before we discuss the definitive therapy of hemophilia The dose of factor replacement (in units) is calculated
there are certain general precautions, do’s and dont’s which as follows:
the treating physician, the parents of the patient and the • Hemophilia A:
patient himself can follow. All patients and their relatives – Each unit of factor VIII infused/kg body weight is
need to be educated about the disease, precautions and assumed to yield a 2 percent rise in plasma factor
preventive strategies for bleeding. VIII complains of fever since 1 day back levels.
Chapter-28 Diagnosis and Management of Hemophilia Patients 287
– Units of factor VIII to be given = (Desired level – Acute hemarthrosis invariably recurs from time to
patients level) × weight in kg/2. This dose is given time. The first episodes are generally relatively mild and
stat and is followed by half the dose at 12 hourly the joint may regain normal function. However, with
intervals as required, as the biological half life of each recurrence, the synovium becomes invariably more
factor VIII is 8 to 12 hours thickened and vascular. Synovial folds and frond like villi
– In case cryoprecipitate is used, the number of bags form, which become trapped and crushed by joint action,
to be given is calculated according to the amount leading to further bleeding and synovial enlargement.
of factor VIII in each bag of cryoprecipitate. This There is a simultaneous weakening of the periarticular
information is available with the respective blood supporting structures which leads to joint instability and
bank. further predisposes the joint to recurrent bleeding.
• Hemophilia B: Thus a vicious cycle of bleeding—synovitis—bleeding
– Each unit of factor IX infused/kg body weight, is sets in, because of the profusion and fragility of the vessels
assumed to yield a 1 percent rise in plasma factor within the tissue.
IX levels Ultimately, there is a gradual conversion of the
– Units of factor IX to be given = (Desired level – synovium from friable hyperemic tissue to fibrotic scar
patients level) × weight in kg tissue.
– This dose is given stat and is followed by half the This is followed by subchondral and synovial ischemia
dose at 24 hours intervals as required, as the which results in progressive loss of hyaline cartilage. As
biological half life of factor IX is 18 to 24 hours. the joint cartilage progressively degrades, deterioration
of joint function occurs leading to limited and painful
HEMOPHILIC ARTHROPATHY movements.
Hemarthrosis is the most common, most painful and most Finally, the stage of chronic hemophilic arthropathy
physically, economically and psychologically debilitating is reached. The cartilage becomes pitted and destroyed.
manifestation of hemophilia, occurring in 90 percent of There is loss of joint space; bony necrosis and cyst formation
severely affected patients. may occur. This may lead to complete destruction of the
In fact most of the physical, psychosocial and financial joint with fibrous or bony ankylosis.
problems in a severe hemophilia patient are caused by the Over a period of time, osteoporosis may develop as a
effects of recurrent hemarthrosis and chronic arthritis. result of disuse and immobilization of the joint.
This hemorrhage may occur spontaneously or as a result
of trauma.
Clinical Features
Pathophysiology Hemarthrosis occurs in 90 percent of patients with
There is a complex relationship between recurrent severe hemophilia. Though any joint may be affected,
bleeding, synovitis and the development of arthritis in a weight bearing joints are more prone to bleed. The knee
patient with hemophilia. joint is the most commonly involved and the most often
Bleeding into the joint originates from the richly permanently crippled.
vascular synovial plexus, developing spontaneously or The joints affected in order of frequency are knee >
as a result of imperceptible/trivial trauma. Following the elbow > ankle > shoulder > wrist > hip. The spine is rarely
development of the hemarthrosis, the synovial space is involved.
distended with blood; the joint becomes swollen, hot, tense The first episode of hemarthrosis usually occurs when
leading to muscular spasm and restriction of movement. the child begins to walk or crawl. There is often a “target
This hemorrhage will ignite an inflammatory response with joint”, one which is more prone to repetitive bleeding. The
the release of kinins and macrophage interleukin-1 (IL-1). onset of bleed is often heralded by an “aura”, which may
Absorbance of the intra-articular blood is usually be a feeling of vague warmth/tingling sensation/a sense of
incomplete, as the synoviocytes can absorb only a limited mild restlessness/or anxiety.
amount of iron and their capacity is reached easily. The A hemophilic patient may present in various ways:
excess clot formed is, therefore, unlikely to be totally removed
by the fibrinolytic system and organization of the remaining Acute Bleed in a Relatively Normal Joint
clot leads to development of fibrous adhesions. The retained
blood produces a chronic inflammation and hyperemia of The earliest definitive symptom is excruciating pain in
the synovial membrane and the joint may remain swollen, the affected joint. It is swollen, warm and tender, with
painful and tender even in the absence of bleeding. restriction of movement and muscle spasm.
Subacute or Chronic Synovitis • Factor replacement: Early factor replacement is the main
stay of treatment
In these patients, in spite of no active bleed, the joint • Prompt replacement.
appears swollen, tender and inflamed with a boggy
synovium and some restrictions of movement (Figs 1A
Prompt Replacement
and B).
• It reduces duration of bleed
Chronic Hemophilic Arthropathy • It reduces joint damage, absenteeism
• It reduces overall amount of factor required and cost.
If untreated, symptoms of chronic arthropathy typically Doses: 25 to 30 percent correction, with factor VIII is
develop by the second or third decade. In chronically given IV, 12 hourly. Though these are standard textbook
damaged joints due to the thickening of the articular recommended doses, one can generally use less. Often
capsule, external evidence of bleeding may not show. 10 u/kg can be given, and repeated if required.
These patients may present with pain in the joint due to: The duration is decided on the basis of the patients’
• The degenerative arthritis symptoms. Once the pain subsides and there is no increase
• Referred pain due to bleeding in adjoining structures in the swelling, further factor need not be given. Thus most
• Actual bleeding in the joint, which may not be apparent patients may require 1 to 3 doses only. It is important to
clinically note that the swelling itself may take a while to subside
It is also important to keep in mind that HIV positive and in situations where there is a shortage of factor and
hemophiliacs are prone to develop septic arthritis, the finances are tight, one need not continue with the factor.
features of which may mimic an acute bleed.
Home Therapy Programs
Practical Approach to Management of
Wherever possible it is advantageous for the patient or a
Hemophilia Patients in India family member to be taught to administer the factor.
Optimal treatment of acute hemarthrosis involves:
• Factor replacement Advantages
• Relief of pain
• Rest • Improved preservation of joint function, as no time is
• Supervised rehabilitation wasted in getting the factor
A B
Figs 1A and B Severe hemophilia showing chronic synovitis of the knee joint
• Decreased hospital care and cost • However, energetic physiotherapy should be done only
• Decreased absenteeism from school and work in conjunction with small doses of factor replacement
• Patient is more active, mobile and independent • Orthopedic devices—splints/braces/traction/corrective
• Self-reliance improves the patients’ confidence and footwear—may be used as per the patients needs (Figs 2
family interaction. to 4).
Relief of Pain Prophylactic Therapy

• Analgesics • Prophylaxis is the treatment by intravenous injection
• Ice application of factor concentrate to prevent anticipated bleeding.
All patients should be given analgesics to relieve • The goal of prophylactic therapy is to convert a severe
the pain. The drugs which can be safely used in these hemophilia patient to a moderate hemophilic by
patients are dextropropoxyphene (proxyvon), paraceta maintaining trough levels of factor above 1 percent
mol (crocin), opiods and Cox 2 inhibitors. Nonsteroidal and thereby preventing spontaneous bleeds.
anti-inflammatory drugs (NSAIDs) which affect platelet • This prevents bleeding and joint destruction and helps
function should be avoided. to preserve normal musculoskeletal function.
We usually recommend that the patient applies • In patients with repeated bleeding, particularly into
crushed ice or an icepack to the joint, over a wet towel target joints, short-term prophylaxis for 4 to 8 weeks
intermittently for periods of 5 minutes to achieve a 10 to can be used to interrupt the bleeding cycle. This
15°C lowering of temperature in the deeper tissues. The may be combined with intensive physiotherapy or
efficacy of this however, is not definitely proven. synoviorthesis.
• Prophylactic administration of clotting factor
concentrates is advisable prior to engaging in activities
Physical and Rehabilitative Therapy
with higher risk of injury.
• Early physical therapy should be instituted as soon • Prophylaxis is best given in the morning to cover
as the acute hemarthrosis subsides, i.e. once the pain periods of activity.
subsides. Exercises should be increased gradually.
A B
Figs 2A and B Severe hemophilia A with chronic synovitis undergoing dual force stretching and exercise.
The patient recovered completely in two weeks time
A B
Fig. 3 Customized device for footdrop. This patient also Figs 4A and B Customized traction system for Volkmann’s ischemic
developed this drop as a result of compartment syndrome contracture in a patient of severe hemophilia A. The patient is now
fully functionally and is an active member of the society
Primary Prophylaxis Administration and Dosing Schedules

Regular continuous treatment initiated in the absence of Prophylactic Therapy Dose
documented osteochondral joint disease, determined by Factor VIII 25 to 40 u/kg thrice a week
physical examination and/or imaging studies, and started Factor IX 25 to 40 u/kg twice a week
before the second clinically evident large joint bleed and These dosage schedules are expensive. Therefore,
age 3 years different countries follow different protocols for prophy
laxis, and the optimal regimen remains to be defined.
Secondary Prophylaxis Thus we now have the concept of low dose prophylaxis,
using 10 u/kg twice a week, which is gaining popularity in
Regular continuous treatment started after 2 or more developing countries.
bleeds into large joints and before the onset of joint
disease documented by physical examination and imaging
studies. Muscle Bleeds and Hematomas (Figs 5 and 6)
• Seventy-five percent of patients present with soft tissue
Tertiary Prophylaxis bleeds
• The most common site is the ileopsoas muscle
Regular continuous treatment started after the onset of
• This leads to reflex muscle spasm and joint flexion
joint disease documented by physical examination and
deformity
plain radiographs of the affected joints.
• It may lead to complications such as compression of
adjacent nerves, vessels; muscle atrophy; contractures
Intermittent (Periodic) Prophylaxis • Large bleeds may produce fever, hyperbilirubinemia
Treatment given to prevent bleeding for periods not and neutrophilia.
exceeding 45 weeks in a year.
Continuous is defined as the intent of treating for 52 Treatment
weeks per year and receiving a minimum of an a priori
defined frequency of infusions for at least 45 weeks (85%) • Factor correction—soft tissue bleeds generally require
of the year under consideration. more factor correction, about 50 to 60 percent. One
Large joints = ankles, knees, hips, elbows and can try with lesser doses if availability or finances are a
shoulders. problem.
• They may be subdural, intracranial or subarachnoid

• They require full factor replacement for a longer
duration.
Treatment
• It should be noninvasive as far as possible; surgical
intervention is rarely required except in those patients
with neurological deficit or an altered sensorium.
• Aggressive 100 percent factor correction should be
given and continued for at least 8 to 10 days.
• Antifibrinolytic drugs such as trenexamic acid, may be
given for 6 to 8 weeks.
Gastrointestinal Bleeds
Fig. 5 Muscle hematoma in a severe hemophilia patient.
• They are seen in 15 to 20 percent of patients
Responded to conservative management
• Bleeding may be exacerbated by an underlying peptic
ulcer; in fact peptic ulcer disease is more common in
hemophiliacs than in the general population
• The patient must also be investigated for an underlying
disease such as ulcer, chronic liver disease, GI
malignancy, etc. especially in patients who are HbSAg
or HCV positive.
Treatment
• Factor concentrates of about 40 to 60 percent correction
may be required
• They are given till the bleeding stops
• H2 receptor blocking agents may be given simul
taneously
• Antifibrinolytic drugs can also be given.
Urinary Tract Bleeding

Fig. 6 Scrotal hematoma in a severe hemophilia patient. • Sixty-six to ninety percent of patients will experience at
Responded to conservative management least one episode of hematuria
• Most patients present with spontaneous painless
hematuria
• Patients may require the factor to be given for at • All patients must also be investigated to rule out an
least 3 to 4 days or more, depending on the response. underlying organic cause, e.g. calculus, malignancy.
However, once the patient responds, further factor
need not be given.
• Once the pain subsides the patient must be given
Treatment
supervised physical therapy to prevent complications. • R ecommended first line treatment for hematuria is to
increase fluid intake to 2 to 3 liters per day, either oral
or parental
CNS Bleeds
• Most patients will respond to conservative therapy
CNS bleeds are a frequent occurrence in hemophiliacs • If hematuria persists, factor correction 50 to 80 percent
and one of the major causes of mortality. should be given, till bleeding stops
• They develop in 10 to 20 percent of patients • Antifibrinolytic drugs are contraindicated in hema
• However less than 50 percent give history of trauma turia.
Mucous Membrane Bleeds Both the above procedures are not recommended
today as nonsurgical synovectomy is a much better
• These commonly present as epistaxis, gum bleeds, treatment option.
tongue bleeds or from the frenulum in infants, and in • Nonsurgical synovectomy: This is done by the intra-
children following loss of decidous teeth, etc. articular injections of drugs or radioactive material
• Whenever a clot forms, the body attempts to break Chemical: Rifampicin
it down within the vessel so that blood flow can be Radioactive: Au 198 colloidal gold, Y colloidal yttrium,
restored. This process of clot breakdown called fibrino Re colloidal rhenium, P 32 colloid
lysis is very active on mucous membrane surfaces. In Of these using intra-articular rifampicin is a safe and
people with hemophilia, this process can prevent a effective means of causing a chemical synovectomy.
bleed from stopping.
• Thus these bleeds especially those following injury are Pseudotumors (Figs 7A to D)
often difficult to treat and may end up consuming a lot
of factor. Hemophilic pseudotumors are large encapsulated
• The problem is worse in children as they tend to hematomas that represent progressive cystic swelling
continuously disturb the clot with their tongue. from persistent bleeding and incomplete resorption. This
serious complication is evident in approximately 1 to 2
Treatment percent of severely affected patients. The pseudotumor is
composed of clot and necrotic tissue.
Fibrinolysis can be inhibited by drugs, and tranexamic Three types of pseudotumors predominate in hemo
acid is the most widely used drug for this. philia.
• It is advisable to treat with local measures such as 1. The most common type arises from repeated hemorr
EACA application to the site of bleeding or fibrin glue hage and inadequate clot resorption. They are
where feasible since the drug is absorbed from the usually confined within facial and muscle planes.
buckle mucous membrane and then secreted into the Radiologically, they appear as simple cysts.
saliva 2. The second type involves large muscle groups, such as
• Oral EACA tablets or a mouthwash prepared by the gluteus maximus and iliopsoas. These lesions are
crushing or dissolving the tablets can be used especially problematic because they may gradually
• If this does not arrest the bleed, then factor replacement enlarge, develop a fibrous capsule, and eventually
30 to 40 percent correction is given till the bleed stops. destroy adjacent underlying structures by pressure
necrosis. Skeletal fractures and bony deformities
Chronic Hemophilic Arthropathy produced by cortical erosion may result.
3. The third and rarest type of pseudotumor arises from
This is a common problem we face in our country, due to within bone itself, often secondary to subperiosteal
inadequate treatment of an acute hemarthrosis. Patients bleeding. This lesion typically is observed in the long
often come with deformed joints and marked restriction bones of the lower extremities and pelvis but has been
of movement. Treatment of this can be conservative or reported to occur within the calcaneus, cranium, and
surgical, depending on the individual patient. mandible.
Most such pseudotumors arise in adults and occur
Conservative Treatment in proximal skeletal structures; distal lesions occur more
frequently in children before skeletal maturity and are
• Factor replacement with gradual supervised physical
associated with a better prognosis.
therapy
• Synovectomy to control bleeding and prevent pro
Treatment
gressive destruction of the articular cartilage.
Distal pseudotumors respond well to conservative treat
Types of Synovectomy ment consisting of aggressive clotting factor replacement
and cast immobilization. Because conservative treatment
• Conventional arthrotomy: Articular debridement and has not been nearly as successful for lesions of the proximal
synovectomy musculoskeleton, and because complete regression is
Disadvantage: Loss of range of motion. very rare, this approach has been reserved primarily for
• Arthroscopic synovectomy: patients with high-titer inhibitors.
– Low morbidity High- and low-intensity radiotherapy regimens have
– Early rehabilitation been successful in eradicating pseudotumors in the long
– Better range of motion bones and may offer an alternative conservative approach.
A B
C D
Figs 7A to D Pseudotumors of hand and foot in hemophilia A
Surgical extirpation is the most effective therapy for • Thus it is best to carry out surgeries in hemophilia
pseudotumors and is the treatment of choice when it can patients only at specialized centers where all facilities
be carried out in major hemophilia centers. Nevertheless, are at hand.
the operative mortality rate approaches 20 percent.
Before operating on a patient certain pre-requisites must
Depending on the size and location of the pseudo
be ensured:
tumor, percutaneous evacuation of the cavity and sub
• Accurate diagnosis
sequent introduction of fibrin sealant or cancellous bone
• A baseline factor level
can be considered.
• Rule out presence of an inhibitor
• Adequate stocks of factor
Surgery in Hemophilia It is best to do the surgery in the morning as that
• Surgeries in hemophilia patients are a major challenge gives adequate time to take care of any unforeseen post-
in our country due to the poor availability of factor and operative complications.
the expense, as majority of our patients may not have
access to it or are unable to afford the factor. Recommended Dose of Factor
• Optimal care requires cooperation between the
hematologist, surgeon, blood bank and physical ther Using 100 percent factor correction as recommended
apist is not always financially feasible for our patients. Thus,
we need to modify our strategies of treatment to suit the

patients’ finances and also give him optimum treatment.
• Depending on the type and site of the operation one
would preoperatively raise the factor levels to 70 to 100
percent and try to continue the dose for about 48 hours
in the perioperative period.
• Subsequently, levels may be maintained around 50
percent or even less, for 7 to 15 days, depending on the
type of surgery.
• There is generally no need to routinely measure factor
levels in the postoperative period. Factor levels should
be measured only, if required.
• Adjuvant use of antifibrinolytic drugs where possible,
considerably reduces the requirement for factor.
• Also intraoperative use of local agents such as anti
fibrinolytic drugs/fibrin glue can minimize bleeding.
Fig. 8 Hematoma following
tooth extraction without factor correction
Use of DDAVP in Hemophilia A
• Infusion of desmopressin—a synthetic analog of vaso the adjunctive therapy in hemophilia. It is especially
pressin causes a 3 to 5 fold rise in factor VIII levels by valuable in controlling bleeding from mucosal surfaces
release of von Willebrand factor (vWF) from endog (e.g. oral bleeding, epistaxis, menorrhagia).
enous stores in the endothelial cells. Dose: Oral tablets are freely available.
• It is effective in mild/moderate hemophilia and there – For an adult 1 g is administered every 6 hours
fore it can only be used in patients those patients. – For a child is 20 mg/kg.
• Fibrin sealant: Fibrin sealant has hemostatic, sealing,
Disadvantages and healing properties. It is made by mixing fibrinogen
and thrombin, which mimics the last step in the blood
• Variability in response, and needs to be individually coagulation cascade. A semirigid to rigid fibrin clot
assessed in each patient consolidates and adheres to the application site and
• Transient flushing/headaches/palpitations acts as a fluid-tight sealing agent able to stop bleeding.
• Fluid retention, hyponatremia Fibrin sealant can be used for:
• Tachyphylaxis – Dental extraction (Fig. 8)
• Seizure activity in infants – Circumcision
• Thrombocytopenia in Type 2B and platelet type vWD – To stop bleeding from mucous membranes
• It is contraindicated in patients with hypertension/ – Postoperatively over suture lines, etc.
CAD/elderly persons. – Commercially available fibrin sealants are prohi
bitively expensive.
Route of Administration • Cryoprecipitate and plasma: Cryoprecipitate, fresh
frozen plasma (FFP), and cryo-poor plasma are
• Spray: The intranasal dose for patients <50 kg is 150 μg
sometimes the only affordable treatment options in
and for patients over 50 kg—300 μg.
many developing countries. However, they are usually
• IV—The usual dose is 0.2 to 0.3 μg/kg IV in a volume of
not treated to eliminate blood-borne viruses. Because
50 to 100 mL infused over 30 minutes.
of the risk of transmitting disease, the use of plasma and
• Increase in factor levels is seen within 15 to 60 min
cryoprecipitate which has not been viral inactivated
• Effect intranasal lasts for 6 to 8 hours
should be considered a temporary measure until
• It may be given daily for 2 to 3 days.
adequate amounts of factor can be made available.
Pharmacologic Options for
PREVENTION
Controlling Bleeding
• Tranexamic acid: Tranexamic acid is an antifibrinolytic
Carrier Detection and Prenatal Diagnosis
agent that inhibits the activation of plasminogen to In developed countries, where hemophilia care has
plasmin. It promotes clot stability and is useful as progressed to such an extent that a child can live a near
normal life with safe and effective therapy, the need 4. G Richard Lee, et al. Wintrobe’s Clinical Hematology.
for carrier detection and prenatal diagnosis may not be 11th edn. Inherited coagulation disorders. Williams and
important. Wilkins 1999.p.1619.
However, these services are necessary in developing 5. Ghosh K, Shetty S, Jijina F, Mohanty D. Role of epsilon
amino caproic acid in the management of hemophilia
countries so that individuals and families can be evaluated,
patients with inhibitors. Hemophilia. 2004;10:58-62.
informed of their carrier status, and be allowed to make an
6. Ghosh K, Jijina F, Pathare AV, Mohanty D. Surgery in
informed choice on whether they will risk having a baby hemophilia: experience from center in India. Hemophilia.
with hemophilia or not. 1998;4:94-7.
If there is an affected family member then the antenatal 7. Ghosh K, Jijina F, Shetty S, Madkaikar M, Mohanty D. First
diagnosis for hemophilia A and B is now available for time development of Factor VIII inhibitor in hemophilia
those parents who wish to opt for it and has more than 99% patients during postoperative period. Hemophilia. 2002;
accuracy. It can be done at 10–11 weeks of gestation on 8(6):776-80.
chorionic villous samples or at 18–19 weeks on cord blood 8. Ghosh K, Nair AP, Jijina F, Madkaikar M, Shetty S, Mohanty
samples. D. Intracranial hemorrhage in severe hemophilia: pre
valence and outcome in a developing country. Hemophilia.
2005;11:459-62.
Take home message
9. Jijina F, Ghosh K, Madkaikar M, Mohanty D. Ophthalmic
The key to success in hemophilia management lies in early surgery in Hemophilia. Hemophilia. 2001;7:464-7.
diagnosis and treatment. This will reduce the morbidity and 10. Madkaikar M, Ghosh K, Jijina F, Gandhi S, Shetty S,
mortality from a disease which is treatable. Though factor Mohanty D. Open heart surgery with mitral valve replace
accessibility remains a major issue due to financial constraints, ment—ordeal of an undiagnosed hemophilia patient.
even with a limited amount of factor concentrates, it is Clinical and Laboratory Hematology.
possible to improve the lives of people with hemophilia 11. Ronald Hoffman, et al. Hematology Basic Principles
in developing countries through education, prevention, and Practice, 5th edn. Clinical aspects and therapy of
and ancillary care. Hemophilia services must emphasize on hemophilia. p. 1883.
education, physiotherapy, laboratory diagnosis, and simple 12. Shetty S, Colah R, Gorakshakar A, Bhide A, Ghosh K,
measures to manage bleeds, along with a supply of safe Pathare AV, Jijina FF, Mohanty D. Prenatal diagnosis of
concentrates. Hemophilia—A preliminary report. The National Medical
Journal of India. 1998;11(5):218-9.
13. Seminars in thrombosis and hemostasis (Impact factor:
4.22). 2005;31(5):495-500.
BIBLIOGRAPHY 14. Srivastava A, Chuansumrit A, Chandy M, Duraiswamy G,
1. Agarwal MB, Patnaik M. Recombinant activated factor VII Karagus C. Management of hemophilia in the developing
japi.org/august 2005/U-717. world. Department of Hematology, CMC Hospital, Vellore,
2. Beutler E, et al. Williams Hematology; 5th edn. McGraw India. Hemophilia. 1998;4(4):474-80.
Hill, Inc. 1995.p.1413. 15. Srivastava A, Brewer AK, Mauser-bunschoten EP, Key NS,
3. Chandy, Mammen: Management of Hemophilia with Kitchen S, Llinas A, Ludlam CA, Mahlangu JN, Mulder
Minimal Factor Replacement in Developing Countries: K, Poon MC and A. Street; treatment guidelines working
Role of Ancillary Therapy Semin Thromb Hemost. 2005; group on behalf of the world federation of hemophilia.
31:495-500. Guidelines for the management of hemophilia.
C H A P T E R 29
von Willebrand Disease and
Other Rare Coagulation Disorders
Among the hereditary coagulation disorders, hemophilia inherited as an autosomal dominant or recessive pattern
is the most common in which one of the clotting factors has in patients with normal platelet counts which is due to an
either quantitative deficiency or absent or has qualitative abnormality, either of quantitative (Type 1 and Type 3)
(functional) abnormality, as a result of other medical and/or qualitative (Type 2)of the von Willebrand factor.4
conditions, it can also be acquired.
Pathophysiology
COMMON HEREDITARY COAGULATION
DISORDERS Bleeding occurs due to abnormalities in platelet adhesion
and aggregation, and decreased factor VIII levels, because
The three most common hereditary bleeding (coagulation) of decrease in quantity or a dysfunction of von Willebrand
disorders are: factor (vWF) in this disease.
1. Hemophilia A (factorVIII deficiency) Extremely large multimeric glycoprotein complex; low,
2. Hemophilia B (factor IX deficiency) intermediate, and high molecular weights of multimers are
3. von Willebrand disease.1 the constituents of von Willebrand factor. It is synthesized
Factors other than factor FVIII, FIX and von Willebrand in endothelial cells and megakaryocytes and after cleavage
factors are classified as rare coagulation factor disorders2 of a large propeptide, is released as a series of multimers,
apart from inherited deficiencies of coagulation. including ultralarge forms that are rapidly cleaved to a
Rare coagulation factor (deficiencies) disorders include: slightly small size.
• Fibrinogen
• FII
• FV (parahemophilia) Type of Hemostasis
• FV+FVIII Primary hemostasis: vWF in the subendothelium and
• FVII plasma bind to the platelet receptor glycoprotein Ib (GPIb)
• FX and to subendothelial structures, such as collagen, during
• FXI (Hemophilia C) normal hemostasis consequent to an injury, and serve as a
• FXIII bridge between platelets and subendothelium in damaged
Kashyap et al.3 reported a comprehensive study of 24 cases
vessels.
with rare coagulation disorders from India of which factor
Higher the molecular weight of the multimer, higher
X deficiency was found in 8 patients, 7 had factor XIII
would be the number of platelet-binding sites and
deficiency, fibrinogen and factor VII deficiency was found
adhesive properties. Each multimeric subunit has binding
in 4 cases each and factor V deficiency was found in 1 case.
sites for the receptor glycoprotein Ib on nonactivated
platelets and the receptor glycoprotein IIb/IIIa on
von WILLEBRAND DISEASE (VWD) activated platelets, this facilitates both platelet adhesion
In 1926, Erik von Willebrand first described a unique and platelet aggregation, making high molecular weight
bleeding disorder, in a 5-year-old girl from Finland, multimers most important for normal platelet function.
Chapter-29 von Willebrand Disease and Other Rare Coagulation Disorders 297
Secondary hemostasis: von Willebrand factor protects Other end of the spectrum when associated with
factor VIII from degradation in secondary hemostatis angiodysplasia vWD, serious gastrointestinal bleeding can
and delivers it to the site of injury. Binding enhances the occur.7 Abnormally heavy bleeding during menstruation
localization of platelets as fibrin is formed at the site of the (menorrhagia), blood loss during childbirth, hematuria,
injury promoting aggregation. hemarthroses, intramuscular, intracerebral and
The small multimers function mainly as carriers for FVIII: retroperitoneal hemorrhages, cephalhematomas in
The size of the multimers determines the binding of vWF. newborns (more common in Type 3 vWD). 8 Severe internal
Patient might have a bleeding diathesis in spite of a normal or joint bleeding is uncommon (except for in vWD type III).
concentration of vWF when there is a decrease in the more In between are the children who present with symptoms
functional large vWF multimers. Thrombus formation usually involving skin and mucosal bleeding like: Easy
as in thrombocytopenic purpura can happen when the bruising, excessive nose bleeds (epistaxis) bleeding
ultra large vWF forms that initially released are sticky from the gums, bleeding during procedures like tooth
and are capable of binding the platelets in the circulation extractions, tonsillectomy, menorrhagia, etc.
spontaneously.
Due to decrease in quantity or a dysfunction of von Classification
Willebrand factor (vWF) there will be abnormalities in
platelet adhesion and aggregation and decreased factor International Society on Thrombosis and Haemostasis’s
VIII levels, thus causing bleeding in this disease. The small (ISTH) classification.
multimers function mainly as carriers of FVIII. There are four types of hereditary vWD, described are
type 1, type 2, type 3 and pseudo or platelet-type.
• vWD Type I
Prevalence
• vWD Type II-4 subtypes-A, B, M, and N
The prevalence of clinically significant cases of vWF in • vWD III
humans are 1 per 10,000. It is detected more in women and • Pseudo or platelet-type
adolescent girls whose bleeding tendency shows during • Within the three inherited types of vWD there are
menstruation. Most forms of vWF are mild. The level of various subtypes
vWF varies depending upon the blood group, for instance • Platelet type vWD is also an inherited condition
people with type “O” are approximately 25% less affected • Most cases are hereditary, but acquired forms of vWD
than those of the other blood groups.5 vWF is reported have been described.
to affect animals including dogs (especially Doberman
Pinschers), rarely it affects wine, cattle, horses and cats. Type 1 vWD
• This is the most common type which accounts for
Inheritance
approximately 75–80% of patients and is characterized
In types I and II of von Willebrand disease is inherited in by quantitatively decreased and qualitatively and
an autosomal dominant pattern. In type III and sometimes structurally vWF
in type II it is inherited in autosomal recessive pattern. • Many patients are typically asymptomatic or may have
However penetrance may widely vary in a single family. mild to moderate bleeding
Also on different occasions even the clinical and laboratory • Often incidentally identified when other medical
findings may vary in the same patient.2 procedures requiring, a blood work-up is done.
CLINICAL PRESENTATION Laboratory Assays

Clinical symptoms have a wide spectrum. One end of the • Decrease in vWF activity and antigen levels
spectrum many children with von Willebrand disease • Decrease in the ristocetin-inducecl platelet aggregation
(vWD) are asymptomatic and are diagnosed as a result and vWF multimers show normal distribution, due
of a positive family history or during routine preoperative to lower vWF concentration their intensity may be
screening. vWD is of various types and each type is diminished
presented with varying degree of bleeding tendency. • Decreased binding of vWF to platelets factor VIII is
Due to its wide range and severity of symptoms, vWD decreased or in the low normal range
patients may present at any age. In majority of the cases • A normal multimer size distribution, though vWF
the bleeding is of mild to moderate severity reflecting the bands with abnormal migration may be present.
predominance of type 1 vWD.6 Individuals with decreased Abnormal multimers may be seen (Vicenza variant)
or qualitatively abnormal vWF function, usually present • Caused by a defect in the von Willebrand factor
earlier in life. Type 3 vWD is more serious in nature. gene that produces decreased or absent binding to
platelet glycoprotein 1b. The vWF gene is located vWF activity assays compared with antigen. There is
on chromosome twelve (12p13.2). It has 52 exons an absence of high-molecular weight multimers on
spanning 178 kb agarose gels
• By performing a binding assay for factor VIII that uses • Decreased RIPA
the patient’s vWF as the binding partner, the diagnosis • The factor VIII may be normal or decreased.
is established.
TYPE 2B von WILLEBRAND DISEASE
von Willebrand Disease Type 2 • Type 2B accounts for approximately 5% of vWD
Type 2 vWBD is associated with primarily qualitative • Inherited either autosomal dominant or autosomal
defects of von Willebrand protein and is most common. Its recessive pattern
incidence is 20–30% and vWBF levels are normal. Bleeding • The removal of platelets aggregates with bound vWF
tendency can vary between individuals as it is a qualitative may result in hemostatic defect caused by qualitatively
defect, however subgroups of large or small multimers abnormal vWF and intermittent thrombocytopenia.
may be absent or structurally abnormal multimers are The ability of the qualitatively defective von Willebrand
present. factor to bind to glycoprotein 1 (GP1) receptor on the
platelet membrane is abnormally enhanced, leading
Type 2A von Willebrand Disease Includes Four to its spontaneous binding to platelets and subsequent
Subtypes (A, B, M and N) rapid clearance of the bound platelets and of the
large vWF multimers. From the circulation, large vWF
• Type 2A. In type 2A vWD the qualitatively defective multimers are reduced or absent.
vWF’s ability to bind to glycoprotein 1 receptor on the
platelet membrane is enhanced abnormally, leading Laboratory Assays
to spontaneous binding to platelets and rapid and
subsequent clearance of the bound platelets and • Low concentrations of ristocetin in ristocetin-induced
of the large vWF multimers. There are chances for platelet (RIPA) show an increased reactivity, similar to
thrombocytopenia. vWF antigen assay is normal but type 2A, vWF ristocetin cofactor activity shows marked
qualitatively defective and decreased capability at decrease than in antigen level and high molecular
multimerization. Large vWF multimers are reduced or weight multimers of vWF are decreased
absent from the circulation • Thrombocytopenia may be present in some patients.
• Large vWF multimers are reduced or absent and Low or normal factor VIII
ristocetin cofactor activity is low. Substitution within a • Bleeding symptoms may be moderate to severe
normal cleavage site in the A2 domain of vWF is the • Platelet-related vWF function is normal
effect of the majority of mutations in 2A. Some make
it more susceptible to proteolysis by the vWF cleaving Type 2M von Willebrand Disease
protease (ADAMTS13)
Type 2M is very rare type of vWF and is a qualitative
• The mutations in type 2A vWD may either cause a defect
defect. In this type of vWD there is normal capability
in intracellular transport (2A, type 1) or render the
at multimerization and a decreased ability to bind to
molecule more susceptible to proteolysis (2A, type 2).
glycoprotein (GP1) receptor on the platelet membrane.
This is characterized by a decreased platelet-directed
Laboratory Findings function which is not because of the decrease of high-
• Its ability to bind to the glycoprotein 1 (GP1) receptor molecular weight multimers. Though vWF bands with
on the platelet membrane is reduced. This results abnormal migration may be present and abnormally large
in abnormally low ristocetin cofactor activity and multimers may be seen, there would be a normal multimer
decreased platelet adhesiveness and aggression. The size distribution (Vicenza variant).
defective vWFs ability to coalesce and form large Caused by a defect in the von Willebrand factor gene
vWF multimers is also impaired which would lead that produces decreased or absent binding to platelet
to decreased quantity of large vWF multimers and glycoprotein 1b characterized by its decreased ability
detected in the circulation are only small multimer to bind to glycoprotein 1 (GP1) receptor on the platelet
units membrane and normal capability at multimerization.
• Because of the loss of high molecular-weight multimers This results from mutations affecting the A1 domain in a
which are more functional there is a decrease in different area from those mutations in type 2B.
Decreased ristocetin cofactor activity and decreased Platelet-type vWD (pseudo-vWD)6

vWF antigen and activity, and high molecular weight large
vWF multimers are present in the circulation. • Pseudo-vWD is another name of platelet type vWD
Laboratory studies show that there is normal vWF • It is a genetic defect of the platelets which is autosomal
function and antigen, and RIPA and multimer distribution, dominant
however shows decreased factor VIII which is between 2% • The vWF is qualitatively normal
and 10%. • No mutational alteration is revealed while genetically
testing the von Willebrand gene
• Thrombocytopenia, and diminishing or absence of
Type 2N von Willebrand Disease large vWF multimers result as large platelet aggregates
• The cause of type 2N vWD is by mutations in the amino and high molecular weight vWF multimers are
terminus of the mature vWF monomer and this is an removed from the circulation. The ristocetin cofactor
uncommon variant. This may be initially confused with activity and loss of large vWF multimers are similar
hemophilia A as patients may have mild to moderate to vWD type 2B and can be distinguished from type
bleeding which is related to the decreased factor VIII. 2B vWD by mixing studies with patient platelets and
The presence of affected females in the family is a vital normal plasma using RIPA
indication that this diagnosis should be considered • Because of the genetic mutation in factor VIII binding
• Decrease binding for factor VIII result in rapid clearance region of vWF it is characterized by a marked decrease
of factor VIII. This is a deficiency of the binding of vWF affinity of vWF for FVIII. Bleeding is related to the
to coagulation factor VIII. Soft tissue and joint bleeding decreased factor VIII. Symptoms include as soft tissue
are apparent symptoms, as expected with decreased and joint bleeding and with decreased factor VIII
factor VIII may initially be confused with hemophilia A. This is
• Normal platelet-related vWF function suspected when patients with mild FVIII deficiency and
• Laboratory studies show decreased factor VIII (2–10%) a bleeding disorder which is not clearly transmitted as
and normal vWF function and antigen, RIPA and X-linked disorder or when patients do not completely
multimer distribution respond to hemophilia.
• Performing a binding assay for factor VIII that uses the A significant indication is the presence of affected females
patient’s vWF as the binding partner is the diagnosis to in the family. It is usually presented before adulthood with
establish type 2N vWD. symptoms such as moderate to severe bleeding in affected
patients.
Type 3 von Willebrand Disease
Laboratory Findings
• Type 3 vWD is characterized by complete absence of
production of vWF and it is the most severe and rare Due to the loss of the more functional high molecular
form of von Willebrand disease. Manner of inheritance weight multimers, laboratory testing shows more marked
of type 3 vWD is homozygous or doubly heterozygous. decrease in vWF activity assays as compared with antigen.
Consanguinity is common. A variety of mutations, • Decreased RIPA
including larger deletions are the causes of type 3 vWD • High molecular weight multimers on agarose gels are
• From proteolytic degradation, the vWF protects absent
coagulation factor VIII, extremely low factor VIII level • Normal-to-reduced plasma levels of factor VIIIc and
is caused due to total absence of vWF, severe clinical vWF (in moderate/severe disease it is abnormal).
bleeding which is similar to severe hemophilia, e.g.
hemarthrosis, intramuscular bleeding, etc., would be DIAGNOSIS OF von WILLEBRAND DISEASE
the clinical presentation
• It is identified by marked deficiencies of both FVIIIc Accurate Personal and Family Bleeding
in the plasma and vWF, the absence of vWF from both
History
endothelial cells and platelets and lack of response to
DDAVP, therefore platelets cannot clot. It is difficult to • Increased or easy bruising and bleeding from wound
stop bleeding in patients with type 3 vWD as bleeding is • Gingival bleeding and recurrent epistaxis
severe. Patients may present with cephalohematomas in • Menorrhagia, postpartum bleeding
newborns, hemarthroses, hematuria and intramuscular, • Postoperative bleeding (particularly after tonsillectomy
intracerebral and retroperitoneal hemorrhages. or dental extractions):
Clinical Evaluation Genetic Testing–Mutation in the Patient’s

vWF
Laboratory Assays Tests for vWD Include
The gene defect in the majority of type 1 patients is
• Routine screening test for coagulation disorders like unknown. Specific gene defect in type 2 and type 3 vWD
• CBC, platelet count patients is available in specialized laboratories and
• Bleeding time: Bleeding time tests are not sensitive and research centers.
are not done as often as they once were
Specialized laboratories and research centers are
• Prothrombin time, APTT: Characteristically there is
available where genetic testing for diagnosis of specific
marked prolongation of the PTT, PT and the thrombin
gene defect in type 2 and type 3 vWD patients are available.
time (TT)
Direct sequencing of the suspect area of the patient’s gene
• vWF antigen level: vWF antigen (vWF Ag) is a
quantitative test that is usually carried out in an ELISA is done to identify the specific defect. Usually genetic
format using antibodies specific for vWF testing is not usually performed in type 1 patients as it is
• vWF multimer distribution by gel assays and RIPA: This unknown at that time.
occurs in type 2B vWD
• The patient’s plasma which is the source of vWF and Medical Care
platelets are used in RIPA. To assess whether the
Treatment for von Willebrand disease depends on type
platelet aggregation is present or absent, different
and severity of the disorder. vWD, the patient has dual
concentrations of ristocetin are added to aliquots of
defect of hemostasis, i.e.
the platelet-rich plasma of the patient. Aggregation will
cause in patients with type 2B vWD if, concentrations of • Defect in platelet adhesiveness and aggregation which
ristocetin is approximately below 0.6 mg/mL, but will can be corrected by raising the level of von Willebrand
not cause aggregation in normal subjects. The “gain of factor
function” can be assessed primarily through RIPA • Low factor VIII activity.
• Ristocetin-induced platelet aggregation (RIPA), collagen
binding: Ristocetin is the antibiotic that promotes the Treatment
binding of vWF to platelets. In the presence of ristocetin
The three major treatment modalities used for patients
the vWF have the functional ability to bind to platelets.
with vWD is detailed in Table 16
If concentration of ristocetin is approximately below
• Desmopressin acetate (DDAVP)
0.6 mg/mL, it will cause aggregation in patients with
type 2 vWD. “Gain of function” mutation in patient’s • Replacement therapy with plasma-derived factor VIII
vWF is primarily assessed through RIPA. vWF concentrates
• Adjunctive therapies such as antifibrinolytic agents
and topical therapies.
Factor VIII Activity
Specific treatment is not required if there is minor
In moderate and severe disease factor VIII would be bleeding problems, such as bruising or a brief nose bleed,
abnormal, otherwise it would be normal or decreased. etc., in patients with vWD. The main aim in case of serious
Factor VIII activity is usually performed in the traditional bleeding is to limit the patient’s bleeding, by medications
coagulation factor assay. that can raise the vWF level.
• vWF multimer study and ristocetin—Additional tests
useful in classifying the type of vWD include–ristocetin Desmopressin Acetate (DDAVP)
induced platelet aggregation (RIPA) performed in a
platelet function analyzer. In this test, the patient’s Desmopressin is a synthetic analog of antidiuretic
own platelets and vWF are employed, so the test is not hormone. It is considered that for patients with mild
specific for vWF abnormalities.9 vWD, the primary treatment and mainstay of therapy
• To assess whether high molecular weight multimers is desmopressin [1-deamin-8-d-arginine vasopressin
are decreased or absent, vWF multimer gels are used (DDAVP)]. It stimulates the release of vWF from the
as they provide visualization of the size distribution of Weibel Palade bodies of endothelial cells (storage site). In
vWF multimers in plasma type 1 vWD patients who have normal vWF in storage sites
• vWF is visualized using antibodies to vWF and DDAVP (desmopressin acetate) is most effective.
immunofluorescence end point by performing It may not be effective in vWD type 2M and is hardly
electrophoresis on diluted plasma in agarose gels and effective in vWD type 2N and in severe forms of vWD 1 and
the proteins are transferred to a membrane.10 2. It is totally ineffective in vWD type 3.
Table 1 Classification and treatment of von Willebrand disease

Type Description vWF activity Ag RIPA Multimer pattern Treatment
Type 1 Partial quantitative deficiency ↓ ↓ ↓ Uniform ↓ DDAVP 0.3 μg/kg IV in 50 mL
of vWF saline over 20 minutes, or
nasal spray 300 μg for weight
>50 kg or 150 μg for <50 kg.
Replacement vWF concentrate
at 20–30 IU/kg q12h
Type 2 Qualitative vWF defect
2A Decreased vWF-dependent ↓↓ ↓ ↓ ↓ Large and DDAVP as in type 1.
platelet adhesion with intermediate Replacement vWF concentrate
selective deficiency of high
molecular weight
multimers
2B Increased affinity for platelet ↓↓ ↓ ↑ ↓ Large Possibly DDAVP (may worsen
GPIb thrombocytopenia) as in type 1.
Replacement vWF concentrate
2M Decreased vWF-dependent ↓ ↓ ↓ Normal DDAVP as in type 1.
platelet adhesion Replacement vWF concentrate
without selective deficiency of
high molecular weight
multimers
2N Markedly decreased binding N N N Normal DDAVP as in type 1.
affinity for FVIII Replacement vWF concentrate
Type 3 Virtually complete deficiency ↓↓↓ ↓↓↓ ↓↓↓ Undetectable Replacement vWF concentrates
of vWF platelet transfusions if
inadequate response to vWF
replacement
Abbreviations: DDAVP: Desmopressin acetate; N: Normal; IV: Intravenous; RIPA: Ristocetin-induced platelet aggregation; vWD: von Willebrand disease;
vWF: von Willebrand factor.
DDAVP indirectly causes release of vWF and factor of the bleeding episode, through intranasal dosing the
VIII from storage sites, increasing the levels of both factors patient can have rapid access to medication at home. It is
2- to 5-fold within 45 minutes following intravenous recommended that DDAVP be administered at 8–12 hours
administration; the effect usually lasts about 6 hours. interval for 2–3 doses and then at intervals of 48 hours in
case of tachyphylaxis and serious hyponatremia, where
Route and Mode of Administration and Dose bleeding can occur even after repeated doses.
Dosage: Fixed doses of 300 μg in adults and 150 μg in
Administration of DDAVP can be either intravenously children by intranasal spray.11
or intranasaly or subcutaneously. If administered slowly
through intravenous infusion or subcutaneous mode, the Common Side Effects Include
recommended dosage is 0.3 μg/kg.
Frequent side effects of intranasal spray include transient
headache, facial flushing and mild tachycardia, but are
Intranasal Preparation usually well-tolerated by patients. More often in infants
If DDAVP is administered through intranasal mode in and in young children severe symptoms such as cerebral
the form of nasal spray the levels peak approximately 2 edema and seizures are reported as a result of water
hours after intranasal delivery.12 A high concentration intoxication. Water retention and dilutional hyponatremia
preparation (i.e. stimate 1.5 mg/mL) available and this with consequent convulsion can occur due to over use of
allows home treatment for bleeding symptoms. At the start DDAVP.
Type 2B patients are at risk for worsening can be used as prophylaxis to treat patients with vWD
thrombocytopenia after DDAVP and in type 2B patients, who do not respond to DDAVP. It can also be used for
platelet count should be evaluated along with vWF levels. patients with vWD scheduled for surgery, patients with
rare types 2B or 3. vWD and cases of vWD complicated
vWF Concentrates by clinically significant hemorrhage
• However, most available FVIII concentrates do not
When there is more severe bleeding and could not
contain sufficient von Willebrand factor to be used
controlled by DDAVP then vWF concentrates should be
in von Willebrand disease e.g. Humate-P, Alphanate,
used. They are also given prophylactically and following
Wilate Alphanate and Koate also contain vWF in
surgery or trauma for 2–14 days, as dictated by the clinical
high molecular weight form. These concentrates
situation.
are especially useful in types 2B and 3 vWD and are
Intermediate-purity plasma-derived factor VIII
available commercially for prophylaxis and treatment
concentrates, administered intravenous mode in an
interval time of approximately 12 hours, contains vWF of vWD. Insignificant quantity of vWF is present in
(not recombinant or monoclonally purified factor VIII monoclonally purified factor VIII concentrates and
concentrates). recombinant factor VIII, hence are not clinically useful.
In 10–15% of patients receiving human derived
medium purity factor VIII concentrates development of
Cryoprecipitate
alloantibodies occur. Other side effects include allergic
Due to lack of viral inactivation, this product is generally reactions including anaphylaxis and increased risk of
not recommended. venous thromboembolic complications.
Nonreplacement Therapy
Cryoprecipitate Contains Multimeric von
Aminocaproic acid and tranexamic are both drugs Willebrand Factor
that steady the clots formed by platelet by preventing
fibrinolysis. The antifibrinolytic agents epsilon amino In general, the dosage of cryoprecipitate or FVIII to
caproic acid and tranexamic acid are useful adjuncts in be used is calculated on the basis of FVIII units. Other
the management of vWD complicated by gum bleeding, blood products are rarely required for patients with von
bleeding from mucous membrane, menorrhagia, etc. Willebrand disease.
Common side effects include nausea, vomiting, and clot Type 3 vWD patients or platelet-type vWD who do not
complications. respond to vWF containing concentrates or cryoprecipitate
Estrogen-containing oral contraceptive medications may be benefitted by platelet transfusion.
are effective in reducing the frequency and duration of Other supportive line of treatments includes blood
the menstrual periods for women with heavy menstrual transfusions for hypotension secondary to hypovolemia to
bleeding. Estrogen compounds available for use in the correct anemia. For correction of hemorrhage associated
correction of menorrhagia are: with platelet type vWD, infusion of platelet concentrates is
recommended.
Ethinyl Estradiol and Levonorgestel (Levona,
Nordette, Lutera, Trivora) Adjunctive Therapies
Antifibrinolytic agents such as epsilon amino caproic acid
Stabilization of the endometrial surface of the uterus is
and topical agents such as topical thrombin, gelfoam, and
done by administration of ethinyl estradiol which in turn
fibrin sealant are used as adjunctive therapies.
diminishes the secretion of luteinizing hormone and
For dental procedures, epsilon amino caproic acid
follicle stimulating hormone from the pituitary.
may be helpful.
Use of Topical Thrombin
Acquired von Willebrand Disease
Are effective adjuncts for correction of hemorrhage from
Acquired von Willebrand syndrome (AvWS) is a rare
wounds.
bleeding disorder and is distinguished from the congenital
form by several factors such as age at presentation, absence
Replacement Therapy Plasma Products of personal and family history of bleeding disorders.13
• Plasma derived-derived factor VIII (FVIII) concentrates Acquired vWD can occur in patients with
• Human derived medium purity factor VIII autoantibodies. Antibodies, binding mechanism
concentrates, contain von Willebrand factors. This responsible for decreased vWF, proteolysis, decreased
production of vWF are the mechanisms responsible factors like fibrinogen, FII, FV, FV+FVIII, FVII, FX, FXI,
for decreased vWF.14 There is a rapid clearance of vWF FXIII.
antibody complex from the circulation.
FIBRINOGEN DEFICIENCIES (F1-5)
Often Associated with Underlying Diseases
Like Fibrinogen deficiencies are inherited disorders of
fibrinogen defect.2 They may be classified as:
• Lymphoproliferative (48%)
• Myeloproliferative disorders (15%) Quantitative Defect
• Neoplasia (5%)
• Immunological (2%) • Hypofibrinogenemia, with fibrinogen levels lower than
• Cardiovascular (21%), aortic valve stenosis, left 1.5 g/L
ventricular assist device (LVAD) • Afibrinogenemia, characterized by the complete
• Miscellaneous disorders including hypothyroidism deficiency of fibrinogen.
(9%), Wilms’ tumor and mesenchymal dysplasias.
Qualitative Defect of the Circulating
Laboratory Findings Fibrinogen
Laboratory features are the same as in congenital von • Dysfibrinogenemia
Willebrand disease. Only the measurement of vWF or
propeptide (also known as vWF Ag II) has been suggested • Both hypo/dysfibrinogenemia
as helpful to discriminate between congenital and Congenital fibrinogen disorders are relatively rare.
acquired vWD. In AvWS, the propeptide levels remain
normal because vWF synthesis is normal or higher and it Clinical Features
is not targeted by antibody and not consumed or bound.
In less than one-third of the cases, antibodies are found • Symptoms in afibrinogenemia are not as severe as
and are difficult to demonstrate in the laboratory. those seen in classic hemophilic disorders
• Congenital afibrinogenemia is an autosomal recessive
disorder. Hereditary dysfibrinogenemia are usual
Treatment autosomal dominant
Treatment of vWS has two main objectives: • It may manifest in the neonatal period with
1. To control the bleeding episode. gastrointestinal hemorrhage or hematoma
2. To treat the underlying associated disease. (cephalhematoma) after normal vaginal delivery
Initial treatment is usually administration of • Thrombosis is another cause of concern. Patients with
DDAVP, however, if the response is not adequate, either congenital fibrinogen disorders may paradoxically
replacement therapy (FVIII/vWF concentrates) or suffer from severe thrombotic episodes. The
intravenous immunoglobulin (IVIg) is used.14 Despite the etiopathogenesis is poorly understood except for a
possible presence of antibodies to vWF, the response to few cases having a severe thrombophilic disorder
replacement therapy is usually satisfactory. concomitantly.15
• The clinical complication which is common in case of
Drugs to Avoid fibrinogen deficiency is pregnancy loss.
As aspirin and nonsteroidal anti-inflamattory drugs can
increase bleeding complications, these drugs are to be DIAGNOSIS16,17
avoided. These children must inform about their health • There is marked prolongation of the PT, PTT and the
problems to health providers, including their dentists of thrombin time (TT)
their condition as well as teachers in the school, family • Fibrinogen is the ligand for the glycoprotein IIb-
members and close friends. IIIa receptor which enables platelet aggregation.
In fibrinogen deficiency, the bleeding time and the
Rare Coagulation Disorders platelet aggregation tests are abnormal
Rare coagulation disorders due to deficiencies of • The best screening tests are the TT and the reptilase
coagulation factors other than factor FVIII and FIX and time, which measures the time required for the
von Willebrand disease, include deficiency of coagulation conversion of fibrinogen in plasma to a fibrin clot.
Unlike the TT, the reptilase time is unaffected by Clinical Phenotypes

heparin treatment
• The bleeding phenotype is difficult to predict even by the Two clinical phenotypes are recognized.
characterization of the molecular defects responsible 1. Hypoprothrombinemia (type I deficiency), in which
for afibrinogenemia or hypofibrinogenemia prothrombin antigen and activity levels are reduced
• Congenital fibrinogen disorder patients may concomitantly.
paradoxically suffer from severe thrombotic episodes, 2. Dysprothrombinemia (type II deficiency), in which
at times independent of any fibrinogen substitution. prothrombin activity is reduced but antigen levels are
Few cases having a severe thrombophilic disorder normal.
concomitantly.
Clinical Features
Management • Hemarthrosis and muscle hematomas are most
During bleeding due to congenital fibrinogen deficiency, frequent bleeding manifestations in this group of
fibrinogen levels should be increased and maintained patients
above 1.0 g/L until hemostasis is secured and it should • Intracranial bleeds and umbilical bleeding have been
be maintained above 0.5 g/L until wound healing is reported in neonates
complete.18,19 A dose of 50 mg/kg is required to increase • Postoperative bleeding and mucosal bleeding are
the fibrinogen concentration to 1 g/L. other manifestations.
Fresh Frozen Plasma or Cryoprecipitate Diagnosis

• The plasma half-life of fibrinogen is between 2 days and • High index of suspicion and family history helps in
4 days and the availability of fibrinogen concentrates is early diagnosis. Prolonged PT and a normal TT is
rare. Treatment with either fresh frozen plasma (FFP) or diagnostic criteria although both PT and APTT may
cryoprecipitate is effective. Each bag of cryoprecipitate be prolonged in FII deficiency. It should also be noted
contains 100–150 mg of fibrinogen. Efficiency of viral that the degree of abnormality may be minimal and
inactivation process is not that effective as it is for results can be within the normal range
fibrinogen concentrates. Transfusion-related acute • A specific FII assay confirms the diagnosis
lung injury or TRALI complication can be caused due • In premature and young neonates where vitamin K
to cytotoxic antibodies contained in the infused plasma deficiency may complicate assessment, the diagnosis of
• To treat mucosal bleeding and to prevent bleeding mild prothrombin deficiency is difficult. Reassessment
following procedures, e.g. dental extraction, after vitamin K replacement may be necessary.
antifibrinolytic agents may be given
• Agents such as tranexamic acid should also be Management22,23
considered There are no specific prothrombin concentrates available.
• For treating superficial wounds or wounds following
dental extraction fibrin glue will be useful Prothrombin Complex Concentrates are
• For selected patients gene therapy could be the future.20 therefore Treatment of Choice
• As a basis for dosage usually approximately 1 unit of
FACTOR II—PROTHROMBIN DEFICIENCY prothrombin per unit of FIX and can be used. Doses
of 20–30 IU/kg seem to be effective as relatively low
Factor II (FII) deficiency also called hypoprothrombinemia levels are required for normal hemostasis. The plasma
or prothrombin deficiency and is a rarest coagulation prothrombin level is estimated to rise by 1 IU/dL with
disorder first, identified in 1947 by Dr Armand Quick. one unit of prothrombin
Prevalence of 1:2,000,000 in the general population.21 • An alternative source of prothrombin is fresh frozen
The mode of inheritance is autosomal recessive. At plasma (FFP). Around 72 hours is the half-life period
least 32 different mutations have been identified. of prothrombin. This eases comparatively occasional
FXa activates prothrombin on the surface of platelets dosing, usually every 2–3 days. Depending on the
in the presence of FV and calcium. frequency and type of bleeding, prohylaxis should be
used in older children. To prevent the development • A potential complication of hereditary FV deficiency is
of chronic arthropathy, prophylaxis should be used in the development of alloantibodies to FV in FFP
cases where recurrent joint bleeding is a feature. • It is suggested that low-level inhibitors can be used to
neutralize large amounts of FFP in case of bleeding
FACTOR V DEFICIENCY (PARAHEMOPHILIA) problem27
• Immunoglobulin administered intravenous may be
In 1943, a Norwegian patient was found to have factor V effective in eliminating FV inhibitor.28
deficiency and was reported by Dr Paul Owren in 1947.
Factor 5 deficiency is also called as parahemophilia,
COMBINED DEFICIENCY OF FACTORS
Owren’s disease, labile factor deficiency and proaccelerin
deficiency. V AND VIII
Clotting factor: FV is a large glycoprotein of molecular In 1954, Oeri et al. first described combined FV and FVIII
weight 249 kDa, is synthesized by hepatocytes and deficiency, which is a rare autosomal recessive disorder.
megakaryocytes. Encoded by a gene on chromosome 1, History of consanguinity present. It is likely to be due to a
hereditary FV deficiency is a very rare autosomal recessive single gene defect (located on the long arm of chromosome
condition. The prevalence of the homozygous state is 18), leading to deficiency of a transport protein, rather
approximately 1 per million. than due to coinheritance of separate defects of the FV and
FV is activated by thrombin and the resulting FVIII genes.
heterodimer FVa acts as a cofactor for FXa in the conversion Affected individuals have reduced plasma levels of
of prothrombin to thrombin. both FV and FVIII.29
Shetty et al. from India reported nine patients from five
Clinical Features unrelated families of combined FV and FVIII deficiency,
youngest being an 8-year-old girl.20
Homozygous deficiency is associated with a moderately
severe bleeding disorder with easy bruising and mucous
membrane bleeding, epistaxis and oral cavity bleeding24, Clinical Features
postoperative, postdental extraction and postpartum Mild bleeding symptoms, such as easy bruising and
bleeding, etc. Hemarthroses and muscle hematomas are epistaxis are present. In 9 patients from India, the most
often related to trauma rather than being spontaneous. common manifestations observed were prolonged
Gastrointestinal bleeding and hematuria may occur bleeding from cuts, easy bruisability, bleeding gums
rarely. Intracranial bleeding especially in the antenatal and postdental extraction bleeding. Following a dental
and neonatal periods have been reported.25 extraction or a surgery, bleeding is common phenomena.
In affected woman menorrhagia and postpartum
Diagnosis hemorrhage is seen.
Factor V deficiency is characterized by prolongation of
both the PT and APTT but a normal TT. By mixing with Diagnosis
normal plasma, both PT and APTT are corrected. By The combined deficiency disorder is associated with
FV assay or by immunological assessment of FV levels, a prolongation of both the PT and APTT, with the APTT
deficiency of FV is confirmed. FV assay has to be performed prolongation disproportionate to that of the PT. By using
on individuals with reduced FV levels to exclude combined normal plasma, both the test times are corrected.
FV and FVIII deficiency. APTT-based activity assays and antigen assays reveal
levels of between 5 IU/dL and 20 IU/dL for both FV and
Management FVIII.
• There is no FV concentrate available
• FV replacement is done in patients presenting with a Management
bleeding episode by administering a dose of 15–20 mL/ • Both FVIII concentrates and FFP are to be used
kg of FFP for treating patients with combined FV and FVIII
• Use of agents such as tranexamic acid should also be deficiency who have spontaneous bleeding episodes.
considered (as a source of FV)
• In patients who are not responding to FPP, use of • FVIII levels should be raised to at least 30 IU/dL for
recombinant activated factor VII (rFVIIa) should also minor bleeding episodes and for more severe bleeding
be considered26 atleast 50 IU/dL with rFVIII concentrate
FFP should be administered for patients with FV happen if the blood samples collected for the determina
deficiency, in order to increase the FV level to at least 25 tion of FVII:C are stored at 4°C. An enzyme-linked
U/dL. immunosorbent (ELISA) assay or immunoradiometric
Rather than intramuscular vitamin K, affected babies assay (IRMA) assay and monoclonal or polyclonal
should receive oral vitamin K.18 antibodies are used frequently to measure FVII antigen
There is no indication for routine prophylaxis with (FVII:Ag). Such assays can detect as little as 0.01 IU/dL
plasma and FVIII. Neonatal intracranial hemorrhage has of FVII. Functional FVII assay should be preferred over
not been described in this condition. immunological assay. Due to the low physiological levels
of the neonate, diagnosis of factor VII deficiency may be
FACTOR VII DEFICIENCY difficult in neonates. Age- and gestation-related reference
ranges must be used in such cases for this reason.
Also known as proconvertin, deficiency, Alexander’s
disease.
Management
Among the rare inherited coagulation disorders
the most common is factor VII deficiency. Factor VII Current therapeutic options to manage patients with FVII
is a vitamin K-dependent glycoprotein with a MW of deficiency include fibrinolytic inhibitors (tranexamic
approximately 50 kDa. acid), plasma, intermediate purity FIX concentrates
FVII deficiency is inherited in an autosomal recessive (prothrombin complex concentrates), FVII concentrates
manner. and recombinant factor VIIa (rFVIIa).
Estimated prevalence of FVII deficiency 1:300000– Plasma FVII has a short in vivo half-life of approximately
1:500000. First recognized in 1951, circulates in plasma 5 hours; plasma infusions may not achieve adequate levels
in two forms—the majority in a single chain inactive form for normal hemostasis. With levels of FVII:C in the range
with a concentration of 10 nmoles/L (0.5 μg/mL) and a of 10–15 IU/dL, efficient hemostasis can be achieved.
much smaller amount (approximately 10–110 pmoles/L For patients requiring replacement therapy due to FVII
as the active two-chain form. deficiency, rFVIIa is recommended.22
Clinical Features FACTOR X DEFICIENCY

• Common manifestations include; epistaxis, gum It is an autosomal recessive disorder which is also
bleeding, menorrhagia and other mucous membrane- called severe (homozygous). In general population its
type bleeding.30 Increased risk for developing incidence is 1:1,000,000. Factor X or Stuart-Prower factor,
intracranial hemorrhage is reported in neonates who deficiency was first identified in the 1950s in the US and
are diagnosed with factor VII deficiency England in two patients: Rufus Stuart and Audrey Prower.
• Although it is not a consistent finding joint bleeds are Kumar et al. from India reported three pediatric cases
reported in some patients with severe FVII deficiency with FX deficiency and two out of them were product of
• Bleeding into the central nervous system is common in consanguineous marriage.32
patients with severe FVII deficiency (FVII:C <2 IU/dL), In the coagulation cascade, factor X occupies a unique
and is reported to be between 15% and 60% position, as the first enzyme in the common pathway of
• Although the mechanism is unclear, thrombosis in thrombus formation. Following secretion into plasma, FX
association with FVII deficiency is also reported.31 synthesis occurs in the liver, at a concentration of 10 μg/
mL.
Diagnosis Either a quantitative deficiency or a dysfunctional
molecule can be the cause of this deficiency. Systemic
Characteristic features of factor VII deficiency is finding amyloidosis, although rare in children, may be associated
of a prolonged PT, which corrects, unless an inhibitor is with factor X deficiency owing to the adsorption of factor X
present, in a 50:50 mix with normal plasma. on the amyloid protein.
FI concentration, APTT and TT are found to be normal.
Before making the diagnosis of FVII deficiency, it is critical
Clinical Features
to exclude vitamin K deficiency or any other clotting
disorder which is acquired. FX deficiency may present at any age in individuals. With
A therapeutic trial of vitamin K may be of value. umbilical stump bleeding, factor X deficiency may be
Using a one-stage PT-based assay, the functional present in the neonatal period too. Easy bruising may be
FVII activity (FVII:C) is measured. Cold activation of FVII experienced in patients who are mildly affected by factor
and substantial overestimation of the true FVII level can X deficiency. Epistaxis is the most frequent symptom
in patients with FX deficiency and other mucosal-type or by autoactivation. FXIa promotes coagulation by
bleeding is less frequent. Women of reproductive age may activating factor IX (FIX).
present with menorrhagia. Rarely reported presentations This disorder is divided into two categories:
include central nervous system hemorrhage, hemarthroses 1. Type I deficiency, which corresponds with low activity
and severe postoperative hemorrhage. Severe arthropathy and levels of FXI antigen.
may be due to recurrent hemarthroses. 2. Type II deficiency, with low activity and normal FXI
Bleeding only after hemostatic challenge is seen antigen levels.35
in moderately affected patients (FX:C 1–5 IU/dL), for FXI deficiency has been described in all racial groups.36
example, trauma or surgery. Gene defect is seen in the F11 gene, located on the
During routine screening or family studies, mild long arm of chromosome 4 [4q35]. This gene encodes
FX deficiency (FX:C 6–10 IU/dL) may be identified an 18-amino acid signal peptide and a 607-amino acid
incidentally. mature protein. More than 100 mutations causing FXI
deficiency have been reported which are distributed all
Diagnosis over the gene.37
Following the finding of a prolonged PT and APTT, which
corrects in a 50:50 mix with normal plasma, the diagnosis Clinical Features
of FX deficiency is suspected. By measuring the plasma
FX levels, the FX deficiency diagnosis is confirmed. the It can affect both men and women and is associated with
one-stage PT- and APTT-based assays, a chromogenic mild to moderate bleeding, especially after trauma or
assay, an assay employing Russell viper venom (RVV) surgery.
and an immunological assay are the five different assays There are no reports of presentation of spontaneous
available for measuring plasma FX levels. However, for the bleeding in the neonatal period. No instances of neonatal
diagnosis of FX deficiency, one-stage PT- or APTT-based intracranial hemorrhage resulting from FXI deficiency
assay are sufficient. Before the diagnosis of FX deficiency have been reported. Epistaxis, soft tissue hemorrhage,
is made, it is crucial to exclude other deficiencies such as and bleeding after dental extraction may occur, but
vitamin K and other acquired causes of a clotting disorder. hemarthroses and concomitant arthropathy are not seen.
However, a therapeutic trial of vitamin K may be of value.
Diagnosis
Management
The APPT is prolonged in factor XI deficiency, whereas the
Management of patients with FX deficiency includes PT is normal.
fibrinolytic inhibitors, plasma and intermediate purity
FIX concentrates (prothrombin complex concentrates).
To treat acquired FX deficiency secondary to amyloidosis Management
rVIIa is used.33 Even in the immediate postoperative Minor surgeries can be controlled with local pressure;
period, for hemostasis, factor levels of 10–20 IU/dL are dental extraction can be monitored closely and the patient
generally sufficient. For management of the acute bleed treated only if hemorrhage occurs. Fibrinolytic inhibitors
and the treatment of choice is prothrombin complex (Tranexamic acid-15 mg/kg, 8 hourly) are useful. The IV
concentrates. The biological half-life of FX is 20–40 hour, preparation is given orally in this situation although this is
so infusion of approximately 20 mL of FFP per kg of body not a licensed use of the product.18
weight followed by 6 mL/kg every 12 hours increases Plasma infusion of 1 mL/kg body weight can increase
the level FX sufficiently to achieve hemostasis for minor the circulating factor by about 1.5 U/dL.
bleeding episodes.34 A loading dose of 15–20 mL of plasma/kg will result in
plasma level of 20–30 U/dL, a level that is usually sufficient
FACTOR XI DEFICIENCY (HEMOPHILIA C) to control moderate hemorrhage. The half-life of FXI is 48
Factor XI (FXI) deficiency or hemophilia C was described hours or greater.
for the first time in 1953 in a Jewish family in the United
States by Dr Rosenthal. It is particularly common in FACTOR XIII DEFICIENCY (FIBRIN
Ashkenazi Jews in whom the heterozygote frequency is 8 STABILIZING FACTOR DEFICIENCY)
percent. Factor XI deficiency is generally transmitted as
an autosomal recessive trait, and both sexes are affected; Inherited factor XIII (FXIII) deficiency is an autosomal
however, cases of dominant transmission have also been recessive bleeding disorder, mostly because of defects in
reported. FXI can be activated to FXIa by FXIIa, thrombin, the FXIII-A gene resulting in FXIII-A deficiency.38
Jayandharan et al. reported nine mutations in dilute monochloroacetic acid or acetic acid. To determine
coagulation factor XIII A gene in eight unrelated Indians FXIII activity quantitatively, measuring the incorporation
and five out of them were novel.39 of fluorescent or radioactive amines into proteins is
adopted. Specific ELISA tests are required to assess FXIII-A
Clinical Manifestations and FXIII-B antigen lvels.
Factor XIII deficiency is charecterized by delayed

Management
hemorrhage. Patients develop a bruise or hematoma
after delay of some time interval of injury. Bleeding Replacement therapy for FXIII deficiency is highly
from the umbilical stump in the first few days of life with satisfactory because of the small quantities of FXIII
delayed separation is common. It is characteristic that needed for effective hemostasis (5%) and the long half-
prolonged bleeding following trauma, after an intracranial life of FXIII (10–14 days). Prophylactic therapy with
hemorrhage, ecchymoses, hematomas.40 Hemarthroses plasma-derived, virus-inactivated FXIII concentrate at a
and bleeding into the muscles are less common than dose of 10–20 U/kg every 5–6 weeks has been successful
in hemophiliacs. Delayed wound healing also occurs. in achieving normal hemostasis.41 A new recombinant
In affected females, habitual abortions are commonly FXIII-A2 (rFXIII-A2) concentrate appears to be safe and
observed. It may either be due to intrauterine bleeding or appropriate for monthly prophylactic administration in
impaired formation of cytotrophoblastic shell leading to patients with FXIII-A deficiency42. Characteristic features
detachment of the placenta and miscarriage. of rare coagulation disorders are shown in Table 2.
Diagnosis CONCLUSION
The normal factor XIII deficiencies are PT and APTT. The most frequent bleeding disorders are the von Willebrand
Due to the failure of cross linking there is an increased and disease, hemophilia A and B. Inherited deficiencies of
solubility of clot and screening tests for factor XIII coagulation factors other than factor (F) VIII and FIX, the
deficiency are based on this observation. FXIII deficiency so-called rare coagulatuion disorders (fibrinogen, FII, FV,
is demonstrated by increased clot solubility in 5 M urea, FV+FVIII, FVII, FX, FXI, FXIII deficiencies), generally leads
Table 2 Characteristic features of rare coagulation disorders

Disorder Inheritance Clinical features APTT PT TT
Factor I Autosomal recessive Predisposition to thrombosis; may suffer from Prolonged Prolonged Prolonged
deficiency 4q23-34 little, moderate or severe bleeding
Factor II Autosomal recessive Umbilical cord bleeding and intracranial bleeds Prolonged Prolonged Normal
deficiency 11p11-q12 in neonates; bleeding after trauma or surgery;
easy bruising
Factor V Autosomal recessive Bleeding into the skin; nose bleeds; bleeding of Prolonged Prolonged Normal
deficiency 1q21-25 the gums; prolonged/excessive bleeding with
minor injuries, surgery or trauma
Factor VII Autosomal recessive Spectrum variable. Bleeding of mucous Normal Prolonged Normal
deficiency 13q34 membranes; excessive bruising; bleeding into
muscles and/or joints; CNS bleeding
Factor X Autosomal recessive Umbilical stump bleeding. Mucous membrane Prolonged Prolonged
deficiency 13q32 bleeding; bleeding into joints; muscle
bleeding; CNS bleeding
Factor XI Autosomal recessive Mild-to-moderate bleeding. Prolonged/ Prolonged Normal
deficiency 4q35 excessive bleeding with surgery or trauma;
bruising; hematuria; delayed bleeding
Factor XIII Autosomal recessive Delayed bleeding; bleeding from umbilical Normal Normal Normal
deficiency A-subunit- 6p24-25 stump after birth with delayed separation;
B-subunit- 1q31-32 prolonged bleeding from trauma; delayed
wound healing
to lifelong bleeding disorders. These disorders are largely 13. Alvarez MT, Jimenez-Yuste V, Gracia J, Quintana
inherited by autosomal recessive genetics. As these are not M, Hernandez-Navarro F. Acquired von Willebrand
well-characterized clinically in comparison to common syndrome. Haemophilia. 2008;14:856-8.
bleeding disorders, they do not have well-established 14. Veyradier A, Jenkins CS, Fressinaud E, Meyer D. Acquired
von Willebrand syndrome: from pathophysiology to
treatment strategies. High index of suspicion is required
management. Thromb Haemost. 2000;84:175-82.
to diagnose rare coagulation disorders and a sophisticated
15. Hayes T. Dysfibrinogenemia and thrombosis. Arch Pathol
laboratory support is essential to confirm the clinical Lab Med. 2002;126:1387-90.
diagnosis. 16. Roberts HR, Stinchcombe TE, Gabriel DA. The
dysfibrinogenaemias. Br J Haematol. 2001;114:249-57.
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6. Nichols WL, Hultin MB, James AH, Manco-Johnson MJ, 22. Hunault M, Bauer KA. Recombinant factor VIIa for the
Montgomery RR, Ortel TL, Rick ME, Sadler JE, Weinstein treatment of congenital factor VII deficiency. Semin
M, Yawn BP. von Willebrand disease (vWD): evidence- Thromb Hemost. 2000;26:401-5.
based diagnosis and management guidelines, the National 23. Keeling D, Tait C, Makris M. Guideline on the selection
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7. Siragusa S, Malato A, Lo Coco L, Cigna V, Saccullo G, Haemophilia Center Doctors’ Organisation (UKHCDO)
Abbene I, Anastasio R, Caramazza D, Patti R, Arcara M, Guideline. Haemophilia. 2008;14:671-84.
Di Vita G. Gastrointestinal bleeding due to angiodysplasia 24. Lak M, Sharifian R, Peyvandi F, Mannucci PM. Symptoms
in patients with type 1 von Willebrand disease: report on of inherited factor V deficiency in 35 Iranian patients. Br J
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8. Mishra DK, Chaturvedi A, Sharma A, Subramanya H, 25. Salooja N, Martin P, Khair K, Liesner R, Hann I. Severe
Kumar H, Varadarajulu R, Anand KP. von Willebrand factor V deficiency and neonatal intracranial haemorrhage:
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2004;60:337-41. 26. Borel-Derlon A, Gautier P, Le Querrec A, L’Hirondel
9. Fressinaud E, Veyradier A, Truchaud F, et al. Screening JL. Severe congenital factor V deficiency: successful
for von Willebrand disease with a new analyzer using high management of bleedings by recombinant factor VIIa.
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10. Krizek DM, Rick ME. A rapid method to visualize 27. Di Paola J, Nugent D, Young G. Current therapy for rare
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31. Perry DJ. Factor VII deficiency. Br J Haematol. 2002;118: stabilization. In: Colman RW, Marder VJ, Clowes
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42:1240-2. 39. Jayandharan GR, Viswabandya A, Baidya S, Nair SC,
33. Boggio L, Green D. Recombinant human factor VIIa in the
George B, Mathews V, Chandy M, Srivastava A. Mutations
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34. McMahon C, Smith J, Goonan C, Byrne M, Smith OP. The
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in children with severe factor X deficiency. Br J Haematol. PM. Pattern of symptoms in 93 Iranian patients with
2002;119:789-91. severe factor XIII deficiency. J Thromb Haemost.
35. Franchini M, Veneri D, Lippi G. Inherited factor XI 2003;8:1852-3.
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36. Peyvandi F, Lak M, Mannucci P. Factor XI deficiency in factor XIII deficiency. Curr Opin Hematol. 1998;5:372-5.
Iranians: its clinical manifestations in comparison with 42. Lovejoy AE, Reynolds TC, Visich JE, et al. Safety and
those of classic hemophilia. Haematologica. 2002;87:512-4. Pharmacokinetics of recombinant factor XIII-A2
37. Fard-Esfahani P, Lari GR, Ravanbod S, Mirkhani F, administration in patients with congenital factor XIII
Allahyari M, Rassoulzadegan M, Ala F. Seven novel deficiency. Blood. 2006;108:57-62.
C H A P T E R 30
Acquired Inhibitors of Coagulation
Acquired inhibitors of coagulation are antibodies that develop against coagulation proteins when there is either a congenital
deficiency of coagulation proteins or when there is an underlying disease process that precipitates the formation of these antibodies.
The resultant functional deficiency of coagulation factors causes altered coagulation profiles leading to, at times, severe bleeding
disorders.
Coagulation inhibitors are basically antibodies against natu- acquired hemophilia differs quite significantly from that of
rally occurring clotting factors and may occur as alloanti- hereditary hemophilia.
bodies in patients with congenital factor deficiencies or as
autoantibodies in patients with previously normal coagu- Pathophysiology
lation who have associated underlying diseases.1 Bleeding
Acquired hemophilia is a spontaneous autoimmune
occurs from multiple sites and is often compounded by the
disorder in which patients with previously normal
simultaneous deficiency of natural clotting factors. As ac-
hemostasis develop antibodies against clotting factors,
quired inhibitors of coagulation are quite rare, a high index
most frequently FVIII.4 The development of antibodies
of suspicion is required to recognize and treat the disorder
against FVIII leads to functional FVIII deficiency, which
effectively. Multiple mechanisms are involved in the patho-
results in insufficient generation of thrombin through the
genesis and hence treatment modalities need to attend to all
intrinsic pathway of the coagulation cascade (Flow chart 1).
the mechanisms in order to be effective.
Patients with this disorder are thus at an increased risk of

Among all the clotting factor inhibitors, the most
both spontaneous as well as post-traumatic bleeding.
commonly occurring inhibitor is against factor VIII and
At the chromosomal level, the most common sites
the resultant clinical syndrome is referred to as “acquired
coding for the development of these offending antibodies
hemophilia”.2 Autoantibody inhibitors against factor II,
appear to occur in the A2 and A3 domain on the heavy
factor V, factor VII, factor IX, factor X, factor XI, factor XIII
chain of FVIII and in the C2 domain on the light
and the von Willebrand factor proteins have also been
chain.3,5,6 These sites when activated produce anti A2,
reported.3
A3 and C2 antibodies which are alloantibodies in nature
in primary coagulation deficiencies and autoantibodies
ACQUIRED HEMOPHILIA when associated with underlying disease.
Inhibitors to FVIII are the most common in clinical Anti-A2 and Anti-A3 antibodies impede the binding of
practice, but the diagnosis of acquired hemophilia is FVIII to activated factors X and IX of the ‘Factor X activation
difficult owing to its rarity and because the patient does Complex’ in the intrinsic pathway while Anti-C2 antibodies
not have the usual precedent personal or family history inhibit the binding of FVIII to phospholipids and may also
of bleeding as seen in congenital hemophilia.2 Moreover, interfere with the binding of FVIII to Willebrand factor
the clinical signs and symptoms as well as the severity of protein.7
Flow chart 1 Coagulation cascade cantly lower than reported with acquired hemophilia
A. There is no known association between the tendency
to develop these acquired antibodies and ethnicity and
these inhibitors have been seen with no specific genetic
inheritance pattern in all racial groups.
ETIOLOGY
Acquired hemophilia results from the development
of antibodies (mostly of the IgG1 and IgG4 subclasses)
directed against various clotting factors.3,8,11
Numerous conditions that have been associated
with acquired inhibitors to FVIII (Table 1) include:
• Frequent blood transfusions (as in hemolytic anemias)
• Pregnancy
• Autoimmune disorders
• Inflammatory bowel disease
• Dermatologic disorders
• Respiratory diseases
• Diabetes mellitus
• Infections
• Malignancies
• Rarely, FVIII antibodies arise as an idiosyncratic
reaction to medications.
However, in approximately 50 percent of cases, no
underlying or precipitating factor can be found.2,12
Among the antibodies, the mechanism of factor CLINICAL FEATURES

VIII inactivation differs.8 For example, alloantibodies
inactivate FVIII activity in totality according to type 1 History
kinetics and this total inactivation is independent of the Unlike patients with hereditary hemophilia, patients
titer or concentration of circulating antibody. In contrast, do not have a personal or family history of bleeding
autoantibodies typically exhibit more complex type II episodes.2 About half of the cases are associated with other
kinetics causing an initial rapid inactivation of factor VIII conditions, such as autoimmune disease, and cancer2,12
followed by a slower inactivation reaction and results in and history may often reflect the underlying disease.
some residual FVIII activity which can be detected in the
blood but is not useful clinically to prevent bleeding.8,9 The
Physical Findings
end result is that severe or partial deficiency of Factor VIII
occurs leading to its associated clinical syndromes. In these patients, instead of the intra-articular bleeding
episodes, which are typical in congenital FVIII deficiency,
EPIDEMIOLOGY hemorrhages occur into the skin, muscles, soft tissues and
mucous membranes.2 Bleeding episodes are often more
Acquired hemophilia has a worldwide distribution. In frequent and severe than in congenital hemophilia.
the United Kingdom, the incidence has been reported
to be 1.48 per million persons per year10 while in the
On Examination
United States, it is 0.2 to 1.0 case per million persons
per year. These figures may, however, underestimate Typical signs include epistaxis, gastrointestinal and
the true incidence of the disorder given the difficulty urological bleeding and rarely cerebral hemorrhage.2,7,11
in making the diagnosis.2 Further, some patients with Spontaneous bruising and muscle hematomas are also
acquired hemophilia and low titers of inhibitors may not quite frequent.3 Other manifestations include prolonged
be diagnosed, unless they bleed after surgery or trauma.2 bleeding following trauma or surgery and iatrogenic

The incidence of acquired inhibitors to clotting bleeding, particularly following attempts to insert
factors other than FVIII is unknown, although it is signifi intravenous lines.2
Chapter-30 Acquired Inhibitors of Coagulation 313
Table 1 Conditions associated with acquired inhibitors to factor VIII

S. No. System involved Disease state
1. Frequent blood transfusions Hemolytic anemias (Thalassemia)
2. Autoimmune disorders Rheumatoid arthritis
Systemic lupus erythematosus
Autoimmune hemolytic anemia
Goodpasture syndrome
Myasthenia gravis
Grave’s disease
Autoimmune hypothyroidism
3. Inflammatory bowel disease Ulcerative colitis
4. Dermatologic disorders Psoriasis
Pemphigus
5. Respiratory diseases Asthma
6. Drugs Penicillin and its derivatives
Sulfonamides
Phenytoin
Chloramphenicol
Methyldopa
Interferon-Alfa
7. Malignancies and premalignant conditions Solid tumors
Chronic lymphocytic leukemia
Non-Hodgkin lymphoma
Waldenström macroglobulinemia
Myelodysplastic syndrome
Myelofibrosis
Erythroleukemia
8. Infections and vaccinations Acute hepatitis B infection
Acute hepatitis C infection
BCG vaccination
9. Idiopathic
INVESTIGATIONS with normal FVIII levels and no bleeding symptoms or

history.14,15
• An isolated prolongation of the activated partial • Acquired hemophilia can occasionally be confused
thromboplastin time (aPTT) that is not corrected when with disseminated intravascular coagulation because
the patient’s plasma is incubated with equal volumes of its clinical presentation and a prolonged aPTT;
of normal plasma in a mixing study is pathognomonic however, the absence of a prolonged prothrombin
of acquired inhibitors to factor VIII.2,11 Because the time (PT), low fibrinogen, elevated fibrin degradation
action of the inhibitor is often delayed, incubation for 2 products and D-dimers and thrombocytopenia7
hours at 37°C is required before the correction study is should help distinguish between the conditions.
initiated.13 • Among the common causes of isolated prolonged
• Bleeding time, prothrombin time, and platelet aPTT is lupus anticoagulant.16 Presence of lupus
counts are normal. anticoagulant is suggested when aPTT values during
• Reduced factor VIII levels and evidence of a factor the mixing study are similar at time zero and after
VIII inhibitor are diagnostic. Although acquired incubation at 37°C8 and can be confirmed by specific
hemophilia A is a rare condition, FVIII inhibitors in tests, such as the dilute Russell viper venom time and
very low concentrations and not typically detected the kaolin clotting time.17,8
by screening coagulation assays have been detected • As heparin administration can also prolong aPTT,
by specific assays in 17 percent of healthy individuals a thorough treatment history and relevant tests
for heparin effects are indicated. The presence of underlying disorder or the discontinuation of an offending
heparin is suggested by a prolonged thrombin time in drug may be all that is required.7
association with a normal reptilase time.8
• The levels of other intrinsic pathway factors (factors IX,
XI, and XII) may also be reduced by antibodies against MANAGEMENT OF BLEEDING
these factors in patients with acquired hemophilia A.14,12 Management of Mild Bleeding
Therefore, it is important to repeat factor assays8 using
increasing dilutions of patient plasma to establish the Patients with mild or minimal bleeding rarely require
specificity of the inhibitor. Once detected, the acquired specific treatment to control bleeding and require only
inhibitor should be quantified to assess the severity of immunosuppressive therapy for the inhibitors. Studies
the disorder and the risk of hemorrhage. Methods used have shown that there is no correlation between the titer of
for quantifying factor VIII inhibitors are the Bethesda the inhibitor and the severity of bleeding hence treatment
assay and the Nijmegen modification of the Bethesda should be based on symptomatology rather than on the
assay.18 One Bethesda unit (BU) is the quantity of inhibitor titers.10
inhibitor that inactivates 50 percent of factor VIII in
normal plasma after incubation at 37°C for 2 hours. Management of Moderate-to-Severe Bleeding
However, both the Bethesda assay and the Nijmegen
In patients with moderate-to-severe bleeding, the manage
modification may underestimate the potency of
ment depends upon the inhibitor titer.10 In patients
the inhibitor due to its nonlinear complex reaction
with very low titer inhibitors (<3 BU) treatment with
kinetics.18 As a result of its kinetic profile, the recovery
Desmopressin has been found to be useful in augmenting
and half-life of exogenous FVIII may be considerably
residual factor VIII activity.7 IV infusion of desmopressin
reduced, even in patients with low inhibitor titers. This
(0.3 mcg/kg) may result in a 2- to 3- fold temporary increase
has significant implications for therapy.
in plasma levels of FVIII and von Willebrand factor.19
• Imaging studies: MRI, CT scan, and ultrasound may
However, in many patients, Desmopressin treatment
be needed to localize, quantify, and serially monitor
alone will not ensure hemostasis.7
the location of bleeding and response to therapy.
In cases where the inhibitor titer is between 3 and
Other imaging tests can be used as needed to diagnose
5 BU, increasing the levels of factor VIII in the plasma by
associated diseases.
factor VIII infusions may suffice to control the bleeding.7,12
• Other tests: Testing patients with pregnancy-associated
These patients may need a higher than usual dose of
acquired hemophilia, for autoimmune disorders such
factor VIII, as much as double or triple the dose compared
as lupus and rheumatoid arthritis is recommended
to patients of congenital hemophilia of the same body
because the presence of an autoimmune disorder may
weight.19,20 An arbitrary dose of FVIII 200 IU/kg IV bolus
require a change in therapeutic approach.19
every 8–12 hours has been recommended.21 There are no
published studies on the use of human FVIII in acquired
DIFFERENTIAL DIAGNOSES hemophilia to guide its dosing.14
• Lupus anticoagulant Patients in whom the levels of Factor VIII inhibitor is
• von Willebrand disease higher (> 5 BU) require other modalities of treatment to
• Disseminated intravascular coagulation control bleeding:
• Dysfibrinogenemia • Recombinant activated factor VII (FVIIa): Here the
• Heparin administration requirement of factor VIII in the coagulation cascade
• Congenital hemophilia is bypassed by FVIIa binding to activated platelets and
promoting thrombin synthesis, thereby controlling
bleeding. Studies have shown that there is dramatic
MANAGEMENT control of bleeding in more than 90 percent of cases
Management of acquired inhibitors involves three within a few hours of infusion.22 FVIIa has also been
strategies: found to have very few side effects, is free of anamnestic
1. Management of acute bleeding reactions and does not transmit blood borne diseases.
2. Eradication of the inhibitor • Activated prothrombin complex concentrates:
3. Management of the etiological cause Activated prothrombin complex concentrates (APCCs)
The approach to these objectives usually depends on are also used to manage bleeding episodes in acquired
the natural history of the disease, the clinical presentation, hemophilia. The mechanism of action is by bypassing
and the titer of the inhibitor. Frequently, treatment of the the requirement of factor VIII and promoting thrombin
synthesis in the coagulation pathway. Studies in adults embolization), thermal (electrocautery, cryotherapy)
have shown a response rate of 86 percent.23 No studies or chemical (fibrin glues, micronized collagen). 27
are yet available on children. There is a potential risk
of anamnestic reaction and transmission of blood- ACQUIRED INHIBITORS TO
borne diseases with APCCs which has restricted their WILLEBRAND FACTOR
von
universal use.
• Immunoadsorption/plasmapheresis: Selective removal Acquired inhibitors to the von Willebrand factor (vWF)
of the inhibitor using immunoadsorption has been are infrequently encountered and have been seen in
found to be useful especially in cases where there association with:
is severe hemorrhage, no response to the above • Autoimmune disorders, monoclonal gammopathies,
modalities of therapy or when the concentration lymphoproliferative diseases
inhibitors is very high. After the inhibitors are removed, • Epidermoid malignancies, Wilm’s tumor
factor VIII infusions are given to control the bleeding. • Hypothyroidism
• Myeloproliferative disorders
ERADICATION OF THE INHIBITOR • Certain medications.
The incidence of these acquired antibodies is
Eradication of the inhibitor is achieved by stopping especially high in children with Wilm’s tumor, which
the production of the inhibitor by immunosuppressive makes identifying such patients important due to the
therapy. inevitable associated hemorrhagic complications that
The various modalities available are: occur during surgery. The exact mechanism of synthesis
• Steroids: These have been used as first line therapy in of this antibody is unclear, however, an interaction
the eradication of the inhibitors. Methyl prednisolone between a plasma factor secreted by the tumor and the
(oral or IV) or oral prednisolone are the drugs of choice naturally occurring vWF in the blood is thought to result
and have shown response in 60 to 70 percent of adult in premature clearance of the vWF.28 Treatment options
cases. include Desmopressin, infusion of FVIII that contains
• Cytotoxic drug therapy with cyclophosphamide vWF (cryoprecipitate), platelet transfusions, intravenous
and azathioprine has been used to control inhibitor immunoglobulin and plasma exchange. Acquired anti-
production. However, the side effects of these drugs vWF antibody usually disappears after treatment of the
and their low safety profiles restrict their universal use. tumor.
• Other drugs which have also been used are mycophe-
nolate mofetil, vincristine and 2-chlorodeoxyadeno
sine. ACQUIRED INHIBITORS TO FACTOR V
• Cyclosporine has also been found to have good Acquired inhibitors to factor V are rare and are seen
immunosuppressive effect on the inhibitors, however in lymphoproliferative disorders, adenocarcinoma,
due to its toxicity and side effects, its use in children is tuberculosis, prolonged aminoglycosides (particularly
restricted. It has been tried both as monotherapy and streptomycin) usage and topical exposure to bovine
as an adjuvant drug to steroids, showing best results in thrombin.29 The clinical presentation of children with this
cases of systemic lupus erythematosus.7 inhibitor is varied, some children bleed while others do not
• Immunoglobulins have been used as a second which is probably because patients with antibodies that
line treatment option in patients not responsive to bind to the factor V present on the platelets bleed more
other modalities.7 However, studies have shown an profusely than those where the antibody binds to factor V
equivocal response of immunoglobulins in eradicating present in the plasma.30 Treatment is by transfusing fresh
inhibitors, and have found best response in those with frozen plasma and recombinant factor VIIa.
low titers of the inhibitor.15
• Biological therapy-Rituximab, an anti-CD20
ACQUIRED INHIBITORS TO PROTHROMBIN
monoclonal antibody, has shown promising results
in eradicating inhibitors in acquired hemophilia.7,24-26 Acquired inhibitors to prothrombin occur in patients
Given in the dose of 375 mg/m2 on D1 and D15, it has with systemic lupus erythematosus, in children treated
shown excellent results in refractory cases. with bovine fibrin glue after surgery for congenital heart
• Surgical management: May be required in cases disease or exposure to procainamide. A small number are
where there are life-threatening bleeding episodes. idiopathic in origin. In children in whom the concentration
Treatment options vary according to the site of bleeding of lupus anticoagulant is high, acquired inhibitors to
and can be mechanical (ligature placement, selective prothrombin are also present. These inhibitors are difficult
Table 2 Conditions associated with acquired inhibitors to other clotting factors11, 31

Coagulation factor Associated disorders
VII Bronchogenic carcinoma, idiopathic
IX Systemic lupus erythematosus, acute rheumatic fever, hepatitis, collagen vascular diseases, multiple
sclerosis, and postpartum
X Amyloidosis, carcinoma, acute nonlymphocytic leukemia, acute respiratory infections, fungicide exposure,
idiopathic
XI Autoimmune diseases, prostate carcinoma, chronic lymphocytic leukemia, chlorpromazine
XIII Idiopathic, isoniazid, penicillin
to measure as they do not neutralize coagulant activity in 3. Boggio LN, Green D. Acquired hemophilia. Rev Clin Exp
activity dependent inhibitor assays and hence do not cause Hematol. 2001;5(4):389-404.
clinically significant spontaneous bleeding. However, 4. Von Depka M. Novoseven: mode of action and use in
they may cause bleeding during surgery or after trauma. acquired hemophilia. Intensive Care Med. 2002;28(Suppl
2):S222-7.
Treatment is by using fresh frozen plasma or activated
5. Knobe KE, Villoutriex BO, Tengborn LI, Petrini P, Ljung
prothrombin complex concentrates. These inhibitors RC. Factor VIII inhibitors in two families with mild
usually disappear spontaneously within 7 to 21 days or hemophila A: structural analysis of the mutations.
else can be treated using plasmapheresis, corticosteroids Hemostasis. 2000;30(5):268-79.
and immunosuppression. 6. Barrow RT, Healey F, Jacquemin MG, Saint Remy JM,
Lollar P. Antigenicity of putative phospholipid membrane
ACQUIRED INHIBITORS TO OTHER binding residues in Factor VIII. Blood. 2001;97(1):169-74.
7. Ma AD, Carrizosa D. Acquired factor VIII inhibitors:
FACTORS VII, IX, X, XI, XIII pathophysiology and treatment. Hematology Am Soc
Inhibitors to these factors occur in various conditions Hematol Educ Program. 2006.pp.432-7.
8. Franchini M. Acquired hemophilia A. Hematology. 2006;
(Table 2) and are not common in children. Treatment
11(2):119-25.
options include fresh frozen plasma, activated prothrom 9. Green D, Blanc J, Foiles N. Spontaneous inhibitors of
bin complex concentrates, recombinant factor VIIa along factor VIII: kinetics of inactivation of human and porcine
with inhibitor eradication using plasmapheresis, corticos- factor VIII. J Lab Clin Med. 1999;133(3):260-4.
teroids and immunosuppression. 10. Collins PW, Irsch HS, Baglin TP, Dolan G, Hanley J, Makris
M, et al. Acquired hemophilia A in the United Kingdom:
CONCLUSION a 2-year national surveillance study by the United King-
dom Haemophilia Centre Doctors’ Organisation. Blood.
Acquired inhibitors to naturally occurring clotting factors 2007;109(5):1870-7.
are commonly encountered in clinical practice and must 11. Cohen AJ, Kessler CM. Acquired inhibitors. Baillieres Clin
be considered in the differential diagnoses of any child Haematol. 1996;9(2):331-54.
with an altered bleeding profile not responding to the 12. Hay CR. Acquired haemophilia. Baillieres Clin Haematol.
standard therapy. Acquired inhibitors are associated with 1998;11(2):287-303.
13. Green D, Lechner K. A survey of 215 non-hemophilic
numerous common underlying conditions and require to
patients with inhibitors to Factor VIII. Thromb Haemost.
be managed aggressively in order to prevent mortality and 1981;45(3):200-3.
morbidity. Early recognition of the presence of inhibitors 14. Collins PW. Management of acquired haemophilia A—
helps to institute appropriate management to control the more questions than answers. Blood Coagul Fibrinolysis.
bleeding as well as prevent further episodes. 2003;14(Suppl 1):S23-7.
15. Algiman M, Dietrich G, Nydegger UE, Boieldieu D, Sultan
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hemophilic factor) in healthy individuals. Proc Natl Acad
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of coagulation factors: part II. Semin Thromb Hemost. 16. Chng WJ, Sum C, Kuperan P. Causes of isolated prolonged
2012;38(5):447-53. doi: 10.1055/s-0032-1305779. Epub activated partial thromboplastin time in an acute care
2012 Jun 27. general hospital. Singapore Med J. 2005;46(9):450-6.
2. Franchini M, Gandini G, Di Paolantonio T, Mariani G. 17. Kazmi MA, Pickering W, Smith MP, Holland LJ, Savidge
Acquired hemophilia: a concise review. Am J Hematol. GF. Acquired haemophilia A: errors in the diagnosis.
2005;80(1):55-63. Blood Coagul Fibrinolysis. 1998;9(7):623-8.
18. Verbruggen B, Novakova I, Wessels H, Boezeman J, 25. Wiestner A, Cho HJ, Asch AS, Michelis MA, Zeller JA,
van den Berg M, Mauser-Bunschoten E. The Nijmegen Peerschke EI, et al. Rituximab in the treatment of acquired
modification of the Bethesda assay for factor VIII C factor VIII inhibitors. Blood. 2002;100(9):3426-8.
inhibitors: improved specificity and reliability. Thromb 26. Aggarwal A, Grewal R, Green RJ, Boggio L, Green D,
Haemost. 1995;73(2):247-51. Weksler BB, et al. Rituximab for autoimmune haemo-
19. Delgado J, Jimenez-Yuste V, Hernandez-Navarro F, Villar philia: a proposed treatment algorithm. Haemophilia.
A. Acquired haemophilia: review and meta-analysis 2005;11(1):13-9.
focused on therapy and prognostic factors. Br J Haematol. 27. Shobeiri SA, West EC, Kahn MJ, Nolan TE. Postpartum
2003;121(1):21-35. acquired hemophilia (factor VIII inhibitors): a case
report and review of the literature. Obstet Gynecol Surv.
20. Rizza CR, Matthews JM. Effect of frequent factor VIII
2000;55(12):729-37.
replacement on the level of factor VIII antibodies in
28. Coppes M, Zandvoort S, Sparling C. Acquired von
haemophiliacs. Br J Haematol. 1982;52(1):13-24.
Willebrand disease in Wilm’s tumor patients. J Clin Oncol.
21. Kessler CM. New perspectives in hemophilia treatment.
1992;10:422-7.
Hematology Am Soc Hematol Educ Program. 2005.pp.429- 29. Savage W, Kickler T, Takemoto C. Acquired coagulation
35. factor inhibitors in children after topical bovine thrombin
22. Hay CR, Negrier C, Ludlam CA. The treatment of bleeding exposure. Pediatr Blood Cancer. 2007;49:1025-9.
in acquired haemophilia with recombinant factor VIIa: a 30. Neisheim M, Nichols W, Cole T. Isolation and study of an
multicentre study. Thromb Haemost. 1997;78(6):1463-7. acquired inhibitor of human coagulation Factor V. J Clin
23. Sallah S, Wan JY. Efficacy of 2-chlorodeoxyadenosine Invest. 1986;77:405.
in refractory factor VIII inhibitors in persons without 31. Bolton-Maggs PH, Perry DJ, Chalmers EA, Parapia LA,
hemophilia. Blood. 2003;101(3):943-5. Williams MD. The rare coagulation disorders: review with
24. Kain S, Copeland TS, Leahy MF. Treatment of refractory guidelines for management from the United Kingdom
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C H A P T E R 31
Immune Thrombocytopenic
Purpura—Diagnosis and Management
Immune thrombocytopenic purpura (ITP) in children is an acquired hemorrhagic disorder occurring in an apparently healthy child,
usually due to transient postviral autoimmune phenomenon, characterized by acute onset of petechiae, bruising and mucosal
bleeding. It is associated with isolated thrombocytopenia (platelet count <1,00,000/cumm) with normal or increased megakaryocytes
in an otherwise normal marrow without evidence of concurrent abnormality or disease process that might account for the
thrombocytopenia. Despite major advances in our understanding of the molecular basis of many blood disorders, despite major
advances in our understanding of basic underlying pathophysiology for more than 50 years, the diagnosis of ITP still remains one of
exclusion and currently no confirmatory clinical or laboratory diagnostic parameters exist.
There is no single simple test for the diagnosis of ITP. Hence, there are many unresolved issues pertaining to its diagnosis and
management of this disease.
Immune thrombocytopenic purpura (ITP) was first Acute ITP is common after the age of 3 months through
described as Morbus hemorrhagicus maculosus by childhood with a peak incidence between 2 and 5 years
German physician Paul Gottlieb Werlof in 1735.1–3 of age which also is the age at which children are most
Immune thrombocytopenic purpura (ITP) is most susceptible to viral infections, a major etiological factor.4-9
common acquired autoimmune bleeding disorder and is Acute ITP is generally a benign and self-limiting
characterized by a low platelet count and mucocutaneous condition, with 90 percent of them making an uneventful
bleeding.1-9,3a recovery within 3 weeks to 6 months with or without
specific treatment. Only 10 percent of ITP cases progress
to chronic ITP. 65-95 percent of prepubertal children who
CLASSIFICATION develop ITP have the acute form of the disease. Of these
ITP may be classified as:4-9 55 to 75 percent in < 1 month, 80 to 90 percent in < 4 to
• Acute 6 months resolve, only 10 percent of ITP cases progress
• Chronic to chronic ITP.2-6 ITP occurring for the first time before
• Recurrent. the age of 10 years is predominantly acute whereas after
These differ especially in respect to patient’s age at 20 years of age chronic ITP is almost the rule, though the
onset, sex, medical events preceding the onset, duration disease is not limited to any age.7,9 Although there is no sex
of thrombocytopenia and response to treatment. It predilection for ITP in childhood, in the adult form of the
is not possible to distinguish them at the onset of disease, there is 3:1 predominance in women.
symptomatology. However, in the age group above 13 In recurrent ITP there is recurrence of thrombocy
years, the incidence of chronic ITP is higher. In this age topenia after a sustained normal platelet count. It is
group 80 to 90 percent cases continue to have active precipitated each time usually following a viral infection.
disease for 6 months to one year.4-9 Boys and girls are equally affected.
Chapter-31 Immune Thrombocytopenic Purpura—Diagnosis and Management 319
DIAGNOSIS OF ITP1-9,12-19 Table 1 Incidence of symptoms in ITP10

There is no single simple test for the diagnosis of ITP. The Common bleeds UK survey Our series
diagnosis of ITP is still a clinical one, based on the patient’s Total 132
history, physical examination and complete blood cell Bruising and 386 (90%) 122
count as well as examination of peripheral blood smear and skin bleeds
bone marrow examination (If done.).12-16 There is no “gold Nose bleeds 95 (20%) 31
standard” test that can reliably establish the diagnosis. ICH 1% 1
The usual presentation is sudden onset of mucocutaneous
bleeds (Figs 1A and B) in an otherwise healthy child (the Mouth, gum, 68 (16%) 35
tongue bleed
child may be considered healthy in the absence of fever
and any systemic abnormality). The diagnosis is more by GI bleed 10 (2%) 5
exclusion. A typical case is a child with ITP, characterized Conjuctival 7 (2%) 5
by isolated thrombocytopenia, with normal counts in hemorrhage
otherwise healthy child without any hepatosplenomegaly, Hematuria 6 (1%) 7
no lymphadenopathy and bony tenderness and may be Heavy periods 3 (0.7%) 2
preceded by viral infection 2 to 3 weeks earlier. However,
Bleeding ear/ 2 (0.5%) 3
patients with risk factors for human immunodeficiency
eye
virus, should be tested for HIV antibodies.
It is usually a benign disorder. No bleeding 8 (2%) 1
symptoms
ITP may be (Table 1):4-12 Preceding 245 (57%) 30
• Asymptomatic: Incidentally detected low platelet viral infection/
count. Systemic examination is usually normal. No immunization
bleeding manifestations may be present.
• Mild symptoms: Bruising/petechiae, minor epistaxis,
little/no interference with daily living. • If child is clinically ill, with moderate or massive
• Moderate symptoms: Skin bleed, epistaxis and menorr splenomegaly, sternal tenderness/bony tenderness
hagia, mucosal bleeds more troublesome. or joint pains then it suggests an alternative cause,
• Severe symptoms: Severe bleeding episodes include sinister causes like malignancy, leukemia.
ICH requiring hospitalization and/or transfusions. • Constitutional symptoms, such as fever or weight loss,
GI bleeding, severe epistaxis, hematuria, prolonged hepatomegaly or lymphadenopathy might indicate
menorrhagia. underlying disorder such as HIV, viral infection, syste
Usually there is no hepatosplenomegaly; spleen may mic lupus erythematosus (SLE), or a lympho-prolife
be just palpable in 10 to 12 percent of the even normal rative disease.
children. • Anemia disproportionate to severity of bleeding, is
unlikely to be due to ITP and should consider aplastic
anemia, leukemia.
• Atypical rash should lead to suspicion of an alternate
diagnosis like viral infections.
• If child is clinically ill, then it is unlikely to be ITP,
and should consider other etiology like infection—
meningococcal infection, septicemia and other sinister
diseases like aplastic anemia, leukemia, etc.
• Presence of significant lymphadenopathy, hepato-
splenomegaly should lead to the suspicion of infectious
diseases like EBV or CMV or any other alternative
diagnosis like leukemia.
• Response to specific therapy, for example, intravenous
A B
immunoglobulin (IVIg) and intravenous anti-D is
Figs 1A and B Mucocutaneous bleeds (hematoma, petechiae supportive of the diagnosis, but a response does not
and purpura) exclude secondary ITP.
On examination, these children may have petechiae,

purpura and/or bruises. Serious bleeding is rare. Only 4
percent have serious symptoms such as severe epistaxis,
GI bleeding or hematuria. Less than 1 percent children
with ITP develop intracranial bleeds.
Presence of fever, weight loss, bony pains, hepato
splenomegaly or lymphadenopathy and anemia dispro
portionate to amount of bleeding, would suggest diagnosis
other than ITP (e.g. leukemia, lymphoma, viral infections,
malaria, aplastic anemia, etc.). However, a just palpable
spleen may be normally present in 10 percent of pediatric
population.
When platelets are reduced in number (Thrombocyto-
penia) or defective in function (thrombasthenia), bleeding
may occur. Bleeding typically involves skin and mucous
membranes including petechiae, purpura, ecchymosis Fig. 2 Normal blood smear with normal platelet count
and epistaxis, hematuria and gastrointestinal hemor-
rhage. Intracranial hemorrhage can occur rarely.
Most thrombocytopenia in children are the result
of increased platelet destruction. The bone marrow in
such cases responds with compensatory increase in the
rate of production with increased number of immature
megakaryocytes. The increased mean platelet volume
provides supportive evidence of the larger size young
platelets, which are functionally very active, are more
prominent in the peripheral blood smear. The increased
mean platelet volume provides evidence of the larger
size. Normal MPV is 6.0–10 fL.
In disorders with decreased platelet production,
the decreased platelet number is associated with small
sized platelets, a decreased mean platelet volume and
a longer bleeding time relative to platelet number. The
megakaryocytes are decreased in number or absent in
bone marrow aspirate.
In the diagnostic evaluation of thrombocytopenia, Fig. 3 Blood smear with decreased platelet count
it is important first to determine whether other blood
components are involved. Co-existing abnormalities of
the white blood cells or red cells may indicate other causes
of diseases involving bone marrow like aplastic anemia
(Fig. 7), leukemia (Fig. 8).
Abnormalities in coagulation, in association with
thrombocytopenia, suggest disorders of consumption
including DIC, liver disorder.
Platelets are one of the important components in the
first phase of hemostasis and platelet plug formation.
The characteristics of platelets are (Figs 2 to 5):
Number: 150,000 to 400,000/mm3, out of which 2/3rd
circulate in blood stream and 1/3rd located in spleen. Life
span is 7 to 10 days.
Mean platelet volume (MPV)—7.1 fL.
Most thrombocytopenia in children is the result
of increased platelet destruction. The bone marrow in
such cases responds with compensatory increase in the Fig. 4 Blood smear with increased platelet count
Fig. 5 Blood smear with increased platelet count and platelet in Fig. 8 Bone marrow aspiration smear in leukemia
clumps
rate of production with increased number of immature

megakaryocytes (Fig. 6). The large young platelets, which
are functionally very active, are more prominent in the
peripheral blood smear. The increased mean platelet
volume provides supportive evidence of the larger size.
In disorders with decreased platelet production,
the decreased platelet number is associated with small
sized platelets, a decreased mean platelet volume and
a longer bleeding time relative to platelet number. The
megakaryocytes are decreased in number or absent in
bone marrow aspirate.
Platelet count less than 100,000/cumm is called
thrombocytopenia
Fig. 6 Bone marrow aspiration smear—increased • 80 percent of children with acute ITP have platelet
megakaryocytes count less than 40,000/cumm.
• In chronic ITP, platelet count at the time of presentation
is usually higher (20,000–75,000/cumm).
Thrombocytopenia is evident on peripheral smear
and is accompanied by bizarre shaped or giant forms of
platelets (Fig. 3).
In chronic ITP both mean platelet volume (MPV)
and number of large platelets are significantly increased
compared to control values. However, these values are
unchanged in acute ITP.
The presence of low or normal MPV in a case of
thrombocytopenia suggests aregenerative thrombocy
topenia, i.e. bone marrow suppression or marrow infil
tration.
Platelet distribution width (PDW) may be more
discriminating than MPV in detection of compensated
Fig. 7 Bone marrow aspiration smear in aplastic anemia thrombocytopenic states.
Leukocyte Count differential diagnosis, associated with hepatospleno

megaly/Lymphadenopathy, (Sternal) bone tenderness
The total leukocyte count is usually normal though mild or painful joint, atypical rash
to moderate lymphocytosis with increased number of • Abnormal leukocyte count (leukopenia and leuko
atypical lymphocytes may be seen especially when pre cytosis) and/or abnormal (premature) cells on periph-
ceded by viral infection. Mild peripheral eosinophilia may eral smear
be seen in 20 percent of children but is of no diagnostic or • Anemia (Low Hb) disproportionate to amount of
prognostic value.2 bleeding.
• Prior to the initiation of the corticosteroid therapy or
Anemia blood transfusion for presumed ITP as this may lead to
Anemia because of blood loss is seen in about 20 percent a temporary remission and may mask the presence of
of children with ITP. However, if the degree of anemia is blast cells
disproportionate to amount of bleeding seen then, other • Lack of response to specific therapy like IVIg /Anti-D
sinister conditions like leukemia, aplastic anemia or occult globin.
blood loss, Evan’s syndrome should be kept in mind. Bone • Prolonged thrombocytopenia (> 6 months)
marrow aspiration, trephine biopsy, Coomb’s test, etc. are A possibility of missing the diagnosis of rare
of immense value in confirming the diagnosis and ruling conditions like a amegakaryocytic thrombocytopenic
out above conditions. purpura should be kept in mind if bone marrow
examination is not done.
• It is not necessary to perform bone marrow aspiration
Antiplatelet Antibody13-25
if IVIg therapy is contemplated.
Understanding of pathophysiology and incite in the
clinical and laboratory aspect started with published series Bone marrow examination in ITP will show normal or
of observation by Harington in 195113 which revealed increased megakaryocytes with normal erythroid and myeloid
transferable plasma factor mediated the disease in many maturation. Cytoplasm of megakaryocytes is decreased, often
patients. This was accomplished by infusion of plasma vacuolated, less granular and stains more basophilic. They may
from ITP patients into healthy volunteers which lead to have increased nuclear lobe count. Platelet production and
turnover is increased up to 8 times the normal.
acute thrombocytopenia in recipient. Shulman et al. and
others subsequently confirmed that this factor as IgG
antibodies. Platelet associated with IgG antibody (PAIgG)
Other Investigations23,30,31
is present in 80 percent of thrombocytopenic children
with ITP. However, PAIgG is also found in other immune Plasma glycocalicin (a fragment of platelet membrane
thrombocytopenic states. Although these tests are highly glycoprotein Ib levels) are significantly below the normal
sensitive they have very low specificity as the patients with range (5–27%) in a regenerative thrombocytopenic
both immune and non-immune thrombocytopenia have conditions like aplastic anemia and amegakaryocytic
elevated PAIgG. In Evan’s syndrome, as it is associated thrombocytopenic purpura. The levels are above the
with autoimmune hemolytic anemia, Coomb’s test is normal range (48–261%) in thrombocytopenia associated
helpful in the diagnosis. with normal or increased megakaryocytes in bone marrow.
Over the last few years platelet survival studies using the
Bone Marrow Examination27-29 radioisotope chromium-51, Indium-111 (In-111) have
become available. There are characteristic patterns of
In a typical case of ITP bone marrow evaluation is unneces platelet recovery and survival. Immune thrombocytopenic
sary. Thus, acute onset of bruising following a viral infec disorders like ITP have nearly normal platelet recovery but
tion in a previously healthy child without any significant a very short platelet survival, whereas markedly reduced
hepatosplenomegaly, anemia not disproportionate to platelet recovery with normal platelet survival is seen in
amount of bleeding, bony tenderness or lymphadenopathy hypersplenism.
and appearance of mega thrombocytes without any
abnormal premature cells on peripheral smear does not Glycoprotein specific acute antibody assay: Early studies
need evaluation of bone marrow aspirate. showed encouraging results with sensitivity of 75-85
percent and specificity of almost 100 percent. Unfortu
nately recent large studies showed low (40–60%) sensitivity
Indication for Bone Marrow Aspiration but high specificity (78–92%) However, patients with
• In patients with atypical features: The clinical presen myelodysplastic and lymphoma with thrombocytopenia
tation suggests leukemia or aplastic anemia as a were also tested positive. Further studies perhaps using
new technology may give better sensitivity and specificity Certain antiplatelet drugs like aspirin, phenacetin,
and needs further study.24 antihistaminics, phenothiazines, nonsteroidal anti-infla
mmatory drugs, etc. should be avoided.
Management of ITP2-9,32-53,57,58, 66,69,70-80 Deep intramuscular injections should be avoided
and if has to be given, then pressure over the injection
The main strategy of treatment of ITP is to administer site should be maintained minimum for 10 minutes,
least amount of therapy “In ITP treat the child and not continuously without trying to see in between whether
the platelet count.” Why certain patients bleed but most bleeding is present or not.
do not, remains unclear. Some patients may have marked Immunization with live viral vaccines (polio, measles,
thrombocytopenia yet normal or near normal hemostasis, MMR) preferably should be avoided during the period of
because of increased young platelets having better severe thrombocytopenia.
functional capacity. Previous studies of ITP have not addressed the risk of
On the other hand some patients (5% of all children hemorrhage during various sport activities. Restrictions of
with ITP) may have impaired platelet function as a result contact sports (football, soccer, kabaddi, etc.) advocated
of antibodies and these children have prolonged bleeding until platelet count is above 100,000. Most noncontact
time and increased bleeding tendency in spite of having sports can be safely enjoyed with platelet count greater
near normal platelet count. Therefore, platelet count as than 30,000/cumm. Serious athletes may need frequent
well as bleeding time estimation is recommended prior to platelet count measurement and treatment during their
the decision regarding management of ITP. participation.
Therapy depends on whether it is acute or chronic ITP.
SPECIFIC THERAPY IN ACUTE ITP
Acute ITP2-9
In general 70 to 80 percent of children with acute ITP will Corticosteroid Therapy (Oral)32-36
have complete remission and permanent recovery without The use of corticosteroids in the management of ITP is a
sequelae with or without treatment. 55 to 75 percent of matter of considerable controversy. In a heterogeneous
those who recover do so within the first month and 80 to 90 disease that usually sooner or later gets better on its own
percent within 4 to 6 months of diagnosis and rest beyond and gives rise to little morbidity and low mortality, it is
6 months to 1 year sometimes even beyond 10 years. difficult to evaluate the modality of the treatment. It has
been estimated that to have statistical significance, a
Chronic ITP randomized trial of corticosteroid versus placebo, would
require some 14,000 patients. A double blind randomized
However, in chronic ITP only 1/3rd go into remission prospective study is more likely to give the truth and
spontaneously, that too usually late in the course of the eliminate both physician and patient bias. Normalization
disease, i.e. between 1 and 10 years after the diagnosis. of platelet counts as well as reduction in prolonged
ITP patients may not need any treatment but bleeding time occurs earlier in the steroid treated group
reassurance. Children with chronic ITP whose platelet as compared to the untreated group. However, it takes
count remains within a relatively safe range (more than 8 to 10 days before significant changes are noticed. In a
10–30,000/cumm) and whose bleeding time is fairly randomized double blind and placebo controlled trial,
normal need no therapy except defensive management. platelet counts reached a level of 30,000/cumm or more
Remission is known to occur in about one-third of these (safe range) significantly earlier, with corticosteroids.
children, sometimes as late as 10 to 20 years postdiagnosis. Platelet survival increased in ITP after steroids.17-21
The treatment of a benign disease like acute ITP
should be decided after balancing the risk of treatment
vs no treatment. The mainstay of treatment in majority Steroids in ICH
of cases of childhood acute ITP hence is a “Defensive No proof exists that use of steroids reduces the incidence
management and nonfrantic watchful waiting.” During of intracranial hemorrhage (ICH) or death.
the initial period following the onset of ITP, restriction of Walker and Walker3 while reviewing the data of ITP in
physical activity and complete avoidance of all contact children from England and Wales noted that 11/12 children
sports and playground activities. Use of helmets to prevent died of ICH, 8 of whom had received corticosteroids.
trauma especially head injury and using knee-cap during Lusher and Zuelzar4 reported ICH in only one child
the phase of thrombocytopenia are indicated. who died despite immediate treatment with steroids.
Table 2 Controversies in ITP in children

No. of No. of Not Platelet History of
% Steroid <1 M 1–6 M >6 M Mortality
patients ICH ITP known count trauma
Lokeshwar
122 1 0.81 0/1 1 - - - 1/1 <20000 1/1
et al.
Walker and
181 1 0.5 1/1 1 - - - - <20000 -
Walker3
Lusher4
465 0 0 - - - - - - - None
et al.
Choi and
413 6 1.4 0/6 2 4 1 - 2/6 <20000 1/6
McClure5
Benham and
132 2 1.5 ½ - 1 - 1 0/2 - 0/2
Taft62,63
Simon et al.7 95 1 1.1 0/1 1 - - - 1/1 <20000 0/1
Lammi and
152 1 0.7 0/1 - 1 1 - 1/1 - 0/1
Lovric6
Zerella et al.6a 183 6 3.3 4/6 2 4 1 - 2/6 <20000 2/6
Imbach et al.64 108 1 0.9 0/1 1 - - - 1/1 - None
8/19
Total 1851 19 1.02 6 8 10 3 1 - 4
42%
Benham and Taft62 reported 132 children with ITP, bleeding, gastrointestinal hemorrhages and fundal hemor
with 2 cases having ICH, one each in steroid treated and rhages and ICH should be treated with steroids. Active
untreated group. young children less than 3 to 4 years with low platelet
Review literature done in 1851 (Table 2) cases showed count also may be treated with steroids, because of fear of
19 cases with ICH (1.026%) and 6 cases had ICH when they trauma induced severe bleeds.
were on steroid therapy. Eight children had within 1 month
of diagnosis and 10 children had intracranial hemorrhage Dose of Steroids
after 1 month of onset of ITP. Few of precipitating factors
included hypertension, aspirin ingestion, platelet count Prednisolone is used in the dose of 2 mg/kg/day for two
less than 20,000/cumm and trauma. to three weeks followed by tapering of dose over the next
Table 2 shows the ICH in children with ITP. week irrespective of platelet count. However, as steroids
are being tapered some patients may develop a drop in
Indications for Steroids in ITP their platelet count. This is usually transitory and not an
indication to step up the dose to previous levels since
Though there is a lot of controversy whether steroids clinically purpura often improves. In severe cases, for
should be given to children with acute ITP or not, there is initial 4 to 5 days, prednisolone may be given in a dose
uniformity in the opinion that large doses of steroids for a of 4 mg/kg followed by reduction in the dose thereafter
prolonged period should not be given, since steroids may to conventional levels.2 A small number of patients with
in fact, suppress platelet production. chronic ITP with recurrent mucosal bleeds or severe
Adverse effects of steroid include: Hyperglycemia, thrombocytopenia can be managed successfully with
hypertension, fluid electrolyte imbalance, psychosis, and small maintenance dose (0.5–1.0 mg/kg/alternate day or
osteoporesis, etc. Hence, the clinician should balance the even less) of corticosteroids.
benefits of the treatment against the risk.
“The child and not the platelet count should be Intravenous Pulse Methylprednisolone
treated.” In children who present with mild illness no
therapy other than purely defensive management is Pulse Therapy37,38,42
required. Pulses of few days’ duration—single dose short course
A child with severe thrombocytopenia with a platelet of methylprednisolone 25 mg/kg/day on 3 consecutive
count of less than 10,000 to 30,000/cumm with generalized days resulted in early response lasting for 3 months or
petechiae and purpura, wet purpura with mucosal more. Lusher et al. (1984)42 have used this therapy to raise
platelet counts prior to splenectomy. One of our patients, bleeding and ICH and during pregnancy as steroids are
16-year-old with chronic ITP for 8 years responded well to 3 contraindicated. In addition, young children below the
doses of methylprednisolone and platelet count increased age of 5 years with severe, recurrent hemorrhage may
from 5,000/cumm to 1,50,000/cumm following which be given IVIgG to postpone/avoid splenectomy. Bussel44
tooth extraction could be done. Similar results have been et al. were able to avoid splenectomy in about 75 percent
reported by other authors. A short intravenous course of of patients with ICH. IVIgG acts by causing temporary
high-dose methylprednisolone is effective as initial reticuloendothelial blockade. This might be due to two
treatment of ITP. Toxicity of long-term treatment with separate effects, a decrease in Fc receptor affinity for
prednisone can be avoided in a number of patients with ITP. platelet associated IgG and competition for Fc receptors
In patients refractory to treatment with methylprednisolone, by the increased serum IgG.
the response rate to second-line treatment with prednisone
was not negatively influenced, since two-thirds of these IVIgG in Chronic ITP
relapsing patients subsequently responded to prednisone. IVIgG is effective for temporarily raising the platelet count
Both IVIgG and methylprednisolone produce a significant in 70 to 80 percent of children with chronic ITP. Platelet
early rise in platelet count that is somewhat greater with count rises within 1 to 3 days of infusion. Permanent
IVIgG. However, the higher platelet counts produced by remission occurs only in minority (0–20%) of these
IVIgG may not justify the additional cost and potential risks children. A safe count however (above 20,000–30,000/
of this agent. cumm) may be achieved with periodic booster doses.
However, for the child with bleeding symptoms and
Intravenous Immunoglobulin43-48 not responding to steroid therapy as whose platelet count
remain precariously low (less than 10,000/cumm) IVIgG
The major goal in the treatment of acute ITP is to restore the now has become the initial therapy of choice prior to
platelet count to relatively safe levels as soon as possible so splenectomy in about 70 percent of patients with chronic
as to prevent ICH and life-threatening hemorrhages. ITP.
Fifty-five to seventy-five percent children with ITP
recover within first month of illness, irrespective of Anti-D in ITP49-53
treatment. An increase in the platelet count to a safer
level of more than 30,000 to 1,80,000/cumm was noted Rh anti-D globulin has been recommended as an
within 1 to 2 days following IVIgG therapy. Bussel et al.44 alternative to IVIgG in treatment of chronic ITP. Rh anti-D
used 1 g/kg/day for 2 to 3 consecutive days followed globulin have been tried in varying doses intravenously.
by maintenance infusion. Imbach et al.43 described Responses are usually slower in onset when compared
randomized multicentric trial in which IVIgG was to IVIgG and are transient. However, in some patients
compared with steroids. Eighty percent of children in each sustained responses have been seen, lasting for 6 months
group responded to the therapy with mean time for the to 3 years.
peak platelet count being 12 days in the group receiving • Splenectomized and Rh-ve patients respond less well
corticosteroids versus 9 days in IVIgG. Thus, the effect of • Though occasionally a complete remission has been
corticosteroids and IVIgG were identical for children who observed after a single course of anti-D globulin,
responded rapidly to the treatment and IVIgG does not repeated booster doses at intervals of more than 3 weeks
offer a major advantage over corticosteroids. However, may be required to maintain platelet count at a safe level.
in steroid nonresponders, IVIgG can produce better • A number of children are able to discontinue the
remissions. Reactions seen in 20 percent of children trivial therapy during the first year of treatment.
such as headache, fever, vomiting, fatigue, etc. But there • The drug is administered slowly in 20 to 50 cc of saline
is a potential problem of transmission of plasma-borne over 2 hours or can be administered fast over 3 minutes.
infections like hepatitis, AIDS and other viral infections. In Though the peak platelet count occur at a mean of 8
addition, the cost is prohibitive. (The total cost for a 10 kg days following initial infusion, platelet counts increase
child for one course will be about ` 30,000/- onwards). As significantly in 72 hours.
the chance of spontaneous recovery is high and chances of • Hypersensitive reactions like for any other plasma
ICH are very low (0–3.3 %) routine administration of IVIgG product are known and may cause shaking and chills.
is not recommended. IVIgG should be considered for any Transmission of HIV and hepatitis after infusion of
patient with ITP in whom rapid rise in platelet count is anti-D is uncommon.
deemed essential such as before surgery, after significant • Hemolysis has been observed and patient may need
trauma, especially a child with head injury, menorrhagia, blood transfusions due to anemia caused by IV anti-D
delivery, life-threatening bleeds like gastrointestinal globulin.
• IV anti-D appears to be useful in treatment of ITP as it hence routine administration of antiplatelet drugs like
is cheaper and effective in steroid-refractory patients aspirin or dipyridamole is not recommended.
prior to splenectomy.
Problems after Splenectomy
Splenectomy in ITP54-65 Risk of postsplenectomy infection is related to age; it is
Spleen is the most important site for the destruction of very high in early infancy and in those with underlying
antibody coated platelets (Graveyard of platelets.). It is disorders. As compared to ITP, the risk of infection is more
one of the major sites of antiplatelet antibody production. when splenectomy is done for diseases like thalassemia
Reported efficacy rate in regard to achieving a stable major, sickle cell disease and Hodgkin’s disease. A detailed
increased platelet count have varied for 40 to 86 percent. review in 1973 estimated the risk of fatal sepsis following
With most reporting approximately 60 percent platelet splenectomy for ITP to be 1 to 4 percent. Pneumococcus
count increased to normal range of 150,000 to 400,000 in was the most common infecting organism with adrenal
5 to 60 days. No response was observed in 21 percent (6– hemorrhages occurring in over 25 percent of fatal cases.
40%) of patients, morbidity of splenectomy 10 percent and Most deaths occur within first 2 to 3 years following
mortality less than 2 percent. splenectomy, though deaths are reported as late as 30 years
after splenectomy. Children rapidly develop progressive,
Indication of Splenectomy ITP overwhelming sepsis presenting initially with acute onset
of fever, nausea, vomiting and then rapidly progressing
• As an emergency measure for life-threatening ICH, and with altered sensorium, confusion and leading to coma
in adolescent girls with chronic disabling menorrhagia. and death within few hours. It may be associated with DIC,
If patient do not afford or failure to IVIg therapy electrolyte imbalance, shock, etc.
• Some clinicians prefer to perform an emergency Parents of splenectomized children should be educated
splenectomy during life-threatening hemorrhages and instructed to seek immediate medical attention
because they believe surgical procedure produces whenever child develops febrile illness. Broad spectrum
more rapid rise in platelet count than IVIgG and occa- antibiotics by intravenous route are recommended after
sionally patient may fail to respond to IVIgG within collecting blood for cultures. Combination of ampicillin/
first 24 hours amoxycillin or 3rd generation cephalosporins should
• Less convincingly perhaps, in those with persistent be administered. Prophylactic antibiotics, particularly
thrombocytopenia to avoid prolonged disruption of during infancy, are recommended for several years
lifestyle caused by limitation of activity and avoidance after splenectomy. Pneumococcal vaccine should be
of contact sports administered at least 2 to 8 weeks prior to the surgery.
• Non responding chronic ITP. Revaccination after 2 to 5 years is recommended. Other
For children with bleeding symptoms whose count vaccinations such as H. influenzae and meningococcal
remains precariously low (less than 10,000/cumm) or vaccine also may be given.
with recurrent mucosal bleeds and who do not respond Thus, splenectomy should not be first treatment
to steroids and IVIgG (medical line of treatment), initiated in the management of patients with ITP
splenectomy is the alternative. Response rate to splenec particularly in children. It should be performed only after
tomy in chronic ITP is about 65 to 88 percent.2,5,6,9,10 There all other therapeutic modalities have been exhausted and
is no definite test by which one can predict response patients have platelet count less than 10 to 25,000/cumm
to splenectomy. But some authors have found that the and has mucosal bleed. It has fairly favorable response
initial response to steroids and thrombokinetic studies rate of 60 to 80 percent. Acute and late morbidity of
could demonstrate predictive relationship.2,26 At the time splenectomy, low rate of mortality of ITP and the chance
of splenectomy the surgeon should look for accessory of late spontaneous remission of thrombocytopenia have
spleens which if missed may result in relapse of ITP. lead to differ splenectomy indefinitely particularly in
Administration of platelet transfusions prior to surgery childhood ITP. Splenectomy is thus differed for as long as
usually is not required as platelet count starts rising possible in pediatric population.
immediately following surgery (Clamping splenic radicles)
reaching as high as 3,00,000/cumm in the immediate post-
Role of Nonsteroidal Immunosuppressant
operative period and reaching a peak within 1 to 2 weeks
and then gradually dropping to normal values by 4 to 8 Drugs in Chronic ITP
months. Though platelet count can rise as high as 1 to 2 A small percentage of children (less than 2%) with severe
millions, there are no reported cases of thrombosis and chronic ITP do not respond either to steroids, IVIgG, anti-D
globulin or splenectomy and continue to have bleeding not responding to steroids, IVIgG or splenectomy. Alpha-
tendencies with a platelet count of less than 10 to 30,000/ 2b interferon is a nontoxic alternative that may modify
cumm. In these patients immunosuppressive therapy is the B-cell activity involved in antibody production. Responses
alternative. Nonsteroidal immunosuppressive agents used were similar in splenectomized and non-splenectomized
are vincristine, vinblastine, azathioprine, 6-mercapto- patients.
purine, cyclophosphamide67,68,68a and cyclosporine,69 etc. Dose used is 2.5 mu/m² 3 times a week given subcuta
Vinca alkaloids act more quickly but cyclophos neously for 12 weeks. The course may have to be repeated
phamides have a more lasting effect. Vincristine (0.025 in some cases. The response is usually transient with a
mg/kg not over 2 mg) or vinblastine (0.125 mg/kg) IV mean duration of fewer than 14 days. Alpha interferon
is given at weekly intervals for 3 to 4 doses. Recently a therapy is well-tolerated and side-effects are mild. It may
constant infusion of vincristine has been tried, however, cause increase in tendency of clinical bleeding. There may
overall advantage of this method has not been established. be a significant fall in granulocyte count. Fortunately this
Target delivery of the drug by infusing platelets has not been associated with an increase in the incidence
incubated with vinblastine has not been found to be of bacterial infections. Flu-like symptoms may develop
effective as binding of vinblastine is easily reversible. Our particularly in older patients for which acetaminophen
observations are discouraging with none out of 5 patients may be used.
in the age group of 3 to 8 years showing any significant Monoclonal anti-FcR III antibody has been found to be
response to Vincristine therapy. Only one child showed effective in 6 to 10 patients with long-term response.54
marginal rise in platelet count and did not show a lasting
improvement in the course of the disease. Rituximab72-76
Studies using azathioprine in chronic ITP refractory Rituximab binds to the transmembrane antigen CD20
to splenectomy show marked variation in the rate of located on Pre B and mature B lymphocytes. CD20 antigen
remission. The dose used ranged from 1 to 4 mg/kg/day found on both normal and malignant cells but not on
which may cause mild neutropenia. Three to six months hematopoietic stem cells, Pro B Cells, normal plasma cells
of treatment may be necessary before maximum response or any other normal tissue.
is observed. The major side effects which are encountered Rituximab in ITP available as Mabthera (Roche) 1 vial
relate to granulocytopenia and other features of bone contain 10 mg in 10 mL or 500 mg in 50 mL.
marrow suppression. Rituximab dose 375 mg per meter square was
Cyclophosphamide67,68 has been used in ITP with administered as IV infusion at weekly interval for 4 doses.
variable success rates. It may be given either orally 1 to 2
mg/kg/day or intermittently intravenously in a dose of 750 Methods of administration: IV infusion should be
to 1000 mg/m² every three weeks. Response occurs 2 to 10 administered through a dedicated line. Administration
weeks after the initiation of therapy. IV push or bolus should not be done.Infusion should
Cyclosporin69 has been recently tried in refractory be administered where full resuscitation facilitates are
ITP with transient increase in platelet counts after the immediately available.
treatment. It is known to modulate cell-mediated immunity Premedication consisting of an analgesic/antipyretic
or alter T-helper cell aspect of humoral immunity. It is (paracetamol) and an antihistaminic drug (e.g. diphen
given in the dose of 8 to 10 mg/kg/day in 2 divided doses hydramine) should always be administered before giving
continued over 2 to 3 months. The drug is usually tolerated each infusion of Rituximab.
well without major side effects but a significant rise in Initially start in micro drip 10 drops/min for 15 minutes
glutamine transpeptidase may be present. Nonsteroidal then 15 drops/min for 15 minutes then 20 drops/min be
immunosuppressive drugs should be used with greater continued.
caution in children as alkylating agents are known to be Adverse events: Common symptoms are fever, chills
mutagenic and increase the risk of subsequent malig rigor, flushing, nausea, vomiting, urticaria, rash, pruritus,
nancy. Secondary lymphomas have been reported. Close angioedema headache, throat pain, abdominal pain,
monitoring of these patients, including WBC count is
myalgia, rhinitis, hypotension, bronchospasm, arthalgia.
necessary.
Prepared solution is stable for 24 hours at 2 to 8° C and
subsequently 12 hours at room temperature. Store the
Alpha Interferon70,71 vials in refrigerator in the carton do not freeze.
Alpha interferon has recently been used to treat refractory Brox et al.75 in 1988 reported beneficial response to
ITP. Interferon alpha-2b therapy is indicated to treat ascorbic acid in 3 out of 11 patients with chronic ITP.56 But,
patient with life-threatening hemorrhage and those patient this has not been confirmed by others.
Since children with chronic ITP can have a spontaneous months period. Since practically none of them in one’s
remission even many years after their initial diagnosis, it is experience go into ICH (less than 1% cases) and morbidity
important to reduce or withdraw the drugs periodically. is minimum, the dictum of the treatment of ITP should be
It has been reported that approximately 0.5 to 3 percent the too frequently forgotten maxim ‘first do not harm’.
of children with chronic ITP will eventually develop auto- In mild ITP with cutaneous bleeding and a platelet
immune diseases and hence it is necessary to evaluate count of more than 50,000/cumm—no drug therapy—
children with chronic ITP particularly adolescent females, defensive management—nonfrantic watchful waiting is
for evidence of concomitant autoimmune disease. the rule.
In severe ITP with mucosal bleeding and platelet count
Thrombopoietin in ITP of less than 20,000/cumm, prompt vigorous treatment
with steroids should be started.
AMG 531 (Romiplostim, Nplate) and Eltrombopag In nonresponders, and during emergency, IVIgG
(Promacta).77,78 should be given. If IVIgG is not available or if the patient
Stimulating the thrombopoietin (TPO) receptor cannot afford, in life-threatening conditions, splenectomy
increases the platelet production has been successful by may be done, particularly in children above 6 to 7 years.
drugs or various agents.79 Intravenous anti-D globulin and intravenous methy
First-generation agents—recombinant human throm- lprednisolone bolus dose may be tried before splenectomy.
bopoietin (rHuTPO) and pegylated recombinant human
megakaryocyte growth and development factor (PEG
rHuMGDF). In Chronic ITP
• First-generation agents “Treat the child and not the platelet count.” No treatment
Recombinant human thrombopoietin (rHuTPO) is required for cutaneous purpura and ecchymosis with
and pegylated recombinant human megakaryocyte a platelet count above 20 to 30,000/cumm. For platelet
growth and development factor (PEG rHuMGDF)-- counts less than 20,000/cumm and mucosal bleeding, 1 to
showed promise, however antibody formation to PEG 2 courses of steroids may be given for 3 to 4 weeks each
rHuMGDF led to the discontinuation of both agents. time. Low minimum required maintenance doses may
• Second-generation agents be continued in partial responders. Avoid long-term high
TPO agonist antibodies--have been developed to dose steroids. Nonresponders may be treated with IVIgG
reduce or eliminate the problem of antigenicity. and booster dose if required or anti-D globulin may be
– TPO peptide mimetics given intravenously along with maintenance booster dose
– TPO non-peptide mimetics. if required. Pulse methylprednisolone therapy is other
– AMG 531 (romiplostim, Nplate) and alternative for nonaffording patients. Avoid splenectomy
– Eltrombopag (Promacta). in children below 5 to 6 years and before 1 year of onset
AMG 531 and eltrombopag are able to stimulate platelet of the disease. In 2 percent of cases who do not respond
production in patients with ITP. to above therapy, immunosuppressive drugs may be used
Clinical studies for some of these agents, such as AMG with caution.
531 (romiplostim, Nplate) and eltrombopag (Promacta), ITP in children are generally benign conditions leading
are demonstrating their relative safety and efficacy in to only in few patients, serious complications and long
increasing platelet counts in patients with ITP. There are term squeal. 10 to 20 percent of children ultimately develop
currently seven second-generation TPO receptor agonists chronic ITP whose platelet count majority of times remains
that have been reported in the literature, representing the in relatively safe range more than 30,000/cumm and whose
potential advantages. bleeding time is fairly normal hence needs only defensive
management and nonfrantic watchful waiting. IVIg or high
Dose dose steroid may benefit some patients who have evidence
of clinical bleeding and severe thrombocytopenia and
AMG 531 (romiplostim, Nplate) and eltrombopag
splenectomy may be of value in patients with chronic
(Promacta) (0.2 to 10 microg - 1 or 3 ug per kilogram of
ITP with recurrent manifestations of bleeding or very low
body weight six weekly subcutaneous injections).
count. In acute ITP with severe bleeding like intracranial
No major adverse reactions.
hemorrhage or severe menorrhagia, splenectomy may be
indicated above the age of 5 years amongst those children
CONCLUSION who cannot afford IVIg or are nonresponsive to steroids. In
‘ITP in children’ constitutes 90 percent cases which some children with recurrent thrombocytopenia periodic
are acute and most of them go into remission in 1 to 6 booster dose of IVIgG or methylprednisolone may be
required so as to keep the platelet count in the safe range. 15. Luiken QA, McMillan R, Lightsey AL, et al. Platelet
However, there is no way to predict which patients are at associated IgG in immune thrombocytopenic purpura.
high-risk for the development of ICH/severe hemorrhage Blood. 1979;50:317-25.
and which patient will respond to which type of therapy 16. Hegde UM, Bower A, Powell DK, et al. Detection of platelet
bound antibodies in autoimmune thrombocytopenia by
and hence the decision to treat the child with ITP is more
enzyme linked assay. Vox Sang. 1981;41:306-12.
often based on entire clinical picture and severity of the
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the platelet count. and genetic study. Ann Intern Med. 1981;94:781-2.
18. Cheung NK, Hilgarnter MW, Schulman I, McFall P, Glader
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44. Bussel JB, Kimberley RP, Inman RD, et al. IVIgG treatment Aust Pediatr J. 1972;8:311.
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45. Mori PG, Mancuso G, Principe DD, Duse M, Miniero R, treatment with steroid and splenectomy. Arch Int Med.
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C H A P T E R 32
Platelet Function Disorders
Shanaz Khodaiji
Platelet function disorders are difficult to diagnose The major structural features of platelets include:
because the laboratory assays are time-consuming, and • Cell membrane which is a bilipid membrane and is
require skilled technical staff to standardize and perform. the site of some complex coagulation activities (Fig.
Mild platelet function disorders such as primary secretory 1). It contains several glycoproteins that function as
defects are rare and are a diagnostic challenge. They may surface receptors. The two membrane systems are the
be missed due to their heterogeneity and failure to perform endoplasmic reticulum or dense tubular system and
the appropriate tests for their diagnosis. the plasma membrane-derived, surface connected
The latest guidelines on platelet function testing have canalicular system (Fig. 1).
been published in 2011, a long time after the previous The glycoprotein Gp Ib is a binding site for von
guidelines, which were published by the British Committee Willebrand (vWF). It is an intrinsic transmembrane
for Standards in Hematology (BCSH) in 1988.1 Lack of protein with a molecular weight of 140 kilodaltons. The
standardization of platelet function tests necessitated the vWF is necessary for platelet adhesion, an important
publication of the new guidelines. The “gold standard” for first step in platelet function.
platelet function testing is platelet aggregometry, which The other glycoprotein GpIIb/IIIa is a prominent
was first described in the 1960s.2 calcium dependent membrane protein complex that
In Germany, Austria, and Switzerland, it is estimated functions as a fibrinogen receptor. Fibrinogen binding
that 2 children per million are affected by this disorder. is necessary for platelet aggregation to occur.
Ethnicity and consanguinity have a role to play in this • Circumferential microtubular system and micro
condition. A study carried out by Israels et al. has shown filaments: Microtubules lying just beneath the platelet
that abnormalities of platelet function are as common membrane form a circumferential band round the
as von Willebrand disease (vWD) in patients with muco- platelet (Fig. 1). The microtubules are composed of
cutaneous bleeding.3 Platelet function disorders are more tubulin which plays a role in cytoskeletal support
common than previously diagnosed as has been reported and in contraction of the stimulated platelet.
by a Canadian registry having 577 cases since 2004 (http:// Closely associated microfilaments contain actin and
www.fhs.mcmaster.ca/chr/data.html). Many of these participate in platelet pseudopod formation
patients have incompletely characterized platelet defects, • Dense tubular system is so named because of the
thus suggesting that these disorders are not as rare as presence of an amorphous electron-dense material.
previously believed. This system selectively binds divalent cations and
serves as the platelet calcium reservoir (calcium flux
PLATELET STRUCTURE is critical to platelet function). The dense tubular
system is also the site of platelet cyclo-oxygenase and
Platelets are discoid smooth surfaced cells, 3–4 µm in prostaglandin synthesis (Fig. 1).
diameter that are present in whole blood in a concentration • Platelet granules: Granules store various substances
of 150,000–400,000/µL. that are secreted during platelet aggregation or are
Chapter-32 Platelet Function Disorders 333
Fig. 2 Interaction between vessel wall and platelets following

injury (adhesion and aggregation)4
Fig. 1 Schematic diagram of morphology of a platelet.

Abbreviations: AG: a granules; CM: Cell membrane; DG: Dense Sequence of events occurring when platelets come in
granule; DT: Dense tubule; GLY: Glycogen; M: Mitochondria; MT: contact with injured vessel wall
Microtubule; OC: Open canaliculus. 1. Collagen and endothelial proteins are exposed on the
internal surface of the affected vessel (Fig. 2).
instrumental to aggregation. The four types of granules 2. Platelet adhesion is initiated when vWF binds to
are: collagen (from exposed endothelium) and Gp Ib/V/IX
1. α granules store a variety of proteins that are complex on the platelet surface (Fig. 2). Simultaneously
secreted by stimulated platelets. These include platelets bind directly to collagen via Gp VI membrane
platelet factor IV, factor V, vWF, fibrinogen, receptor complex and the Gp Ia/IIa integrin which is
β-thromboglobulin, and platelet derived growth a collagen receptor (Fig. 2). This in turn binds to the
factor (PDGF). Various glycoproteins important G chain of fibrinogen and links the platelets to one
to adhesion are also contained in these granules another (Fig. 2). The platelets form a layer on the
e.g. osteonectin, fibronectin and thrombospondin breached surface of the vessel wall causing platelet
(Figs 1 and 2). activation, which leads to the next step of primary
2. Dense granules are electron dense particles that hemostasis (Fig. 2).
contain high concentration of ADP and Ca2+ as well Since Bernard–Soulier syndrome (BSS) is
as serotonin and other nucleotides. It also contains characterized by the absence of the platelet membrane
ATP. These substances are released upon platelet GP Ib complex, normal adhesion to the vWF-A1
stimulation and enhance platelet aggregation (Figs domain cannot take place.
1 and 3). 3. The platelets change shape rapidly from discoid to
3. Lysosomes contain hydrolytic enzymes. round and spread with filopodia and lamellipodia
4. Peroxisomes contain catalase. (stellate form). Platelet adhesion initiates intracellular
• Externally communicating open canalicular system signaling and platelet activation, by which substances
(Fig. 1). such as ADP are released from platelet granules and
Platelets perform many functions and are therefore thromboxane, (TXA2) are generated (Fig. 3).
associated with many pathological processes. 4. Platelets stagnate and transiently stick to (adhere) or
Platelet function in bleeding and clotting: When platelets roll along the vessel wall.
come in contact with a damaged vessel wall, they undergo 5. Platelet aggregation occurs when vWF and fibrinogen
shape change, adhesion, release of secretory granules bind to activated platelets through the Gp IIb/IIIa
and aggregation leading to exposure of the procoagulant complex (Fig. 2). Calcium ions are necessary for this
surface. As a result, a hemostatic plug is formed that process. This is the final step in the formation of the
prevents further blood loss by occluding blood vessels at platelet plug. Absent or severely reduced platelet
the site of injury. If this sequence of events is disturbed, aggregation is seen in glanzmann thrombasthenia
clot formation is impaired and patient is at increased risk (GT), which is due to decreased levels/function of the
of bleeding. Gp IIb/IIIa complex.
such as the lumiaggregometer, which can assess platelet

function from whole blood and flow cytometry, which can
detect the presence or absence of antigens on the surface
of the platelet as well as some secretory pathway defects.
These are also used for monitoring antiplatelet therapy in
patients of coronary artery disease.
Initial Approach to Diagnosing Platelet

Dysfunction
Though there are not many studies comparing platelet
Fig. 3 Platelet response to activation4 function in pediatric age group and adults, all studies
conclude that with the exception of neonates, there is no
difference in aggregation patterns between the two groups.3
The first step in investigating a suspected bleeding
disorder is to take a detailed personal and family history
followed by examination and ordering of appropriate lab
tests.
Clinical Features of Platelet Defects

Primary hemostasis involves formation of the platelet plug
and secondary hemostasis is represented by the initiation
of the coagulation process up to formation of the fibrin clot.
Defects of both these functions have characteristic features,
which can distinguish these 2 conditions (Table 1).
In addition, mucosal membrane bleeding can occur
from the gastrointestinal and genitourinary tracts and
pulmonary sites in patients with platelet functional defects.
The petechiae and purpura are usually symmetrical
and found on the extremities as well as the torso, unlike
the vascular disorders where petechiae and purpura are
Fig. 4 Formation of a procoagulant surface4
seen in dependent areas.
6. The hemostatic plug is stabilized (Fig. 4) by reactions Platelet Function Defects may be Hereditary,
such as activation of Gp llb/IIIa complex, exposure Acquired or Drug-Induced
of anionic phospholipids on the platelet surface and
Acquired platelet function defects are associated with
production of procoagulant microvesicles.
many diseases and are more commonly seen than the
7. As the activation process proceeds, the actin-myosin
hereditary disorders. They often cause clinically significant
complexes within each platelet shorten and draw the
bleeding.
platelet mass together, which results in clot retraction.
8. TXA2 generation induces further platelet aggregation
(Fig. 2) and vasoconstriction, slowing the flow of blood
Table 1 Characteristic bleeding patterns seen in primary and
and increasing the shear forces.
secondary hemostatic defects
9. Platelet function is regulated in vivo by the presence of
nitric oxide. Primary/platelet defect Secondary/coagulation defect
While coagulation screening tests such as the PT and Immediate excessive Delayed bleeding
the APTT are standardized in all labs, and not costly, there bleeding after trauma
are no easy diagnostic screening tests for platelet function Petechiae, epistaxis, Bleeding in the deeper
defects. Owing to the heterogeneity of these disorders it menorrhagia, gingival tissues, joints (hemarthrosis),
is difficult to find a platelet function test which is 100% bleeding (mucocutaneous and intramuscular
sensitive. Moreover, these tests are laborious, expensive bleeds) accompanied by a hematomas are seen
and require trained and experienced persons to perform history of easy, spontaneous commonly
and interpret. Newer instruments are now available bleeding
Flow chart 1 Approach to diagnosis of platelet function disorders in children3
Key: Gray boxes – Investigations; hatched boxes – results; circles – diagnosis; dotted circles - suspected diagnoses.
Abbreviations: BSS: Bernard–Soulier syndrome; CAMT: Congenital amegakaryocytic thrombocytopenia; ATRUS: Amegakaryocytic
thrombocytopenia with radio-ulnar synostosis; FPD/AML: Familial platelet disorder and predisposition to acute myelogenous leukemia;
GT: Glanzmann thrombasthenia; GPS: Gray platelet syndrome; SGD: Storage granule disorder; TAR: Thrombocytopenia with absent radii;
THC2: Autosomal dominant thrombocytopenia; XLT: X-linked thrombocytopenia.
An algorithm for evaluation of children with suspected • Abnormalities of receptors for

platelet disorders is described in flow chart 1. – Thromboxane A2
– P2Y12
CLASSIFICATION OF HEREDITARY – α2-adrenergic receptor
PLATELET FUNCTION DEFECTS3 • Platelet granule defects
– Dense granule defects leading to δ-storage
• Receptor abnormalities affecting platelet adhesion
pool deficiency, Hermansky–Pudlak syndrome,
– Gp Ib–IX–V abnormality causing Bernard–Soulier
Chediak–Higashi syndrome, thrombocytopenia
syndrome or platelet-type vWD.
with absent radii (TAR) syndrome
– Gp IIb-IIIa (αIIbβ3) defect causing Glanzmann
– Alpha granules defects such as Gray platelet
thrombasthenia
syndrome ARC syndrome, Quebec platelet
– Gp Ia-IIa (α2β1) defect
disorder, Paris–Trousseau–Jacobsen syndrome
– Gp VI defect
– α and δ granules storage pool deficiency
– Gp IV defect
• Abnormalities of signal-transduction of primary hemostatic defects both vWD and platelet

– Primary secretion defects dysfunction are found. VWD can also present with
– Abnormalities of the AA or TXA2 pathway thrombocytopenia. Macrothrombocytopenia can be
– Gαq deficiency seen in patients of type 2B vWD.3
– Partial selective PLC-β2 deficiency • Bernard Soulier syndrome (BSS): BSS is a manifestation
– Defects in pleckstrin phosphorylation of a defect in platelet adhesion and is inherited as an
– Defects in Ca2+ mobilization autosomal recessive trait. Heterozygotes are often
• Cytoskeleton related abnormalities asymptomatic. The platelets in this syndrome lack
– Disorders of MYH9 e.g. May–Hegglin anomaly, membrane glycoproteins Ib, V and IX. It mimics vWD
Fechtner syndrome, Epstein syndrome, Sebastian in that the clinical manifestations are similar.
syndrome – Clinical manifestations: Patient can present with,
– Wiskott–Aldrich syndrome epistaxis, menorrhagia, petechiae and purpura.
– X-linked thrombocytopenia Easy bruising, which is sometimes spontaneous
• Membrane phospholipid abnormalities can be the presenting feature.
Scott syndrome – Laboratory manifestations:
These disorders also have thrombocytopenia in addition - Thrombocytopenia (mild/moderate) with
to functional defects.3 macrothrombocytes
Secondary aggregation defects occur more frequently - Abnormal template BT but clot retraction is
than primary or hereditary platelet disorders. Storage pool normal
defects are the most common in the hereditary as well as - Abnormal petechiometer test (in children)
acquired groups. - Reduced aggregation response to ristocetin.
Other rare disorders are: The response may be abnormally increased in
• Quebec platelet syndrome characterized by delayed- type 2B vWD.
onset bleeding, impaired epinephrine induced platelet The template BT is not standardized in children; hence
aggregation. The diagnosis is confirmed by presence of a petechiometer is more useful in children for assessing
platelet urokinase by immunoblotting or ELISA platelet function defects or vascular type bleeding.
• Scott syndrome presenting with mucocutaneous Peripheral blood smear examination shows macro-
bleeding but normal aggregation with all agonists. platelets along with mild thrombocytopenia in most
The confirmatory test is by demonstrating absence patients of BSS but not in vWD. Tests for diagnosing
of annexin A5 binding to activated platelets by flow vWD are the factor VIII coagulant activity (factor VIII:C),
cytometry factor VIII related antigen (factor VIII:RAg) and ristocetin
• Thromboxane A2 receptor defect presents with muco- cofactor activity.
cutaneous bleeding, absent or decreased aggregation Treatment: Platelet concentrates are given only if bleeding
with AA and U46619. The molecular assay used for is life threatening.
confirmation of this disorder is mutational analysis of
TBXA2R gene. GLANZMANN’S THROMBASTHENIA
Secondary or acquired defects of platelet function are
encountered more frequently than hereditary disorders, Glanzmann’s thrombasthenia (GT) and essential
the most common being storage pool defects (SPD). athrombia are very rare primary aggregation disorders.
These divisions are somewhat arbitrary but are Patients of GT lack the membrane glycoprotein Gp IIb/
convenient for categorizing the defects and interpreting IIIa complex.
laboratory tests.
The hereditary disorders are discussed below: Clinical Findings
• von Willebrand disease (vWD): von Willebrand Patients present with history of easy and sometimes
disease can present in children with mucocutaneous spontaneous bruising and petechiae, menorrhagia and
bleeding in the absence of a family history, in which very occasionally hemarthroses. The symptoms of bleeding
case it is difficult to differentiate it from platelet usually disappear as the patient grows older. This feature is
function disorders. Testing for both vWD, and common to most of the hereditary hemostasis defects.
platelet function defects is helpful. Both conditions
are common specially in children, and combined
Laboratory Findings
disorders may be present. Quiroga et al. demonstrated
that in 11.5% of 113 individuals (ages 4–50 years) with The BT is prolonged. Platelet function defects such
mucocutaneous bleeding and laboratory evidence as reduced or absent primary aggregation with ADP,
epinephrine, thrombin and collagen and reduced platelet

factor 3 availability are seen.
Clot retraction is impaired in GT but is normal in
essential athrombia.
Therapy
Treatment for both the disorders is platelet transfusion
only if the bleeding is severe and life threatening. Some
workers prefer to infuse platelets till the bleeding stops
rather than relying on empirical monitoring of aggregation
patterns or the template bleeding time. The vast majority
of patients with hereditary platelet function defect of any
type will stop bleeding immediately with the appropriate Fig. 5 Aggregation pattern in patients on acetylsalicylic acid
use of platelets. (ASA)5
Abbreviations: ADP: shows a disaggregation in the curve; Collagen
HEREDITARY STORAGE POOL DEFECT (SPD) (COL) normal aggregation; Arachidonic acid (ARA), aggregation
shows reduced response.
Clinical Features
These patients have a variable pattern of inheritance and Laboratory Findings
commonly present with mucocutaneous hemorrhage,
These patients have a prolonged BT and show abnormal
hematuria and epistaxis. Petechiae are not so common
collagen adhesion. Secondary aggregation response to
in these disorders as in other platelet function disorders.
ADP (Fig. 5) and epinephrine is absent. Aggregation to
These patients usually present with spontaneous bleeds.
collagen is normal or absent. Aggregation response to AA is
absent (Fig. 5) along with absence of cyclo-oxygenase and/
Laboratory Findings or thromboxane synthetase enzymes. The same findings
Prolonged BT and abnormal collagen-induced aggrega are seen in patients taking aspirin or COX-2 inhibitors.
tion is noted (absent or markedly reduced). Absent
secondary aggregation wave to ADP and epinephrine Therapy
is seen although the primary waves are present. Normal
When clinically significant bleeding occurs, treatment
ristocetin aggregation and usually normal response to AA
consists of infusing platelet concentrates. Steroids have
is observed.
been used in some patients with beneficial effects.
Therapy
ACQUIRED PLATELET FUNCTION DEFECTS
In case of heavy bleeding, patients can be given platelet
Patients with acquired platelet function defects present
transfusion.
with profuse bleeding during surgery or trauma. This is
a common cause of bleeding in adults. Acquired platelet
ASPIRIN-LIKE DEFECTS function defects can be seen in a variety of conditions but
This is an inherited condition which mimics the aspirin- the platelet aggregation abnormalities are not specific to
induced acquired platelet function defect. It is very rare any condition, unlike the hereditary disorders.
and is inherited as an autosomal dominant trait. Causes of acquired platelet function defects:
• Myeloproliferative syndromes7
Clinical Features – Essential thrombocythemia (ET)
– Agnogenic myeloid metaplasia
Like other platelet function defects these patients present – Paroxysmal nocturnal hemoglobinuria (PNH)
with easy, spontaneous bruising and bleeding from – Polycythemia vera (PV)
mucocutaneous areas, epistaxis, menorrhagia, petechiae – Chronic myeloid leukemia (CML)
and purpura. This defect may be due to a hereditary – MDS, RAEB
deficiency of the enzyme cydo-oxygenase or the enzyme – Sideroblastic anemia
thromboxane synthetase. • Uremia
• Malignant paraproteinemias Fibrinogen degradation products: Fragments D and E

• Waldenström’s macroglobulinemia appear to bind to the platelet membrane causing a very
• Multiple myeloma severe bleeding and abnormalities of platelet aggregation.
• Leukemic reticuloendotheliosis Nutritional anemia: Iron deficiency or vitamin B12 and
• Autoimmune disorders folic acid deficiency also demonstrate a platelet function
– Collagen vascular disease defect, with or without clinical bleeding.
– Antiplatelet antibodies
• Presence of FDPs DRUG-INDUCED PLATELET FUNCTION
– Disseminated intravascular coagulation DEFECTS7
– Primary fibrinolysis
• Anemia Numerous drugs cause defective platelet aggregation;
– Severe iron deficiency though not all of them cause significant clinical bleeding.
– Severe folate or B12 deficiency Drugs act through the following pathways:
• Drug induced • Interaction with platelet membrane or receptors.
The drugs acting through this mechanism are
propranolol, ampicillin, penicillin, diphenhydramine,
MYELOPROLIFERATIVE SYNDROMES phenylephrine plus promethazine and alcohol.
Acquired platelet function defects are often seen in these Other drugs included in this class are reserpine,
disorders. However, the platelet aggregation pattern is not nitrofurantoin, dextran and hydroxyethyl starch
characteristic of any one disorder. • Inhibition of prostaglandin pathways. The most
common drugs in this category are aspirin, indometh-
acin, phenylbutazone, ibuprofen, sulfinpyrazone and
UREMIA7
furosemide. Other, not commonly used drugs in this
Almost all patients of uremia have a platelet function category are quinacrine, mefenamic acid, tocopherol,
defect. It is thought that the circulating guanidinosuccinic hydrocortisone, methylprednisolone and cyclosporine
acid and/or hydroxyphenolic acid in these patients cause • Inhibition of phosphodiesterase activity. The most
a platelet function defect by reducing platelet factor 3 common drugs are caffeine, dipyridamole, amino-
activity. The aggregation pattern is not diagnostic of the phylline, theophylline and papaverine
condition. • Drugs with unknown mechanism. Acetazolamine, chlo-
Dialysis can reverse this effect and normalize platelet rtetracycline, hydroxychloroquine and nitroprusside.
function.
Other mechanisms such as altered prostaglandin COMMONLY USED DRUGS WHICH INHIBIT
metabolism have also been proposed in uremic patients.
PLATELET FUNCTION7
Treatment • Anti-inflammatory drugs
• Psychiatric drugs
Platelet concentrates are recommended for life threatening • Cardiovascular drugs
bleeding. Cryoprecipitate, desmopressin and estrogen • Antibiotics
compounds can also be used to correct bleeding time and • General and local anesthetics
control the hemorrhage. • Antihistamines
PARAPROTEIN DISORDERS HYPERACTIVE PLATELETS7

A majority of patients with malignant paraproteinemias Macrothrombocytes are commonly seen in hyper-
have platelet function defects. Paraproteins coat the coagulable states and in patients with thromboses. Large
platelet membrane in malignant and benign states, thereby platelets are immature platelets with more RNA content
impairing their function, which manifests as significant and this makes them prothrombotic.
bleeding and abnormal platelet aggregation curves.
Microvesicles and Platelet Procoagulant
MISCELLANEOUS CAUSES Activity
Autoimmune disorders: Systemic lupus erythematosus, A major advance in the last several years has been the
rheumatoid arthritis and scleroderma. discovery of small, membrane-bound microparticles
which are shed from plasma membrane of activated LABORATORY TESTS FOR PLATELET
platelets. These microparticles are rich in binding DISORDERS
sites for factor Va and Xa, thus conferring on them a
procoagulant property. This shedding is accompanied Diagnosing these disorders in the laboratory is a
by local collapse of the normal asymmetrical distribution challenging task, more so in children because large volumes
of plasma membrane phospholipids and the exposure of of blood are required for testing. Newer testing modalities
phosphatidylserine and phosphatidylethanolamine on are not universally available and need proper validation
the outer surface. especially in the pediatric age group. Standardization of
platelet function assays has recently been undertaken
PLATELET FUNCTION DEFECTS IN INFANTS and this effort is bound to improve the quality of testing
and diagnosis of these disorders. Specialized tests should
AND SMALL CHILDREN1 be made available to those requiring a diagnosis of rare
Hereditary platelet disorders such as GT and BSS are platelet function defects. 3
usually seen in infants and young children. They pose a • Basic clotting tests such as PT and APTT are mandatory
diagnostic dilemma in very young children mainly due to to exclude coagulation defects.
preanalytical errors occurring at this age. Blood collection • Measuring platelet number and size: A peripheral
for platelet function tests should be done on a free-flowing blood smear examination is necessary to assess
venous sample and not form heel or finger prick. At least platelet size, and granularity. Platelet morphology is
20 mL of blood is required for these assays; this may be helpful in diagnosis of gray platelet syndrome (GPS) in
8-10% of the total blood volume of a neonate and could which platelets are large and appear gray in color due
result in hypovolemic shock. Generally, 19-21G needles to lack of granules, or a borderline thrombocytopenia
are used in adults but these are too big for use in infants as with large platelets suggests BSS. The diagnosis of
they are likely to cause significant trauma to subcutaneous platelet function defects can be excluded in acute
tissues if a severe platelet disorder is present. For blood leukemias which present with petechiae or purpura
collection in infants and smaller children, a 23G needle is by examination of the peripheral smear. Accurate
used. The control sample should also be collected with a platelet counts are now possible with introduction of
23G needle.1 fluorescence flow cytometry in hematology analyzers,
There is a paucity of data regarding aggregation by improving the ability to distinguish large platelets
patterns in normal neonates due to difficulty in collecting from RBCs. The international reference method (IRM)
large volume of blood form healthy children. Few for platelet counting is by flow cytometry. If the platelet
studies conducted in this group of patients suggest that count is normal, but signs of platelet dysfunction
the platelets of infants are hyporeactive to all agonists are present, the differential diagnosis lies between
except ristocetin and sometimes collagen. Later, platelet a platelet function defect and a vascular defect, both
aggregation patterns and nucleotide release reactions of which may cause a prolonged bleeding time by the
reach adult levels. Therefore, platelet function should be standardized template method
assessed in children above 1 year of age. Family studies are • Global screening tests of platelet function: They are
recommended to assess inheritance pattern. Severe platelet normally performed as first line tests for assessing
function defects such as in GT or BSS show characteristic platelet function. Screening tests should be done to
patterns, which are easy to diagnose. Methods using less exclude the diagnosis of platelet function disorder so
blood volume like PFA-100/200 and flow cytometry can be that further specialized testing can be avoided. Thus,
used in children prior to performing platelet aggregation global platelet function tests can be performed along
assay for confirmation of the disorder. In BSS, PFA reveals with routine coagulation assays such as PT, APTT, and
a severe defect of primary hemostasis and this along with some specialized coagulation tests such as vWF assay.
macrothrombocytopenia and demonstration of absence The vWF panel of tests include vWF:Ag, vWF:RCo
or very low levels of the defective receptor, is sufficient and F:VIII:C assays and an accurate platelet count.
to start appropriate treatment for bleeding. Both GT and The template bleeding time is the simplest screening
BSS show prolonged closure times on the PFA. In severe test for platelet function and is easy to perform in all
unexplained bleeding (intracranial) when an inflicted laboratories. A prolonged BT can be followed up by
injury has to be differentiated from spontaneous bleeding testing on the PFA-100.1
in a patient with a severe bleeding diathesis, this approach Apart from the PFA analyzers, other instruments
is very helpful.1 are available to test platelet function. These include
those that can assess the effect of antiplatelet vWF activity and may therefore be longer in patients with
drugs. Thromboelastography (TEG) and Rotational blood group O. The CEPI-CT is usually prolonged in
Thromboelastometry (ROTEM) are tests of coagulation patients taking COX-1 inhibitors such as aspirin. This is
and platelet function which have found favor with not the case with the CADP-CT,
surgeons. Though used extensively in a surgical
setting, there is no proper validation of these systems, PFA-100 in vWD 1
and their routine use for diagnosing platelet function
defects is therefore not recommended currently.1 Abnormal CT on both CEPI and CADP cartridges is seen
in of vWD types 2A, 2B, 2M and 3 with a sensitivity of
>98%. The overall sensitivity of CT to vWD is lower (85-
Template Bleeding Time 90%) if type 1 vWD is also considered. There seems to be a
The oldest test of platelet function is the BT, which was significant correlation between vWF level and CT. Type 2N
described by Duke in 1910. It was previously recommended vWD shows normal results. The PFA-100 can also be used
for diagnosis of platelet function by the BCSH hemostasis for monitoring desmopressin therapy in vWD.
and thrombosis Task Force and is widely used in the
UK. However, there is lack of standardization of this test PFA-100 in Diagnosis of Hereditary Platelet
between laboratories.1 It is technologist dependent and Function Defects
highly subjective. It varies according to certain patient
characteristics such as age, gender, hematocrit, vascular Nonclosure is typically seen in both cartridges in patients
pattern, skin thickness and skin temperature. Therefore, it with severe platelet function defects such as GT, BSS and
is not reproducible and has a low sensitivity and specificity. platelet type or pseudo-vWD. The PFA-100 CT is not very
Also, it is invasive and for these reasons it is not routinely sensitive for mild platelet dysfunction. A recent study
recommended and performed. showed that the overall sensitivity was 83% and specificity
The template BT is standardized; therefore it possible 89% of the CEPI cartridge for primary hemostatic disorders.
to compare this parameter from different labs. It is CADP sensitivity was lower. The PFA-100 has shown a
performed by tying a sphygmomanometer cuff on the sensitivity of >90% in screening patients with menorrhagia
upper arm of the patient, which is inflated to 40 mm Hg. for vWD and platelet function disorders.
Using a spring-loaded template, an incision 5 mm long However, if the platelet defect is strongly suspected,
and 1 mm deep is made on the extensor surface of the a normal PFA result should be overlooked and specific
forearm. Avoids going into scar tissue and blood vessels, platelet function assays should be performed.
and make the incision within 60 seconds of inflating the
sphygmomanometer. The edges of the wound are blotted PLATELET FUNCTION ASSAYS
by filter paper at 30 second intervals until the bleeding
stops. Normal template BT is 2–9 minutes.1 Platelet Light Transmission Aggregometry
(LTA)1
Platelet Function Analyzer PFA-1001 Platelet aggregometry is considered the gold standard for
platelet function testing. It was invented in the early 1960s.
Assay Principle However, this test varies widely in laboratory practice due
The PFA-100 analyzer is made up of disposable cartridges to lack of standardization of the assays for many years.
containing apertures coated with either collagen- Recently, new guidelines for platelet aggregometry have
epinephrine (CEPI), or collagen-ADP (CADP). Blood been published.
collected in citrate (0.8 mL per cartridge) is aspirated at
high shear rates (5000-6000s-1) into the PFA analyzer. Principle
These agonists on the aperture initiate platelet adhesion,
activation and aggregation leading to rapid closure of the Platelet aggregometry works on the principle of optical
aperture. The end-point for each agonist is time taken to density in which upon addition of agonists the platelets
obstruct the blood flow. Nonclosure of the aperture is seen undergo a change in shape from discs to round structures
if the closure time (CT) exceeds 300 seconds. Since small with extended filopodia. This results in a transient
quantities of blood are required, it is useful for pediatric decrease in light transmission, followed by an increase as
patients too. the platelets aggregate. The increase in light transmission
It has been observed that a low platelet count (<100 (% aggregation) is measured at 370C by a photometer.
× 109/L) and anemia (<20% hematocrit) often cause a A secondary aggregation response curve is seen with a
prolonged CT. The CT also correlates inversely with plasma higher concentration of ADP and epinephrine. This is due
to TXA2 formation and release of the contents of platelet avoid changes in pH, which affect platelet aggregation
granules. Platelet agglutination by ristocetin, which and tests of nucleotide release. It is kept at room
changes the conformation of plasma vWF thereby making temperature till tested. It is stable for about 3 hours
it suitable to bind to the Gp Ib–IX–V complex, can also be • After removing the PRP, platelet poor plasma (PPP)
assessed by platelet aggregometry. for test and control is obtained by centrifugation of the
Platelet aggregometry is carried out with a complete remaining blood sample at 2000 rpm for 20 minutes
panel of agonist and require at least 15 mL of blood, which • Standardization of PRP: The platelet count of the PRP
is a major disadvantage in children. A recently introduced is adjusted to 200 – 400 × 109/L. If it is high, the PRP is
method for testing platelet function is whole blood diluted with the patient’s PPP. A platelet count lower
aggregometry, which requires a smaller blood volume and than 200 × 109/L gives rise to a diminished aggregation
measures platelet aggregation as the change in electrical response. In the case of a low platelet count, further
impedance between electrodes. It is not widely used centrifugation of PRP is not recommended because it
because it is more costly than platelet aggregation with induces platelet activation. The control PRP should be
PRP. A more recently developed multiplate analyzer is diluted to the same count and tested as a comparison.
also available that operates on the principle of electrical
impedance. It requires only 175 mL of blood and is Aggregating Agents1
therefore very useful in pediatric practice.
Five useful aggregating agents (agonists), which are
sufficient for the diagnosis of most functional platelet
Preparation of Patient1 disorders are ADP, collagen, ristocetin, epinephrine/
• The patient and control subjects should be off drugs, adrenaline and arachidonic acid (AA). An extended panel
beverages and foods which may affect aggregation for of agonists can be used to characterize other defects
at least 7 days prior to performing the test not defined by the primary panel. This includes gamma
• Both the patient and the control subject should fast thrombin, thrombin receptor activating peptides (TRAP),
overnight as chylomicrons may interfere with the collagen-related peptide, endoperoxide analog U46619
aggregation pattern and calcium ionophore A23187.
• 20 mL of venous blood is collected in citrate tubes A number of pre-analytical factors can influence the
keeping in mind the anticoagulant to blood ratio of 1:9 results and interpretation of platelet aggregation (Table 2).
• The blood should not be chilled as cold activates
platelets Interpretation (Fig. 6)
• PRP is obtained by centrifugation at room temperature
(18–22 °C) for 10–15 minutes at 1000 rpm ADP: This is tested in two dilutions, 2.5 and 5.0 µmol/L
• The PRP is pipetted out slowly, avoiding mixing of (Figs 6 and 7). A primary or reversible aggregation
cells form the buffy coat or RBCs. The PRP is taken curve is seen (Fig. 7) with ADP in low concentrations
in a stoppered plastic tube filled nearly to the top to (<0.5–2.5 µmol/L). ADP acts by binding to a membrane
Table 2 Technical factors which may influence platelet aggregation tests6

Centrifugation: At room temperature, not at 4°C. Speed should be adjusted so that red cells and white cells are removed but not the
larger platelets. Residual red cells in the PRP may hamper proper aggregation.
Time: Platelets are refractory to the effect of agonists for up to 30 minutes after centrifugation. Progressive increase in reactiveness
occurs thereafter.
Platelet count: Slow and weak aggregation observed with platelet counts below 150 or over 400 x 109/L.
A pH of less than 7.7 slows down aggregation while more than 8.0 increases aggregation.
Mixing speed: <800 rpm or> 1200 rpm slows aggregation.
Hematocrit: >0.55 is associated with less aggregation, especially in the secondary phase owing to the increased concentration of
citrate in PRP. It may also be difficult to obtain enough PPP. Centrifuging twice may help.
Temperature: <35°C causes decreased aggregation except to low dose ADP which may be enhanced.
Dirty cuvette may cause spontaneous platelet aggregation or interfere with the optics of the system.
Air bubbles in the cuvette cause large irregular oscillations even before the addition of agonists.
No stir bar: No response to any agonist obtained.
Note: These are illustrations and not actual tracings.

Fig. 6 Aggregometry patterns in rare platelet function disorders1
receptor resulting in release of Ca2+ ions. A complex with precedes aggregation with collagen. The duration of the
extracellular fibrinogen forms and the platelets change lag phase depends on the concentration of collagen used
shape from discs to rounded structures. This causes a and to the responsiveness of the platelets being tested. The
slight increase in light absorbance. After this, reversible lag phase is shorter with high concentrations of collagen
aggregation occurs when bound fibrinogen participates in and vice-versa. A single wave of aggregation follows the lag
the cell-to-cell interaction. When very low concentrations phase and is caused by activation of the AA pathway and
of ADP are used, the platelets disaggregate after the first release of the granules. A higher concentration of collagen
phase and do not show a secondary aggregation response. (>2 µg/mL) causes a spurt in calcium concentration within
In the presence of higher concentrations of ADP, an the platelet and this results in a direct release reaction
irreversible secondary wave of aggregation occurs owing without causing activation of the prostaglandin pathway.
to activation of the AA pathway (Fig. 6) resulting in release Collagen responses should therefore always be measured
of dense and α-granules. Therefore both concentrations of using 1 and 4 µg/mL concentrations.
ADP should be used as If only the high dose is used primary Ristocetin: It is used in a concentration of 1.4 mg/mL. If
wave defects (which measure the second pathway) will be used in higher concentrations, protein precipitation might
missed. occur in plasma and give rise to false results. Ristocetin
Collagen: This is available as a 1 mg/mL stock solution. A induces clumping of platelets (agglutination) by reacting
short lag phase lasting between 10 and 60 seconds (Fig. 6) with vWF and the membrane receptors. It does not act
Fig. 7 Pattern of platelet aggregation with different doses of ADP5

A - no agonist added or thrombasthenia platelets; B – fIrst wave reverse aggregation seen with low dose ADP or high dose ADP if platelets
inhibited by aspirin; C - normal biphasic aggregation seen with moderate doses of ADP; D – prompt irreversible aggregation stimulated
by high dose ADP and all potent agonists; E - platelets unresponsive to ristocetin as in vWD and BSS; F - normal response to ristocetin
through any of the usual aggregation pathways and not cause clotting), PAR-1 (SFLLRN) and PAR-4 (AYPGKF)
does not initially cause granule release. The response is TRAP peptides (if gamma thrombin is abnormal),
assessed on the basis of the angle of the initial slope (Fig. 6). collagen-related peptide (CRP), calcium ionophore and
Aggregation to ristocetin is tested in high and low doses. In PMA (Phorbol 12-myristate 13 acetate). These tests are
Type 2B and platelet type vWD the aggregation response available in highly specialized labs only.
to high dose ristocetin is normal but response to low dose
(0.5-0.7 mg/mL) ristocetin is hyper-reactive. If with high Aggregation Patterns in Normal Subjects
dose ristocetin the aggregation response is absent, then
an external source of vWF, (e.g. cryoprecipitate, or a vWF Normal platelet aggregation response to ADP varies with
concentrate) is added to the patient PRP and repeat testing the concentration used. It is usually a single reversible
is done to distinguish between a vWF or Gp Ib defect (BSS). primary wave with 1 µmol/mL of ADP or less, a biphasic
response with 2.5 µmol/mL and a single irreversible wave
Arachidonic acid: TXA2 generation and secretion of with 5 or 10 µmol/mL. With 1 and 4 µg/mL of collagen
granule occur with AA even if there is a defect of receptors a single wave is seen with a lag phase of not more than
for binding AA on the membrane or of endogenous release 1 minute. Ristocetin shows a single phase or biphasic
of arachidonate induced by phospholipase (Fig. 6). In case aggregation with 1.2 mg/mL. An abnormal response
of absence and/or inhibition of cyclo-oxygenase (e.g. to low dose ristocetin (0.5 mg/mL) is characteristic of
aspirin effect), the AA induced aggregation is abnormal type 2 vWD (Figs 6 and 7). A single or biphasic response
(Figs 5 and 6). Abnormal AA aggregation requires further is seen with 50–100 µmol/L of AA. Both primary and
testing with 1.0 µM U46619 to look for any thromboxane secondary aggregation curves are seen with 2–10 µmol/L
receptor abnormalities. of epinephrine.
Epinephrine/Adrenaline: It is used in a concentration of Every laboratory performing the test should establish
2.6 µmol/L. Unlike ADP there is no shape change before its own reference intervals.
aggregation but the response later is the same as in ADP The platelet aggregation pattern for some of the most
(Fig. 6). A severely reduced response to epinephrine is common inherited platelet function defects are shown in
noticed in normal people due to natural variations of the Table 3.
adrenoreceptor numbers. These patients are clinically
normal. SUBSTANCES COMMONLY AFFECTING
If abnormalities in the thrombin receptors, Gp VI,
PLATELET FUNCTION1
calcium mobilization and protein kinase C are suspected,
an extended panel of agonists can be used for categorizing • Cyclo-oxygenase inhibitors (irreversible)
the abnormality; such as gamma thrombin (which does Aspirin and all drugs containing acetylsalicylic acid.
Table 3 Diagnostic criteria and LTA patterns in heritable platelet defects1

Plt function Plt number and PFA result Aggregation Nucleotides assay Flow cytometry Remarks
defect morphology pattern results
vWD Type 1, Within normal limits Equally Abnormal response Within normal limits Within normal Confirm with vWF
2A and 3 prolonged to high dose limits panel tests for this
CADP/CEPi. ristocetin corrected subtype
Markedly by vWF addition
prolonged in
2A and 3
Type 2N VWD Within normal limits Both closure Ristocetin Within normal limits Increased vWF Abnormal vWF panel
or platelet type times aggregation binding to in Type 2B vWD/Loss
prolonged normal by adding platelets can be of high MW vWF in
plasma or cryo. measured platelet type vWD
Increased response
with low dose
GT Within normal limits CADP/CEPI Markedly low Within normal limits Reduced copy
both very response to all number of IIb/
prolonged agonists except IIIa
closure times high dose ristocetin
BSS Mild to moderate CADPI/CEPI Low response to Normal to high levels Reduced copy
thrombocytopenia with both very high dose ristocetin number of Gp Ib
large platelets prolonged not corrected by (heterozygotes
closure times adding vWF can also be
measured)
Defect of Reduced or normal count CADP normal Secondary Reduced ADP Reduced Reduced serotonin
dense granule Electron dense granules CT aggregation Increased ATP:ADP mepacrine release seen in
reduced as measured by CEPI response to ADP ratio reduced ATP uptake and Hermansky Pudlak
whole mount EM sometimes and epinephrine is release release and Chediak-Higashi
prolonged decreased syndromes which are
autosomal recessive
Defect of Within normal limit Normal Reduced secondary Normal but with Normal
secretion CADP CT aggregation defective release. mepacrine
CEPI response to ADP Reduced ATP release uptake but
sometimes and epinephrine defective release
prolonged
Aspirin-like Within normal limits CADP normal Absent AA Within normal limits Retest or defer for 10
defect CT response but days if patient is on
CEPI normal to U46619. aspirin or NSAIDs
normally Decreased
prolonged secondary
(NB can be aggregation to ADP
normal with and epinephrine
high vWF
levels)
Thromboxane Within normal limits CADP normal Absent AA and Within normal limits
receptor defect CT U46619 response
CEPI
sometimes
prolonged
Giant platelet Macrothrombocytopenia Sometimes Normal response to Normal/high Normal/
syndrome normal ristocetin High receptor
numbers per
platelet
Contd...
Contd...
Plt function Plt number and PFA result Aggregation Nucleotides assay Flow cytometry Remarks
defect morphology pattern results
Collagen Within normal limits Both Decreased collagen Within normal limits Reduced Gp Ia/
receptor prolonged aggregation IIa or Gp VI levels
defects
P2Y12 defect Within normal limits Both normal ADP-decreased Within normal limits Low P2Y12 Retest or defer
aggregation. number using if patient taking
Reversible response clopidogrel or other
at high doses anti-P2Y12 agents
Reduced secondary
wave
P2Y1 defect ? ? Decreased Within normal limits
response to
ADP-curves not
reversible
Scott Within normal limits Within Within normal Within normal limits Reduced Reduced PCI and ETP
syndrome normal limits limits expression
of phosph-
atidyl serine
on activated
platelets using
Annexin-V
Abbreviations: GT: Glanzmann thrombasthenia; BSS: Bernard Soulier syndrome; PCI: Prothrombin consumption index; ETP: Endogenous
thrombin potential. The result for P2Y1 defect are hypothetical as these are rare disorders and have not been completely studied.
• COX-1 and COX-2 inhibitors (reversible) • Foods and drinks commonly consumed affecting
• (Nonsteroidal anti-inflammatory drugs-NSAIDs) such platelet function are caffeine, alcohol, cumin,
as ibuprofen, indomethacin, mefenamic acid fenugreek, garlic, onion, ginger, ginseng, fish oil,
• Platelet receptor inhibitors such as abciximab, tamarind, turmeric, vitamins C and E and, Chinese
tirofiban, eptifibatide (αIIbb3), ticlopidine, mushroom.
clopidogrel, prasugrel (irreversible), cangrelor Many other substances apart from the ones mentioned
(revesible), ticagrelor (reversible) (P2Y12) above can affect platelet function. It is mandatory to take
• Phosphodiesterase inhibitors such as dipyridamole a drug and relevant dietary history from the patient. Re-
and cilostazole testing is recommended to confirm if the defect is transient.
• Anticoagulants vitamin K antagonists, heparinoids (Reprinted and modified with permission from Kottke-
and direct thrombin inhibitors. Marchant and Corcoran G. The laboratory diagnosis of
• Cardiovascular agents which include β-adrenergic platelet disorders. Arch Pathol Lab Med 2002:126:133-
blockers (propanolol), vasodilators (nitroprusside, 46 with permission from Archives of Pathology &
nitroglycerine), diuretics (furosemide) and calcium Laboratory Medicine. Copyright 2002. College of American
channel blockers Pathologists.)
• Antimicrobials: Antibacterials such as β-lactams
(penicillins, cephalosporins), antifungals FLOW CYTOMETRY1
(amphotericin), antimalarial (hydroxychloroquine)
and nitrofurantoin Flow cytometry is used to quantify glycoproteins in GT
• Chemotherapeutic drugs: L-aspraraginase, vincristine, and BSS. Flow cytometric tests are also available for
plicamycin measurement of dense granules using mepacrine uptake
• Psychotropics drugs: Antidepressants (imipramine), and release, and for measurement of microparticle
phenothiazines (chloropromazine) and local and procoagulant activity. These are used in the diagnosis of
general anesthetic agents (halothane) SPDs. Flow cytometry can be employed to confirm certain
• Thrombolytic drugs: Streptokinase, urokinase, tissue aggregometry findings such as (Gp Ia/IIa and Gp VI) and
plasminogen activator (TPA) PAR-1 receptor densities in collagen defects. Platelet
• Miscellaneous drugs such as clofibrate, dextrans, activation can be measured in response to routinely used
guaifenesin (expectorant) and radiographic contrast agonists, dense granule content, and exposure of anionic
media phospholipids. Citrated whole blood is used for analysis.
Delay in performing the test can cause platelet activation resulting in an increased ATP:ADP ratio. On the other
and give false results. The test is performed by adding hand, a release defect shows normal ADP levels and
fluorescent-labeled antibodies to the blood, incubating ATP:ADP ratio but decreased ADP release.
the tubes at room temperature in the dark, then diluting Serotonin (5-HT) is stored in the platelet dense granules
the samples to a final volume of between 1 mL and 2 mL. and the uptake and release of radiolabelled serotonin into
The tubes should be mixed gently, by tapping to avoid and from the platelets can be measured by ELISA tests.
platelet aggregation. Commercial antibodies/reagents
are available that can quantify the various receptors. WHOLE BLOOD AGGREGOMETRY1
Receptor numbers may be lower in neonates. A limitation
of this method is that receptors with a density of less A lumiaggregometer measures platelet function using
than 500 receptors/platelet cannot be measured, so the electrical impedance in whole blood or optical density
test cannot always be used reliably to detect reduced in plasma (LTA) while simultaneously measuring dense
number of receptors. It is possible to measure platelet granule secretion (ATP release) by the luminescence
procoagulant activity, apoptosis and microparticles by method. It allows study of platelet function in whole blood
incubating samples with high affinity probes against in presence of other cells before decay of labile modulators.
phosphatidylserine (e.g. Annexin-V) and activating the The quantity of blood required is less compared to LTA.
cells with calcium ionophore, collagen-related peptide or The turbidometric features permit the performance of
combinations of thrombin and collagen. The diagnosis of ristocetin cofactor assay and sticky platelet syndrome tests.
Scott syndrome and related disorders though very rare can It can also be used to a) monitor aspirin therapy by looking
be made with these assays. for lack of aggregation response to collagen in whole blood
b) monitor plavix therapy by ADP response in whole blood
c) to monitor DDAVP administration by ristocetin induced
MEASUREMENT OF NUCLEOTIDES1 aggregation in whole blood and d) to monitor Gp IIb/IIIa
Measuring the adenine nucleotides is an important receptor blockers and other anti-platelet drugs.
additional diagnostic method used along with A study carried out by UK NEQAS surveyed 169
aggregometry for detecting deficiency in dense granule hemostasis centers. They found that out of 88 centers
numbers or content such as seen in SPDs or degranulation doing platelet studies, only 4 performed whole blood
or release defects in granules. These defects are easily aggregometry. Very few studies are available which have
missed on platelet aggregometry alone. In spite of validated this new modality clinically and in the lab with
nucleotide measurement being an easy to perform test, the conventional LTA.
in which ATP is measured by simple bioluminescent
assays (using firefly luciferin/luciferase assays), it is rarely OTHER TESTS USED FOR MEASURING
performed in a routine laboratory. Therefore, many PLATELET AGGREGATION
disorders of platelet storage and secretion defects are
being missed. Rapid Platelet Function Assay by Ultegra–
The lumiaggregometer is capable of performing RPFA1
simple assays of released platelet nucleotides in real This is a turbidometric test which works on the principle
time with whole blood or PRP. Assessment of ATP levels of optical detection that correlates platelet aggregation
during platelet aggregation and release of ATP during the with an increase in light transmittance. This test correlates
secondary aggregation phase can be measured by a lumi- well with platelet aggregometry and was originally used to
aggregometer. However, it is not possible to distinguish monitor antiplatelet drugs by measuring the anti Gp IIb/
between SPD and defects in release of secretory granules IIIa complex.
using this approach. Therefore, many laboratories measure
the total content of ADP and ATP of the platelet by making
Hemostasis Analysis System
a lysate of platelets. These assays can be performed on
frozen samples which can be transported. The platelet contractile force (PCF), clot elastic modulus
The platelet contains 2 nucleotide pools: the metabolic (CEM) and thrombin generation time (TGT) are measured
pool and the dense granule or storage pool, which makes in 700 µL of whole blood by this instrument. It is capable
up 60% of the total content. The ATP:ADP ratio is therefore of diagnosing both thrombophilia and bleeding disorders
important as there is a marked difference between the and is also used to monitor treatment in these conditions.
relative concentrations in the two pools. Storage defects It is popularly used as a POC instrument in surgical
have a decreased amount of stored and released ADP settings, cardiology clinics and ICUs.
ELISA, RIA and Western Blot Platelet function testing was a much-neglected area as
the previous guidelines were published in the late 1980s.
Platelet α granule proteins comprising of platelet factor A variety of new tests and techniques have been added
4 (PF4) and β-thromboglobulin can be measured by
to the armamentarium of platelet function testing. These
various techniques such as ELISA, RIA or western
include platelet size distribution profiling and molecular
blot. They are contributory to the diagnosis of quebec
markers of platelet reactivity. Lumiaggregation has added
platelet disorder. However, there are problems with
a significant new dimension to assessment of platelet
reproducibility and interpretation of results. Adenine
function. Flow cytomertry provides a variety of specific
nucleotide and serotonin release from the dense granules
tests that are very useful in diagnosing various defects.
are best measured by a specialist laboratory. The release
Reliable but simple to use whole blood tests that attempt to
from α-granules indicates activation of platelets and
thrombotic tendency. Platelet vWF is measured to simulate in vivo hemostasis can be used to screen samples
diagnose some variants of vWD. If the study suggests rapidly before applying the existing battery of tests. The
a defect in the prostaglandin pathway, TXA2 can be general consensus is that the bleeding time should be
estimated quantitatively by radioimmuno assay. Highly replaced. Many of the simpler platelet function tests
specific assays of various steps in AA metabolism are could be potentially utilized as point of care instruments
available in specialized laboratories.1 for assessing bleeding risk and monitoring antiplatelet
Electron microscopy has made it possible to detect treatment. It is possible that in the near future important
ultra-structural abnormalities in platelets of patients with developments in the platelet genome will be made leading
platelet function defects. Dense granule defects can now be to exciting advances in this field such as platelet specific
diagnosed by whole mount electron microscopy. Substances microarrays which may have a significant impact on
released from the granules can also be measured.1 the diagnosis and management of patients with either
Molecular diagnosis of inherited platelet function thrombotic or hemostatic defects.
defects is confirmatory in affected individuals and their
family members and for antenatal diagnosis. It is easily REFERENCES
done in GT and BSS because the number of candidate
1. Guidelines for the laboratory investigation of heritable
genes is small. Diagnosis for clinically suspected cases of disorders of platelet function Paul Harrison,1 Ian
GT and BSS can be confirmed by direct sequencing of PCR- Mackie,2 Andrew Mumford,3 Carol Briggs,4 Ri Liesner,5
amplified genomic DNA. Individual affected genes can Mark Winter,6 Sam Machin2 and British Committee for
occasionally be identified in patients with mild defects e.g. Standards in Haematology British Journal of Haematology
MYH9 related disorder, CAMT, TAR, WAS, thromboxane 2011;155(1):30-44.
and P2Y12 ADP receptor defects and GPVI defects. 2. Harrison P. Platelet function analysis. Blood reviews
2005;19:III–123.
Molecular analysis of these disorders is mostly done in
3. Israels SJ, Kahr WHA, Blanchette VS, Luban NLC, Rivard
research laboratories. In regions having a prevalence of GE, Rand ML. Platelet disorders in children : A diagnostic
a specific mutation, e.g. 16bp deletion in HPS1 in Puerto approach. Pediatr. Blood Cancer. 2011;56:975-83.
Ricans with Hermansky-Pudlak syndrome, allele specific 4. Rao AK, Gabbeta J, Congenital Disorders of Platelet Signal
mutation detection strategies help in quick detection of Transduction Arterioscler. Thromb Vasc Biol. 2000;20:285-
the molecular defect.1 9.
5. Mani H, Luxembourg B, Kla¨ffling C, Erbe M, Lindhoff-
Last E. Use of native or platelet count adjusted platelet
CONCLUSION rich plasma for platelet aggregation measurements. J Clin
Nonavailability of laboratories to perform platelet function Pathol. 2005;58:747–50. doi: 10.1136/jcp.2004.022129.
6. Laffan MA, Manning RA. Investigation of haemostasis. In:
assays makes diagnosing inherited platelet disorders in Lewis SM, Bain BJ, Bates I, Eds. Dacie and Lewis’ Practical
children very difficult. Screening tests can be done in most Haematology. 9th ed. London: Churchill Livingstone,
routine laboratories. These include platelet count, MPV, Harcourt Publishers. 2001.pp.339-90.
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C H A P T E R 33
Pediatric Thrombosis
Rashmi Dalvi
Thromboembolic disease represents an infrequent event in childhood, however, one associated with considerable mortality and
morbidity. The incidence of venous thromboembolism (VTE) has been reported at about 0.07/10,000 children, 5.3 percent of pediatric
admissions and 2.4 percent of newborns in intensive care units. Neonates are at highest risk, possibly because of physiologically
lower anticoagulant levels and markedly reduced fibrinolytic activity. The incidence of vascular accidents decreases significantly
after infancy, with a second peak during puberty and adolescence, again associated with reduced fibrinolytic activity. Although in the
past, this was more an issue in the adult domain, thrombotic disorders are now an increasingly recognized clinical challenge. This is
due partly to enhanced clinician awareness, advances in pediatric therapeutics and tertiary care, as also to specific identification and
molecular characterization of a number of heritable prothrombotic defects.
PHYSIOLOGIC CONSIDERATIONS in primary hemostasis. When thrombin binds to

thrombomodulin on endothelial surfaces, it no longer
The fluid state of blood is maintained by a delicate and cleaves fibrinogen but activates protein C, which
dynamic balance between the coagulant, anticoagulant
in turn complexes with protein S to proteolytically
and fibrinolytic systems, all regulated through a series of
inactivate factors V and VIII.
feedback mechanisms. Despite all progress in the field,
3. Clot dissolution in vivo is mediated by the enzyme
Virchow’s triad of three basic risk factors for thrombosis
plasmin which is generated from its precursor plas
viz:
minogen by two known activators, tissue plasminogen
1. Stasis, hypercoagulability and endothelial damage are
still central to clinical considerations in thrombosis. In activator (tPA) and urokinase plasminogen activator.
most cases, whether or not an underlying risk factor Inherited or acquired deficiency in these pathways
is identified, clinical thrombosis always results from may foster a hypercoagulable state.
a combination of two or more factors. The natural The evolving hemostatic system in neonates and
anticoagulant system includes three pathways for children is distinctly different from the mature adult.
inhibition of procoagulant activation: Neonates have a hypocoagulable state with decreased
i. Cleavage of factors V and VIII by the protein C and capacity to generate thrombin, which, however, is
protein S system. balanced by the protective effects of deficient circulating
ii. Direct inhibition of thrombin by antithrombin anticoagulants. The physiological immaturity may extend
III (AT III), heparin cofactor II and alfa-2 macro to variable extents through childhood, which perhaps
globulin explains the strikingly low incidence of clinical throm
iii. Inhibition of factor VIIa by the tissue factor pathway bosis. An elegant longitudinal study of normal children
inhibitor/factor Xa complex. from birth to adolescence has shown that during the
2. In addition, the von Willebrand factor (vWF) cleavage newborn period and infancy, although levels of AT
protease ADAMTS13 regulates the size of vWF III, protein C and S are low, thrombin generation is
multimers thereby reducing its functional activity reduced and alfa-2 macroglobulin is increased. Likewise,
Chapter-33 Pediatric Thrombosis 349
throughout childhood, protein C and heparin cofactor In the clinical situation there may be an overlapping
II are low, but alfa-2 macroglobulin is raised and presence of both inherited and acquired factors. Some
prothrombin and factor VII are reduced compared to of the milder forms of inherited thrombophilia, develop
adult values. These physiologic differences bear effect on thrombosis only in the presence of other comorbid states
the incidence and natural history of thrombosis, as well or therapeutic interventions, as is increasingly becoming
as on pharmacokinetics and response to anti-thrombotic evident, many of the so-called acquired thromboses may
drugs, making clinical management a greater challenge. have an underlying thrombophilic defect.
Among newer concepts, is the role of the endothelium
and endothelial heterogeneity in thrombosis and hemo ACQUIRED RISK FACTORS
stasis. The endothelium is now not considered as
merely lining vascular channels, but a highly metabolic, Neonatal Thrombosis
functional and dynamic tissue which uniquely adapts Thrombotic complications occur five times more frequ
to local tissue environment. Thrombotic complications ently in the newborn compared to older children. Though
in various disorders or hypercoagulable states show a they may occur spontaneously, most often are associated
predilection to specific locations, likewise with bleeding with a known event.
too. Research supporting this changing paradigm has
shown heterogeneous distribution of coagulation and
Risk Factors
fibrinolytic factors across organs and tissues. They also
show a variable response to stimuli, e.g. cytokines (TNF- • Indwelling vascular catheters especially umbilical
alfa), blood flow, drugs , toxins. In particular, inflammatory artery cannulation
cytokines upregulate endothelial prothrombotic factors • Malposition venous catheters or
with a switch of homeostatic balance in favor of hemostasis, • Use of hyperosmolar solutions, dehydration
but this too varies with location. • Polycythemia
• Hypoxia
ETIOLOGY AND RISK FACTORS • Maternal diabetes
IN CLINICAL THROMBOSIS • Intrauterine growth retardation, or
• Shock syndromes, as in asphyxia or sepsis
In the context of childhood thrombosis, more of venous • Severe congenital protein C or S deficiency may pre
thromboembolism than arterial is encountered in clinical sent in the newborn period. Abnormal chorionic
practice. Arterial clotting is seen in the presence of vessels occurring in a range of maternal disorders may
abnormal platelet activation or endothelial dysfunction, produce chorionic thrombi which embolize to fetal
whereas VTE is predisposed to by venous stasis, venous pulmonary arteries or portal vein.
endothelial damage, deficiency of anticoagulant or anti • Maternal antiphospholipid antibody syndrome has
fibrinolytic factors or an excess of anticoagulant protein. also been associated with neonatal arterial thrombosis.
As an extension of the traditional Virchow’s triad, another Neonates have a propensity to large vessel thrombosis
researcher, Eberhard Mammen, postulated reduced and present with limb or organ dysfunction depending
mobility as another important factor, which albeit may on the vessel involved. Presentation may be in the form
be more applicable in adults. He proposed that decreased of edema, lower limb cyanosis (Inferior vena cava), scalp
muscle contraction reduced blood flow, with stasis and and facial edema (Superior vena cava, SVC), renal lump
accumulation of blood within intramuscular sinuses, thus with hematuria (renal vein), or seizures. Aortic thrombosis
triggering hypercoagulabilty due to local accumulation of may present with congestive heart failure, feeble femoral
activated clotting factors and simultaneous consumption pulses, necrotizing enterocolitis or renal failure, peripheral
of anticoagulants. artery block may have absent pulses with cold discolored
Microparticles, which are circulating small fragments skin, and cerebral artery thrombosis may have apnea
of cell membranes are found to carry procoagulant and and seizures. Neonates with homozygous protein C or S
anticoagulant proteins and plasminogen system compo deficiency invariably present with purpura fulminans.
nents, are mainly derived from platelets and lesser degree
from leukocytes and endothelial cells. These micro-
particles are believed to have a thrombogenic role, as VASCULAR ACCESS DEVICES
increased circulating microparticles have been associated Central venous catheters are increasingly being used in
with thromoboembolic complications in cancer, anti- critical care settings and for prolonged vascular access
phospholipid syndrome, sickle cell disease, diabetes and in children requiring parenteral nutrition, chemo
systemic inflammatory disorders. therapy, blood transfusions antibiotics. These are today
an important risk factor for pediatric thrombosis. Clinical of phosphatidyl serine. Studies have shown elevated
pointers to such an event include recurrent blockage, plasma levels of thrombin-antithrombin complexes and
frequent sepsis, local pain or swelling, chylothorax, SVC significantly decreased levels of protein C and S in all TM
obstruction or appearance of chest wall collaterals. Cardiac patients without other thrombophilic mutations. Thus, a
catheterization may also be complicated by thrombosis at chronic hypercoagulable state exists in TM, with children
the site of cannulation. and adolescents presenting with CNS thrombosis, DVT,
cardiac thrombosis, pulmonary embolism or recurrent
DEHYDRATION AND SEPSIS thrombophlebitis. Identified risk factors for thrombosis in
TM include: platelet count > 6 lacs</cumm, protein C < 50
These two factors alone or in combination may promote percent, plasminogen< 50 percent, protein S < 50 percent.
thrombosis by causing a hyperviscosity of blood and
cytokine induced endothelial damage in the course of MALIGNANCY
sepsis. Thus, acute gastroenteritis with dehydration is a
common clinical setting where such an event is predis Tumor microparticles, cytokine induced endothelial
posed often affecting cerebral venous sinuses. Suppurative changes, sepsis in neutropenic patients, vascular acce
thrombophlebitis of the internal jugular vein or Lemierre’s sses and drugs such as L-asparaginase may promote
syndrome triggered by fusobacterial sepsis, though a rare thrombosis in cancer.
cause in children, carries a potential risk of CNS morbidity Other risk factors include trauma, surgery, immo
and mortality. bilization, infusion of prothrombin complex concentrates,
use of oral contraceptives in adolescent girls. Acquired
protein C deficiency may also occur in liver disease,
NEPHROTIC SYNDROME
sepsis, disseminated intravascular coagulation (DIC)
Thromboembolic complications can occur in about 25 and especially in purpura fulminans and DIC with acute
percent of patients with nephrotic syndrome presenting meningococcal infection.
with not only renal vein thrombosis but also arterio venous Arterial thrombosis may be associated with sickle
thrombosis at various other sites including CNS and abdo cell disease, vascular malformations/Moyamoya disease,
minal vessels. Factors predisposing a hypercoagulable state APLA, hyperlipidemias, vasculitis.
include AT III protein loss in urine, hyperfibrinogenemia,
increased platelet activation, hyperlipidemia, increased FAMILIAL THROMBOPHILIA
platelet activation, hyperlipidemia, along with a propensity
Inherited prothrombotic states are usually suspected
to intravascular volume depletion, hyperviscosity, use of
in children with an unexplained cause for thrombosis,
diuretics, and relative immobilization.
a positive family history, recurrent thromboembolism,
or thrombosis at an unusual site. Most of them present
ANTIPHOSPHOLIPID ANTIBODY beyond adolescence unless compounded by additional
SYNDROME risk factors. However, severe deficiencies may have
This condition may occur as a primary antiphospholipid spontaneous thrombosis as early as newborn period.
antibody syndrome (APLA) or secondarily with systemic Coinheritance of these susceptibility genes is known and
lupus erythematosus or other connective tissue disorders. the risk of thrombosis increases with multiple coinherited
APLA has most often been associated with CNS stroke in defects. Some of the better understood anticoagulant
children, although arterial thrombosis elsewhere may also defects are discussed briefly here.
occur. APLA is also described in conjunction with viral • Protein C deficiency in its milder phenotype is
diseases such as hepatitis and human immunodeficiency inherited as an autosomal dominant trait with a
virus disease. APLA promotes thrombosis by platelet acti population prevalence of 0.2 percent and having two
vation, release of endothelial platelet factor and inhibition subtypes identified by quantitative and qualitative
of protein C and AT III. defects respectively, the latter being less common.
Milder homozygotes and heterozygotes present with
recurrent thromboembolism usually beyond the
THALASSEMIA second decade and in association with additional risk
A higher than normal incidence of thromboembolic events factors. Severe homozygous protein C deficiency is
has been observed in patients with beta thalassemia usually inherited in an autosomal recessive form.
major (TM). Red blood cells in TM have been shown to • Protein S deficiency has a very similar inheritance
facilitate thrombin formation due to altered symmetry pattern, subtypes and clinical profile as protein C
of membrane phospholipids with enhanced exposure deficiency.
• Factor V Leiden is a form of inherited prothrombotic Approach to Diagnosis

defect characterized by a mutation in factor V (factor
V Arg506Gln) at exactly the site where it is spliced by A high index of clinical suspicion is necessary for an
activated protein C, making it resistant to the action early diagnosis, which is crucial so as to prevent fatal
of the latter. It is thus also called ‘activated protein C complications such as pulmonary embolism, organ
resistance’. This mutation is present in about 8 percent dysfunction and prevent long-term morbidity. Once VTE is
of Caucasians, but < 1 percent Asians and Africans. suspected, there are various laboratory tests and imaging
Deficient individuals have a milder phenotype, and modalities that help confirm the diagnosis and delineate
both homozygotes and heterozygotes may have the extent of thrombosis.
recurrent thromboembolism during childhood or later,
Radiologic Imaging
usually with an associated risk factor. It may rarely
cause arterial thrombosis and has also been implicated • Although venography is the gold standard to demons
in porencephaly, cerebral palsy and Perthe’s disease. trate thrombosis, it is used infrequently as it is invasive,
• Prothrombin gene mutation at the nucleotide 20210A needs specialized radiology, needs multiple peripheral
position estimated to occur with a frequency of 3 to access and is not useful for internal jugular vein as the
4 percent in the population. The mutation results in dye cannot flow in a retrograde fashion.
abnormally high prothrombin levels, which probably • Doppler ultrasound is the most frequently used
contributes to increased thrombotic risk by increased imaging, being noninvasive, easily available and
thrombin generation. Clinical manifestations are mild lower in cost. It has a high sensitivity for lower limb
even in homozygotes. and abdominal vein thrombosis. However, it may be
• Antithrombin III deficiency displays two types of ineffective for upper limb thrombosis due to the chest
mutations, with quantitative and qualitative defects wall lung tissue and the clavicles.
respectively. The homozygous state is incompatible • Magnetic resonance (MR) venography clearly images
with life. Heterozygotes present in early adulthood all large veins and is useful in cerebral sinus throm
with recurrent and/or thromboembolism. Thrombosis bosis, though it is expensive and needs sedation. CT
in adulthood is rare, and its occurrence is usually angiography is also useful, but needs greater contrast,
associated with secondary risk factors. has exposure to radiation.
• Hyperhomocysteinemia is classically due to deficiency Likewise for arterial thromboses, though angiography
of cystathionine biosynthase. However, another more is the gold standard, it is invasive, needs an interventional
common polymorphism in the methyltetrahydrofolate radiologist, and may itself cause thrombosis. Doppler
reductase (MTHFR) gene appears to reduce conversion ultrasound is a good screening and diagnostic modality
of homocysteine into methionine. The MTHFR is not except for the chest region.
an uncommon polymorphism and its homozygosity,
well documented in adults, may increase risk of Laboratory Evaluation
thrombosis especially stroke in children. Diagnostic evaluation for pediatric acute venous throm-
Among other heritable prothrombotic states, yet to be boembolism (VTE) includes a complete hemogram,
clearly defined, are: coagulation tests, comprehensive thrombophilia evalu-
• Elevated factor VIII levels ation. Additional laboratory evaluation would depend on
• Raised lipoprotein associated with lipoprotein coagu associated medical conditions and VTE in specific organ
lation factor Lp(a) systems.
• Dysfibrinogenemia D-dimer assay may be useful as a screening test for
• Thrombomodulin mutations massive or diffuse thrombosis, however, low levels do
• Factor XII deficiency not exclude VTE. Other abnormal tests in VTE include
• Defects in plasminogen and plasminogen activator circulating prothrombin fragment 1.2 and the TAT
inhibitor. complex, however, these are not clearly adapted to the
Mutations causing a deficiency in the ADAMTS13 clinical setting. Prolonged activated partial thromboplastin
protease present as congenital thrombotic thrombocyto time (APTT) with a normal prothrombin time (PT)
penic purpura, with predominantly a microangiopathic may indicate presence of lupus anticoagulant/APLA
hemolytic anemia that worsens with infection, showing or factor XII deficiency. Shortened APTT with normal
marked elevation in D-dimers. PT may be associated with elevated factor VIII levels.
Prolongation of both PT and PTT may be seen with DIC or vascular access poses problems with drug delivery and
dysfibrinogenemia. with monitoring of anticoagulant effect. With most agents,
Hemogram may help identify sickling of red cells, pediatric-specific formulations are not available making
thrombocytosis or thrombocytopenia in massive throm reproducible dosing difficult, especially so in the case of
bosis or DIC. Sickling test and hemoglobin electrophoresis low molecular weight heparins (LMWH) and vitamin K
is recommended in arterial thrombosis. antagonists (VKA) warfarin. Dietary differences especially
Recommended panel for identifying thrombophilic in milk fed patients make VKA dosing difficult. Thus, actual
states as per the Scientific and Standardization Sub clinical experience becomes important and VTE should, if
committee on Perinatal and Pediatric Hemostasis of the possible be treated in a pediatric hematology setting, or if
International Society on Thrombosis and Hemostasis, not by a neonatologist/pediatrician in close consultation
for laboratory evaluation of VTE in children include the with a pediatric hematologist.
following.
Unfractionated Heparin
Acquired or Genetic Standard heparin still remains the most common form
• AT III assay used in pediatric patients overall and acts by enhancing
• Protein C assay AT III mediated inactivation of factor Xa and thrombin,
• Protein S assay hence needs normal ATIII levels to be effective. Its efficacy
• Elevated plasma factor VIII assay is monitored by aPTT or antifactor Xa activity. Newborns
• Blood homocysteine levels have a faster clearance of unfractionated heparin (UFH)
• APLA (including anticardiolipin antibodies, Russell due to a larger volume of distribution and hence need a
viper venom time, antibody to beta-2 glycoprotein 2, higher dose to achieve anticoagulant effect. Its major
APTT–based lupus anticoagulant test). disadvantage is the need for vascular access and repeated
• DIC (including platelet count, fibrinogen levels, monitoring. Risk of bleeding with UFH for deep vein
D-dimer) thrombosis (DVT) is low but may be as high as 24 percent in
• Activated protein C resistance (APTT-based assay). the ICU setting. Osteoporosis with UFH is rare in children.
Heparin induced thrombocytopenia in various cohorts is
reported in 0 to 2.3 percent, at varying ages and in UFH
Genetic
exposures ranging from vascular access device flushes to
• Factor V Leiden polymorphism massive doses in cardiopulmonary bypass surgery.
• Prothrombin G20210A polymorphism
• Elevated plasma lipoprotein(a). Low Molecular Weight Heparin
Despite unproven efficacy, low molecular weight heparin
Therapeutic Approach
(LMWH) have rapidly become a treatment of choice
There are few clinical trials available to guide decision on pediatric patients, both for primary prophylaxis and
making in the treatment of thrombosis. Most published treatment of VTE. Potential advantages of LMWH in
guidelines are based on adult trials, uncontrolled pediatric children include subcutaneous route of administration, no
studies, case series and reports and it is unclear how these significant need for monitoring, minimal risk of bleeding
guidelines are being used. Nevertheless patients today are HIT or osteoporosis and no interference with diet or drug
being diagnosed with thrombosis and must be treated. metabolism. Most available data in children is with the
Therapy is based on methods that are best for restoring use of enoxaparin, although dalteparin and reviparin have
circulation rapidly balanced by the risk of bleeding, with been used as well. Bleeding with LMWH can be treated
an aim to re-establish flow through the occluded vessel, with protamine sulfate, although multiple doses may be
prevent embolization, and arrest the thrombotic process. required. LMWH should be withheld for 24 hours prior to
any procedure, as also it needs dose adjustment in renal
ANTITHROMBOTIC AGENTS failure.
The interaction of antithrombotic agents with the hemo
static system of the young differs from the adult with Thrombotic Agents
respect to a multitude of variables. Pharmacokinetics of For large vessel and massive or extensive thrombosis, or
these agents vary in an age-dependent manner as well pulmonary embolism, thrombolytic therapy should be
as with intercurrent illnesses and medications. Limited considered, after weighing the potential benefits versus
risk of bleeding. It is recommended to ensure that serum or newborns also on indomethacin. Newer agents include
fibrinogen is > 50 mg/dL and platelet count > 50,000/ ticlopidine and glycoprotein IIb-IIIa antagonists such
cumm and that efficacy be monitored by documenting as abciximab, the latter being found to induce faster
increasing D-dimer levels. Thrombolysis carries significant regression of coronary aneurysms in Kawasaki’s disease.
risk in patients who have had surgery in the past 7 days,
premature neonates or with cerebral sinus thrombosis Venacaval Interruption
when intracranial bleed may occur. Minor bleeding with
Inferior venacaval filters have been implanted in some
thrombolysis may be treated with local pressure, but major
cases to prevent pulmonary TE.
bleeds will require stopping therapy and administration of
cryoprecipitate and/or antifibrinolytics. In children there
is no data showing any advantage of local over systemic Surgical Thrombectomy
administration. In fact, except for catheter related TE, Rarely used in children, it may be required in IVC throm
catheter-directed local administration may cause more bosis in Wilms’ tumor, blocked Blalock-Taussig shunt,
harm in view of small vessel lumen size. The thrombolytic massive intracardiac thrombosis following surgery, local
agent of choice in pediatric patients is recombinant thrombosis after vascular access.
tPA, however, it is expensive. Streptokinase is a cheaper
alternative, but may cause severe allergic reactions be less Treatment of Venous Thromboembolism
effective in the presence of plasminogen deficiency as in a
newborn. Such situations will need supplementation with • Initial management: This largely depends on the
fresh frozen plasma to make thrombolytic agents effective. location extent and symptoms. For severe massive
A recent study by Leary et al. has shown low-dose systemic throm bosis, thrombolytic therapy should be con
r-tPA an effective thrombolytic agent in DVT in children, sidered, followed by anticoagulation. Less severe
which may represent a transition between high dose r-tPA DVT, non-occlusive or small thrombi of extremities,
and anticoagulation. simple catheter related thrombi can be treated with
anticoagulation alone, where currently the choice
VITAMIN K ANTAGONIST is UFH or LWMH as a first line. LWMH is preferred
because of its safety and convenience in children. UFH
Warfarin is the commonly used vitamin K antagonist (VKA) which has a shorter half life is preferred when there is a
when long-term treatment is warranted and effects its greater comorbid risk of bleeding, labile acute clinical
anticoagulant action by inhibiting gamma carboxylation status, impaired renal function or with cost constraints.
of vitamin K dependent proteins. Though an oral and Recommended duration of heparinization is 5 to 10
low-cost agent, it has unpredictable pharmacokinetics, days.
numerous drug and food interactions as well as a narrow • Further treatment: Once some flow is restored, further
therapeutic index. It is extremely challenging thus, to anticoagulation is needed to treat residual thrombi,
manage children under 4 years on warfarin. However, prevent embolization and recurrence. Extended anti
currently point of care portable devices to monitor PT and coagulation in a subacute phase would prefer
INR using capillary samples are available. LMWH, though warfarin is also used. For chronic
anticoagulation in an older child warfarin is used.
Alternative Thrombin Inhibitors
Patients with HIT may be treated with alternative agents DETAILS OF ANTITHROMBOTIC AGENT
such as danaparoid, argatroban or hirudin, though ADMINISTRATION (TABLES 1 TO 4)
limited data is available on these children. Antifactor Xa
agents such as fondaparinux act by indirect inhibition of Recommended Durations of Therapy
thrombin.
(Anti-thrombotic therapy in neonates and children:
Evidence-based guidelines, P Monagle et al.)
ANTIPLATELET DRUGS • For idiopathic VTE as a first episode 6 months anti
Aspirin is the most common antiplatelet drug used in coagulation with LMWH or warfarin (INR 2.5).
children at an empiric dose of 1 to 5 mg/kg/day, and • For secondary risk factors which have resolved, 3
second commonest being dipyridamole (2–5 mg/kg/ months of VKA or LMWH.
day). However, clopidogrel (1 mg/kg/day) is found to • For ongoing secondary risk factors (e.g. nephrotic
be effective and safe in children. Bleeding is uncommon syndrome or L-asparaginase) continue anticoagulation
except in situations with added hemostatic abnormalities till risk factor resolution.
Table 1 Administration of unfractionated heparin Table 4 Administration of systemic thrombolytic agents

Loading dose 75 u/kg over 10 minutes for all r-tPA 0.1–0.6 mg/kg/hr over 6 hours—systemic
Maintenance dose 25–30 u/kg for infants; high dose
20 u/kg > 1 year of age 0.5 mg in normal saline volume required to
fill the line—local
APTT monitoring First at 4 hours, then daily and 4 hours
after every change Streptokinase 2000 u/kg bolus, then 2000 u/kg/hr for 6–12
hours
Desired APTT 60–85 sec
(antifactor Xa level 0.3–0.7 u/mL) Urokinase 4400 u/kg bolus, then 4400 u/kg/hr for 6–12
hours
Dose modification A PTT < 50 sec—increase 20%
50–59 sec—increase 10% Monitor PT, PTT, platelet count, fibrinogen for all
80–95 sec—decrease 10%
96–120 sec—withhold heparin for
30 minutes and decrease 10% The above guidelines may be referred to for further
>120 sec—withold heparin details in management of thrombosis in a wide spectrum
60 minutes and decrease 15%
of clinical situations including neonates, venous access
Daily CBC
devices, arterial ischemic stroke, cardiac devices and pros
thesis, cardiac procedures, cardiomyopathy, Kawasaki’s
disease, cancer, cerebral sinus thrombosis, APLA and
Table 2 Administration of LMWH (enoxaparin)
purpura fulminans.
Initial treatment dose 2 mg/kg 12 hourly in neonates
1.5 mg/kg 12 hourly > 2 months age
Neonatal Purpura Fulminans
Maintenance dose 1.5 mg/kg for neonates
1 mg/kg > 2 months age This is seen with homozygous or doubly heterozygous
Antifactor Xa First at 4 hours, 24 hours, 1 week, protein C deficiency, wherein presentation is very early
monitoring monthly in the newborn period large areas of skin necrosis due to
diffuse thrombosis within the skin vasculature. Clinical
Desired antifactor Xa 0.5–1 u/mL
effects may occur even earlier, in the fetus with CNS
thrombosis giving rise to neonatal seizures. Management
of these babies is complicated as, neither heparin nor
Table 3 Administration of warfarin
antiplatelet drugs are effective. Replacement therapy is
Loading dose 0.2 mg/kg for 2–4 days necessary with a source of protein C, usually FFP 10 to
PT-INR monitoring Daily till INR in therapeutic range, 20 mL/kg, or if possible protein C concentrates, 20 to
then weekly 60 u/kg, on a 12 hourly basis. However, protein C has
Desired PT-INR 2.0–3.0 a half life of 6 to 16 hours, and frequent administration
Dose modification a. Loading is limited by development of hyperproteinemia, hyper
INR 1.1-1.3—100% of loading dose volemia, hypertension, loss of venous access, potential
INR 1.4–3 50% exposure to viral agents, infections. These babies are also
INR 3.1–3.5 25% prone to warfarin induced skin necrosis although warfarin
INR >3.5 withhold till <3.5, restart is required for long-term control of thrombotic diathesis.
at 50% Liver transplantation may offer a cure with normalization
b. Maintenance of protein C levels.
INR 1.1–1.4; 120% of previous dose
INR 1.5–1.9; 110%
INR 2.0–3.0; 100%
OUTCOMES
INR 3.1–4.0; 90% Complications of VTE may occur acutely or over a long-
term. Early adverse outcomes include bleeding associated
with anti-thrombotic therapy, thrombotic hemorrhage,
• For recurrent VTE with reversible risk factor, 6 to 12 and organ dysfunction depending on the site/severity
months and resolution of risk. of thrombosis. Long-term adverse outcomes include
• For idiopathic recurrent VTE, 12 months to lifelong. recurrent VTE, renal dysfunction/hypertension, variceal
• For chronic risk factor associated VTE, lifelong anti- bleeding, chronic SVC syndrome and post-thrombotic
coagulation is recommended. syndrome.
Recurrent TE rates are lower than adults, seen in 6 to 8. Hoyer PF, et al. Thromboembolic complications in
10 percent. children with nephrotic syndrome: risk and incidence.
Post-thrombotic syndrome (PTS) seen in nearly Acta Paediatr Scand. 1986;75:804.
1/3rd of DVTs is characterized by edema, visibly dilated 9. Kenet Gili, et al. Bleeding and thrombosis issues in
pediatric patients: current approach to diagnosis and
superficial collateral veins, venous stasis dermatitis and
treatment. Acta Haematol. 2006;115:137.
ulceration. Some patients, both neonatal VTE survivors
10. Kwaan HC, et al. The significance of endothelial hetero
and older children, may have persistent residual geneity in thrombosis and hemostasis. Semin Thromb
thrombosis which in the long-term may cause venous Hemost. 2010;36:286.
valvar insufficiency and hence PTS. Factors predicting 11. Leary SE, et al. Low dose systemic thrombolytic therapy
poor long-term outcome in terms of recurrent VTE, for deep vein thrombosis in pediatric patients. J Pediatr
persistent thrombosis and PTS include, complete veno- Hematol Oncol. 2010;32:97.
occlusion at diagnosis, and more importantly, elevated 12. Lippi G, et al. Pathogenesis of venous thromboembolism:
factor VIII levels >150 u/dL and D-dimer> 500 ng/ when the cup runneth over. Semin Thromb Hemost.
mL at diagnosis and after 3 to 6 months of standard 2008;34:747.
13. Male C, et al. Central venous line related thrombosis in
anticoagulation.
children: association with central venous line location and
insertion technique. Blood. 2003;101:4273.
CONCLUSION 14. Manco Johnson MJ, et al. Laboratory testing for throm
bophilia in pediatric patients. On behalf of the Sub
Thrombotic disorders have emerged as a serious pediatric committee for Perinatal and Pediatric Thrombosis of the
concern and clinical challenge resulting in acute and Scientific and Standardization Committee (ISTH), Thromb
chronic sequel. Though we have come a long way over Hemost. 2000;88:155.
the past decade, many questions remain regarding 15. Manco-Johnson MJ, et al. Diagnosis and management
pathogenesis, natural history, optimal therapy. Advances of thrombosis in the perinatal period. Semin Perinatol.
in ability to predict outcomes may help risk-stratifying 1990;14:393.
16. Manco-Johnson MJ. How I treat venous thrombosis in
therapy and achieve meaningful improvements in long-
children. Blood. 2006;107:21-9.
term outcomes. 17. Middledorp S, et al. Thrombophilia: an update. Semin
Thromb Hemost. 2007;33:563.
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C H A P T E R 34
Disseminated Intravascular
Coagulation in Neonates
VP Choudhary
Disseminated intravascular coagulation (DIC) is a studied and with the better understanding of underlying
syndrome which develops following large number of mechanism has helped to evolve multiple diagnostic
disorders (Flow chart 1). It occurs whenever there is and monitoring tests and appropriate strategies for
systemic activation of coagulation system leading to its management which have improved the survival
generalized uncontrolled formation of fibrin with in the significantly. The mortality in the neonatal period is
blood vessels leading to micro-vascular thrombosis in very high. Early diagnosis and prompt management is
multiple organs along with consumption of platelets and essential. However, it is desirable that the development of
coagulation proteins which results in variable bleeding DIC should be prevented by appropriate measures.
symptoms. Therefore patients of DIC can have symptoms Systemic activation of coagulation system may occur
related to bleeding and thrombosis simultaneously. The in a large numbers of diseases. The activation of the
subcommittee on DIC of the Scientific and Standardization coagulation results in clinical symptoms which may vary
Committee of the International Society of Thrombosis from decrease in platelet count, subclinical prolongation of
and Hemostasis (ISTH) has recently proposed the global clotting time, mild bleeding symptoms to fulminant
following definition of DIC. It is an acquired syndrome disease complex termed as disseminated intravascular
which presents with variable symptoms secondary to coagulation.
intravascular coagulation and it may spread to involve
multiple organs leading to their dysfunction.1,2 PATHOGENESIS OF DIC
First description of DIC appeared in the literature in
1950s.3 Over the years the pathogenesis of DIC have been Many pathogenetic mechanism either singly or
together play a major role in pathogenesis of DIC.4,5
Flow chart 1 Disseminated intravascular coagulation
Fibrin depositions plays the key role in its pathogenesis
which occurs following tissue factor mediated
thrombin generation which exceeds the physiological
anticoagulation mechanism (mainly antithrombin III
and protein C system). In addition fibrin removal is less,
as fibrinolytic activity does not increase proportional to
thrombotic activity. Fibrinolytic activity infact is inhibited
in DIC because of high level of PAI-1 which is a fibrinolytic
inhibitor. Thus there is impairment of endogenous
thrombolysis. All these processes are complex and occur
simultaneously. It is for the better understanding, these
factors have been described separately (Fig. 1 and Flow
chart 2). There is an interaction of these factors at multiple
levels.
Chapter-34 Disseminated Intravascular Coagulation in Neonates 357
on endothelial cells through various cytokines, such

as TNF-α and interleukin (IL)–β.8 These observations
are further supported by improved survival following
administration of activated protein C to patients with DIC.
Tissue factor pathway inhibitor (TFPI) is another major
inhibitor of coagulation system. There are experimental
and clinical evidences which have demonstrated that
administration of recombinant TFPI have blocked the
inflammatory process induced by thrombin generation. In
clinical studies the use of TFPI in pharmacological doses is
capable of reducing the mortality significantly in systemic
infection.9
Fig. 1 Disseminated intravascular coagulation—pathogenesis Impaired Fibrinolysis

Presence of bacteremia and endotoxemia increase the
fibrinolytic activity because of the release of plasminogen
Flow chart 2 DIC—pathogenesis activators from the endothelial cells.10 However, increase
in plasma levels of PAI-1 results in reversal of fibrinolytic
activity.11 Rise in PAI-1 levels occurs as a result of release of
cytokines and endothelial injury (Flow chart 2).
CLINICAL PRESENTATION
Based upon the onset and severity of symptoms, DIC has
been subdivided into acute and chronic DIC.
Acute DIC is of sudden onset and carries very high
morbidity and mortality. It is most common in neonate
period and therefore has been described in detail.
• Neonates with severe acute DIC may manifest with
bleeding from multiple sites such as development
of purpuric and ecchymotic spots, mucosal oozing,
gastrointestinal blood loss and bleeding from venous
access.
• Neonates may progress to hypotension, shock and
often develop fluid and electrolyte disturbances.
• The deposition of thrombin is dependent upon the
organ involvement. Neonates may become lethargic
and neonatal reflexes are depressed or absent,
Dysfunctional Physiologic Anticoagulant comatose, or develop seizures, become unconscious
Pathways as a result of CNS involvement, may develop stroke as
a result of thrombosis in cerebral vessels, which may
Activation of coagulation system occurs through various progress to hemorrhagic infarcts.
regulatory pathways which are impaired thereby leading • Micro thrombi in the renal system may progress
to amplification of thrombin generation which leads to to oliguria, renal failure as a result of acute tubular
increased thrombin formation.6 The antithrombin III(AT- necrosis. Micro thrombi may cause acute abdominal
III) is an important inhibitor of thrombin whose levels are pain simulating clinical picture of acute abdomen or
reduced in patients with severe sepsis. Multiple factors bleeding in the gut as a result of gangrene.
in combination, such as (a) consumption due to ongoing
thrombin generation, (b) degradation by elastase, released
from activated neutrophils and (c) impaired synthesis. Low
DIAGNOSIS OF ACUTE DIC
levels of AT-III results in DIC with increased mortality.7 High index of suspicion is essential. Any neonate, who
This impaired function of the protein C pathway occurs as starts bleeding in pressure of underlying condition should
a result of down regulation of thrombomodulin expression be considered to have DIC and should be investigated
periodically for diagnosis of DIC. Thrombocytopenia is MANAGEMENT OF DIC

an early manifestation of DIC. Patients with acute DIC are
critically ill, and therefore early diagnosis is essential for The heterogeneity of the underlying disorder and clinical
improved survival. Several of sensitive and sophisticated severity is so variable therefore, it is difficult to have a
tests are not readily available in clinical practice even in common therapeutic approach for management of DIC.
the advanced centers. Therefore the diagnosis of DIC is Thus, the treatment of DIC should be individualized,
based upon the platelet count, examination of peripheral based upon the clinical presentation, i.e. bleeding,
smear, measurement of PT and APTT , high level of fibrin thrombosis or both and patients conditions such as
degradation products (FDP) and D-dimers. hemodynamic situation, presence of hypothermia or
Thrombocytopenia is often attributed to consumptive not, electrolyte imbalance and gas exchange along with
processes but underproduction also plays a role in renal, cardiac and neurological status.14 Early diagnosis
presence of severe sepsis which is often the cause of DIC of underlying condition and their prompt appropriate
in neonates. Coagulation on studies when minimally management plays a major role in the outcome of DIC.
damaged, there may be difficulty in distinguishing it as The management of the underlying condition will not be
abnormal as low levels in preterm are expected. Similarly discussed as the underlying conditions are many and have
there is no reliable range of D-dimers. different specific treatment.
Fibrinogen concentration normally may increase Replacement therapy: Platelets and coagulation factors
during the first few days of life. Therefore, at time diagnosis (FFP) are administered to correct thrombocytopenia and
of DIC in neonates becomes very difficult. coagulation factor to control the bleeding at multiple sites.
It has been observed that serial coagulation tests Currently it forms the major form of therapy for treatment
and platelet counts are usually more helpful than single of DIC.15
laboratory results to establish the diagnosis of DIC.12 Low
fibrinogen level is detected only in severe DIC but it is not Transfusion of Platelet Concentrates
a specific marker.3 One to two units of platelet concentrates per 10 kg of
High levels of FDP and D-dimers help the clinicians to body weight are administered if platelet count is below
differentiate other disorders like chronic liver disease with 30 x 109/L in absence of any bleeding. Platelet therapy
low platelet count and prolonged PT and APTT.13 is essential in presence of bleeding manifestation even
The subcommittee on DIC of the International Society if the platelet count is >30 × 109/L. The main objective is
of Thrombin and Hemostasis has recently published a to control the bleeding and there is no need to transfuse
scoring system1 to facilitate the clinicians to establish the platelets to maintain platelet count of >50 × 109/L which is
diagnosis of DIC. In presence of any condition known to considered as a safe level.
cause DIC, algorithm suggested by the subcommittee
should be used to determine the score. A score of 5 or
Fresh Frozen Plasma
more suggests the diagnosis of DIC. However this scoring
system needs to be validated by prospective studies in Fresh frozen plasma (FFP) should be administered
neonatal DIC (Table 1). at a dose of 15 to 20 mL/kg in an attempt to correct the
Table 1 Diagnostic algorithm for diagnosis of DIC

• Risk assessment: Does the patient have an underlying disorder known to be associated with overt DIC?
– If ‘yes’ proceed
– If ‘no’, then do not use this algorithm
• Order for screening coagulation tests (platelet count, PT, APTT, fibrinogen, soluble fibrin monomers or fibrin degradation
products)
• Score screening coagulation tests results
– Platelet count (>100,000=0, <100,000=1, <50000=2)
– Prolonged PT (<3 sec = 0, >3 sec but <6 sec = 1, >6 sec = 2)
– Fibrinogen level (≥1 g/L = 0, <1 g/L = 1)
– Elevated fibrin related marker e.g. soluble fibrin monomers/FDP (no increase=0, moderate increase=2, strong increase=3).
• Calculate score
– If the total score is ≥5, then patient is compatible with overt DIC, repeat scoring daily.
– If the score is <5, it is suggestive (not affirmative) of nonovert DIC, repeat the tests at 1–2 days interval till the patient
recovers or whenever the score exceeds 5, diagnosis of DIC can be made.
Chapter-34 Disseminated Intravascular Coagulation in Neonates 359
multiple factor deficiency and to control bleeding. Newer Agents

Alternatively fibrinogen concentrates (total dose
2–3 g) or cryoprecipitate (1 bag/10 kg body wt) may be Some authors have demonstrated that the administration
administered. Cryoprecipitate (5–10 mL/kg) is a better of recombinant interleukin (IL-10), which is a potent
source of fibrinogen, which should be kept above 1 g/L. anti-inflammatory cytokine, has been demonstrated to
Dose and duration of therapy needs to be monitored moderate the activation of coagulation system in humans
with platelet counts, PT and APTT levels daily. Treatment but not in neonates. Its use completely abrogated the
should be directed to correct the PT and APTT levels and effects of endotoxins on coagulation pathway. Pajkrt
to prevent the further deterioration. and his colleagues24 observed significant improvement
of DIC in patients with sepsis following administration
of monoclonal antibodies against TNF. While Branger et
Anticoagulants al25observed that p38 nitrogen activated protein kinase
The role of heparin in the treatment of DIC remains still inhibitor modified the activation of coagulation, fibrinolysis
controversial.16 However, the present data indicates that and endothelial cells injury following administration of
heparin is effective in treatment of neonate with acute DIC endotoxins in experimental studies. However, all these
having predominant thrombotic symptoms, e.g. purpura modalities are still experimental. There is hope that with
fulminous. Presently role of heparin in chronic DIC is well better understanding of the pathophysiology, newer
established. It has been used effectively in patients with diagnostic tests and development of newer agents, the
recurrent thrombosis such as hemangioma or dead fetus treatment of DIC will improve significantly in near future.
syndrome.17
Heparin is usually given at relatively low doses (5–10 CONCLUSION
U/kg of body weight per hour) by continuous infusion
and may be switched to subcutaneous injection for long- DIC is a syndrome characterized by systemic intravascular
term therapy. Its dose needs to be adjusted with APTT or activation of coagulation in the circulation. It is associated
heparin level. Alternatively low molecular weight heparin with variable clinical manifestations from mild bleeding
may be used. It is preferred these days as it is not essential to organ failure. Better understanding of pathogenesis
to monitor heparin level or APTT. Secondly the incidence has lead to better clinical management strategies. Using a
of heparin induced thrombocytopenia is less when scoring system based on the clinical as well as laboratory
compared with standard heparin. tests, an early and accurate diagnosis of DIC can be made.
Recombinant hirudin which is newer anticoagulant The cornerstone of the management of DIC is the vigorous
has AT III independent inhibitory activity of thrombin treatment of the underlying disorder. In addition, the
has been used successfully to treat DIC in experimental strategies that interfere with the coagulation system, such
studies.18 as replacement, use of antithrombin III and activated
protein C, have improved the survival greatly. However,
larger studies are essential to determine their efficacy
Concentrates of Coagulation Inhibitors and safety of products such as antithrombin III, activated
In the pathogenesis of DIC it has been observed that there protein C, etc. Current research is likely to evolve newer
is deficiency of coagulation inhibitors. Therefore the novel strategies for management of DIC in near future.
normalization of anticoagulation pathway should form a
part of treatment for DIC.19 AT-III is a primary inhibitor of REFERENCES
circulating thrombin, its use in DIC certainly is rational.20
1. Taylor FB, JrToh CH, Hoots K, Wada H, Levi M. Towards a
It has been shown to have beneficial effects in terms
definition clinical laboratory criteria and a scoring system
of correction of coagulation parameters and organ for disseminated intravascular coagulation. Thrombosis
dysfunction.21 and hemostasis. 2000;86:1327-30.
Use of activated protein C and recombinant throm 2. Levi M, Ten Cate H, Van der Poll T. Disseminated
bomodulin has also been shown to reduce the mortality intravascular coagulation: State of the art. Thrombosis and
significantly in patients with severe sepsis.22 The activation hemostasis. 1999;82:695-705.
of coagulation cascade in DIC occurs exclusively through 3. Ratnoff OD, Pritchard JA, Colopy JE. Hemorrhagic states
the extrinsic pathway. Thus inhibition of tissue factor during pregnancy. New England Journal of Medicine.
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De Jongeet al.23 was first to use the tissue factor pathway 4. Van der Poll T, van Denenter SJ, Bulber HR, Sturk A, Ten
inhibitor (TFPI) for treatment of DIC following sepsis. Cate JW. Comparison of the early dynamics of coagulation
activation after injection of endotoxin and tumor necrosis 15. Feinstein DI. Diagnosis and management of disseminated
factor in healthy humans. Progress in Clinical and intravascular coagulation: The role or heparin therapy.
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activity in monocytes of patients with meningococcal Angelo A. Antithrombin replacement in patients with
infection: related to an unfavorable prognosis. Thrombosis sepsis and severe shock. Haematologica. 1999;84:452-60.
and Haemostasis. 1983;49:5-7. 17. Kessles CM, Tang Z, Jacobs HM, Szymanski LM. The su-
6. Egmon CT. The regulation of natural anticoagulant prapharmacological dosing of antithrombin concentrate
pathways. Science. 1987;235:1348-52. for Staphylococcus aureus induced disseminated intravas-
7. Fourrier F, Chopin C, Gordemand J, Hendeycx S, Caron cular coagulation in guinea pigs. Substantial reduction in
C, Rime A, et al. Septic shock multiple organ failure, mortality and morbidity. Blood. 1997;89:4393-401.
and disseminated intravascular coagulation. Compared 18. Bernard GR, Hartman DL, Helterbrand JD, Fisher CJ.
patterns of antithrombin III, protein C and protein S Recombinant human activated protein C (rhAPC)
deficiencies. Chest. 1992;101:816-23. produces a trend toward improvement in morbidity and
8. Conway EM, Rosenberg RD. Tumor necrosis factor 28 day survival in patients with severe sepsis. Critical Care
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8:5588-92. pathway inhibitor dose dependently inhibits coagulation
9. Creasey AA, Chang AC, Feigen L, Wun TC, Taylor FBJ, activation with influencing the fibrinolytic and cytokine
Hinshaw LB. Tissue factor pathway inhibitor reduces response during human endoxema. Blood. 2001;95:1124-9.
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10. Levi M, Ten Cate H, Bauer KA, vander Poll T, Edgington TS, leukemia. Lancet. 1989;2:122-4.
Buller HR, et al. Inhibition of endotoxin induced activation 21. Reinhart K, Waheedullah K. Antitumor necrosis factor
of coagulation and fibrinolysis by pentoxitylliue or by a therapy in sepsis update on clinical trials and lessons
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Hack CE, et al. Plasminogen activator and plasminogen 89:2701-74.
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S E C T I O N 5
Transfusion Medicine
CHAPTERS OUTLINE
35. Blood Components in Pediatric Practice
36. Nucleic Acid Amplification Testing
37. Transfusion Transmitted Infections
38. Noninfectious Hazards of Blood Transfusion
C H A P T E R 35
Blood Components in Pediatric Practice
Availability of blood components has improved the outcome of various childhood hematological disorders, especially the pediatric
malignancies. Every unit of whole blood collected must be subjected to components as one can satisfy the needs of more than one
patient from the same unit of blood. Whole blood has a limited application in clinical practice like during massive blood loss and for
exchange transfusion; and there too one can use reconstituted whole blood instead. The notable components prepared from one
unit of whole blood include packed red blood cells (PRBC), platelets and fresh plasma; which can be further frozen and used as fresh
frozen plasma (FFP). The PRBC can be used to improve the oxygen carrying capacity as well as volume expander in acute blood loss.
Platelets are very useful to treat bleeding due to thrombocytopenia caused by decreased platelet production and also in platelet
dysfunction. Single donor platelet is much more efficacious than random donor platelet but is expensive and not easily available.
Platelets must be kept on a constant agitator and stored at 22°C. Platelets are often misused in clinical practice. Platelets have no role
in immune causes of thrombocytopenia or as prophylaxis in chronic stable thrombocytopenia. FFP has role to play in treating patients
with multiple factor deficiency classically seen in disseminated intravascular coagulation (DIC) or liver disorders. It should not be used
as volume expander or as source of proteins. Leukodepleted blood products are preferred as donor lymphocytes present in the blood
product can lead to severe toxicities like febrile reactions, allosensitization, increased chances of graft rejection in potential transplant
recipients and transfusion associated graft versus host disease. Use of blood filters can prevent these to a large extent; however
tagvhd can be only prevented by using irradiated blood products. The most important is to prevent unnecessary prescriptions of
blood products and that is possible if the center has written policies which are strictly followed by the clinicians and by in-built audit.
Keywords
Blood components, pediatric, guidelines, packed red blood cells, platelets, fresh frozen plasma, leukodepletion, IAP guidelines.
INTRODUCTION newborn. Accordingly the guidelines for the use of blood

components differ in a child than that for an adult and
Availability of blood components has improved the in a newborn as compared to a child. Various recent
outcome of children with malignancies and those in publications are available which define the guidelines
intensive care set up. It has changed the main focus in for the use of blood components specific for children and
cancer therapy complications from bleeding to infections. newborns.
Blood components now allow administration of high dose
chemotherapy which has changed the outcome in some
Why not Whole Blood and why Components?
pediatric cancers.
Child is not a miniature adult and so also a newborn is Each unit of whole blood has at least four basic components
not a miniature child. There are major differences between which include red blood cells, white blood cells, platelets
an adult and a child in the etiology of cytopenias, the and plasma. Each of these components has specialized
effect of cytopenia on the homeostasis, the physiological function. All these functions are not deranged in all the
responses by the body to the cytopenia, the need of patients and hence all the components are not required
various blood components, the choice and the dose of all the time. Blood is always in short supply and making
the blood component used. This is even more true for a components from one unit of whole blood will satisfy the
364 Section-5 Transfusion Medicine
needs of more than one patient from the same unit of blood. Table 1 Choice of ABO blood group of donor components in
Besides, giving whole blood can lead to harmful effects children
like plasma overload; lymphocytes mediated toxicities Patient’s ABO
or allosensitization, etc. Some components can only be Donor ABO group
group
given effectively as component, e.g. platelets, which are Red cells Platelets FFP∆
otherwise, destroyed in refrigerated stored whole blood. O
Some components are better given as component e.g. First choice O O O
clotting factors as one can not achieve effective levels by
Second choice - A A or B or AB
using FFP alone. It is a social crime to use whole blood and
A
waste this rare commodity!
First choice A A A or AB
Second choice O* O*
Which Components?
B
From one unit of unrefrigerated whole blood one can First choice B B# B or AB
make packed red blood cells, platelet pack (random Second choice O* A or O*
donor platelet), granulocytes pack and fresh plasma. AB
Fresh plasma can be further frozen at –30°C and be
First choice AB AB# AB
used as FFP in future. Pooled plasma can be converted
Second choice A or B A A
into further components like cryoprecipitate, albumin,
Third choice O*
gamma globulins, anti-D globulins, plasma proteins,
* Group O component without high anti-A or anti-B titers
etc. One can modify and manipulate these components
should be selected.
and obtain neocyte red cells, frozen red cells, washed red #
Platelet concentrates of B or AB group may not be easily
cells or platelets, filtered red cells or platelets, UV light or
available.
gamma irradiated red cells or platelets. One can select a ∆
Group O FFP should be given only to O group patients and no
specific donor and get CMV negative blood components,
one else. AB group FFP may not be easily available.
HLA matched blood components or blood products from
specific minor blood group compatible donor. Lastly
one can get stem cells from the umbilical cord blood
Table 2 Choice of Rh blood group of donor components in
of a newborn or peripheral blood of an older child for
children
autologous or allogenic bone marrow transplant or rescue
Patient’s Rh
as the case may be. Donor Rh group
group
Red cells Platelets FFP∆
Storage and Shelf Life
Rh positive
Whole blood is stored at 1 to 4°C. Shelf life will depend First choice Rh +ve Rh +ve Rh +ve
upon the type of anticoagulant and additive used. ACD Second choice Rh –ve Rh –ve Rh –ve
is no more used. CPD or CP-2D blood can be kept for 21 Rh negative
days. CPDA1-A2 blood can be kept for 35 days. If one uses First choice Rh –ve Rh –ve Rh –ve
additives like Nutrisol or Adsol, one can keep the blood for Second choice - Rh +ve* Rh +ve
42 days. Packed red blood cell is stored at 1 to 40°C and * If Rh +ve platelets are given to an Rh negative recipient, anti-D
should be used within 24 hrs if packed using open system. globulin in the dose of 250 mcg should be given to the recipient,
Platelets are stored at 20 to 22°C and that too on a constant which will cover up to 5 platelet transfusion for up to next 6
agitator as resting platelets tend to aggregate. The shelf life weeks.
is 3 to 7 days. Granulocytes are kept at room temperature ∆
FFP usually are not labeled as Rh positive or negative.
and should be used within 24 hours of collection. FFP and
cryoprecipitate have shelf life of one year and are stored
at –30°C. Frozen red blood cells can be kept at –70°C and Whole Blood
have shelf life of 5 to 7 years.
Whole blood has all the components, but that is only in the
first 6 to 8 hours that too when stored at room temperature.
ABO and Rh Compatibility
The platelets are the first to disappear in the first 4 to 48
Tables 1 and 2 describe the choice of the ABO and Rh type hours, the labile clotting factors V and VII are the next to
of the donor blood component in various recipient ABO disappear and the other clotting factors go down thereafter.
and Rh settings. On prolonged storage the potassium levels go up whereas
Chapter-35 Blood Components in Pediatric Practice 365
the pH, the 2-3-DGP levels and the ATP levels fall. Hence Table 3 Indications of using PRBC in a > 4-month-old child
for the exchange transfusion one prefers to use less than 7 Acute blood loss of >15–20% blood volume with
days old blood. Whole blood is stored at 1 to 4°C and has a •
hypovolemia
shelf life of 21 to 42 days as discussed before. • Hb <8 gm% with
– Symptomatic perioperative anemia
Indications – Chronic congenital/acquired transfusion dependent
Whole blood is used only when massive transfusions are anemia
required like in exchange transfusion, massive blood – Emergency surgery with anticipated blood loss
loss with at least one volume blood transfused or during – Uncorrectable preoperative anemia
extracorporeal membrane oxygenation (ECMO). One can – Severe infections
use reconstituted whole blood and one should remember – Associated severe pulmonary disease
that there is nothing like ‘fresh’ blood! 10 cc/Kg body • Chronic transfusion dependent states, e.g.
weight of whole blood will raise HCT by 5 percent and Hb
– Thalassemia
by 1 to 1.5 gm percent.
– Other hemoglobinopathies
Packed Red Blood Cells – Bone marrow failure syndrome including Fanconi’s
anemia
Packed red blood cells (PRBC) contains packed red cells • Pediatric oncology
in 22 to 50 percent of original plasma. Ideal HCT for PRBC – Hb <8 gm% with chemotherapy/radiotherapy
is 70 to 75 percent and it should not be too tightly packed. – Hb <10 gm% if
For newborns, while doing exchange transfusion, HCT
i. Intensive chemotherapy planned
can be adjusted to 50 to 55 percent using additional FFP
or albumin. PRBC is used for improving oxygen carrying ii. Presence of febrile neutropenia
capacity as well as volume expander. Advantage of PRBC is iii. Severe lower respiratory tract infection
that it is low volume as compared to whole blood and hence iv. Thrombocytopenic bleeding
does not lead to circulatory overload. It has less plasma – Hyperleukocytosis (partial exchange preferred)
and hence has less citrate related toxicity. It is mainly used
in patients with hemorrhage or chronic anemia needing
recurrent transfusions. However it contains significant all, used in nutritional anemia, if patient has severe
amount of plasma and leukocytes to lead to toxicities anemia with impending cardiac failure or has associated
related to them like allergic reactions, Nonhemolytic cardiorespiratory disease. Lastly it can be used before
febrile transfusion reactions (NHFTR), allosensitization, surgery, where patient is anemic with Hb less than 7 gm%
Graft-versus-host disease (GVHD), etc. Full cross-match and where moderate blood loss is expected during surgery.
for ABO and Rh and screening for abnormal antibody It is most often misused as “top-up” in patients with
should be done before each transfusion. 10 cc/Kg. Body nutritional anemia, or during surgery to keep Hb above
weight of PRBC will raise the HCT by 10 percent and Hb by “10 gm%”. In such cases, it is counterproductive as it can
3 to 4 gm percent. lead to immune suppression of the recipient and delay
healing.
Indications
The ‘cut offs’ used in various indications are shown in
Chronic Anemia
Table 3. It is used for replacement of volume as well as Special precautions are required while transfusing patients
oxygen carrying capacity. It is used in acute hemorrhage with transfusion dependant states like thalassemia. Ideally
where more than 15 to 20 percent blood volume is lost, detailed blood grouping of the recipient should be done
monitoring vitals, blood pressure and CVP. The most before the first transfusion so that in future one can use
common indication of PRBC is chronic transfusion a specific donor if the patient develops intolerance to
dependent anemia as seen in thalassemia, sickle cell some minor blood group antigen. Always use Coombs’
disease, congenital dyserythropoietic anemia, Diamond cross matched, triple saline washed PRBC in the dose of
Blackfan syndrome, Fanconi’s anemia, aplastic anemia, 15 cc/Kg which will raise the Hb by 3 to 4 gm%. Maintain
chronic renal failure, cancer patients, sideroblastic the pre-transfusion Hb above 9.5 to 10 gm% and raise
anemia, etc. It is also useful in episodic transfusions the post Hb to around 12 to 14 gm%. Keep the record of
for acute hemolysis like in G6PD deficiency, malaria, the pre- and post-transfusion Hb levels and the volume
autoimmune hemolytic anemia, etc. It is rarely, if at transfused every time so that one can calculate and keep
a watch on the yearly requirements. If affordable, use a One can use same donor again after 2 to 3 weeks. One can
WBC filter which will help reduce the nonhemolytic febrile select specific donor like CMV negative or HLA matched
transfusion reactions, allosensitization, etc. donor. But SDP is extremely costly and needs sophisticated
cell separator. Also it is not a product available on the blood
Platelet Transfusions bank shelf and has to be preplanned.
Types of Platelets ABO/Rh Compatibility

There are two types of platelets, random donor platelet Whenever possible use ABO and Rh identical platelets. If
(RDP) obtained by centrifugation of a unit of whole blood, not available, use ABO and Rh compatible donor. This is
and single donor platelet (SDP) obtained by apheresis. One shown in Tables 1 and 2. Platelets though are red cells free
can use a HLA matched or CMV negative donor in specific can contain some RBCs or RBC stroma enough to lead to
situation. Use of WBC filters helps reduce leukocytes and Rh sensitization. Hence Rh positive platelets are given to
hence allosensitization and febrile reactions. Rh negative patient only in emergency and in such cases
Random donor platelet one has to give 250 μg of anti-D globulin to recipient to
prevent Rh sensitization, especially in female recipient.
Random donor platelet (RDP) is obtained by centrifugation
of a unit of whole blood within 6 to 8 hours of collection.
Storage
Collected unit of whole blood must be stored at room
temperature till centrifugation as otherwise platelets will Platelets are thermosensitive and become dysfunctional
lose their function if stored in refrigerator. The blood bag if stored at temperature below 20 to 22°C. Hence unlike
is centrifuged first at 200 RPM for 2 to 3 minutes which will all other blood component, platelets are not stored in
separate the whole blood in to PRBC at the top, WBCs in refrigerator but are stored at 22°C. Shelf life of platelets is
the buffy coat and platelet rich plasma at the bottom. The up to 5 days. Platelets have a natural tendency to aggregate
PRBC is siphoned out in another satellite bag and platelet when left standing still making them lose their function.
rich plasma is then spun at 5000 RPM for another 2 to 5 Hence they need to be stored on a constant agitator.
minutes which will leave platelet poor plasma at the top Transport the platelet quickly and infuse the same in
and platelets button at the bottom. Platelet poor plasma 20-30 minutes. Again do not leave platelets in a tray
is then siphoned out in another satellite bag and what lying still while awaiting transfusion. Caretaker should
remains is one unit of RDP. be told to shake gently the platelet bags periodically to
One unit RDP of contains 5 to 6 × 1010 platelets in 50 to prevent aggregation. Use plastic tubes and never use
60 mL of plasma, trace to 05 mL of RBCs or RBS stroma, and glassware as platelet will stick to the glass surface and get
up to 108 leukocytes. One unit of RDP per 10 kg body weight activated. Remember, platelets should never be stored in
will raise the platelet count by 20,000 to 30,000/cumm. a refrigerator! In case they are put in a refrigerator, they
RDP is less costly and easily available from the blood bank should be discarded.
shelf however it is less efficacious than SDP as it contains 6
to 7 times less number of platelets. Hence patients needing Criteria to Transfuse
repeated platelet transfusions may benefit by using SDP
which will reduce the exposure to a fewer donors. This will Platelet transfusions are usually given to those with
also reduce chances of allosensitization which will reduce thrombocytopenia due to decreased production than to
future platelet refractoriness as well as reduce chances of those with increased destruction. Platelet transfusions
rejection in case of future stem cell transplant. are given when they have significant mucosal bleeds.
Only skin bleeds do not warrant platelet transfusion, but
Single donor platelet such patients should be closely monitored for any further
Single donor platelet (SDP) is obtained from a designate single mucosal bleeds.
donor using cell separator or apheresis machine. Compatible It is controversial as to when to give prophylactic
donor is screened for fitness and serology and once found to platelet transfusion. Child with thrombocytopenia usually
be fit is subjected to continuous or discontinuous apheresis does not bleed spontaneously unless the platelet count
and platelets are collected over 4 to 6 hours. SDP contains 2 falls less than 50,000/cumm. The chances of spontaneous
to 3 × 1011 platelets in 250 to 300 mL of plasma, up to 5 mL bleeds increase when the count drops to less than 5000
of RBCs and 106 to 109 leukocytes. Thus it has 6 to 7 times to 10,000/cumm. Hence the decision when to transfuse
more platelets than RDP (and also that much more volume). platelets prophylactically is based on basic disease, type
The donor should be healthy, off medicines like aspirin and of thrombocytopenia, platelet count, and presence of
should have platelet count of more than 1.5 lakhs/cumm. associated coagulation abnormalities. A well child may
be given prophylactic transfusion when the platelet count Table 5 Platelet transfusion guidelines for neonates
is less than 5,000-10,000/cumm. In patients with massive Prophylactic platelet transfusion (nonbleeding) in the
hemorrhage it should be given when the count is less A
neonate:
than 50,000/cumm as most of the circulating platelets are a. Stable preterm neonate with platelet count <20,000
likely to be non-functional platelets of the infused stored b. Stable term neonate with platelet count <10,000
blood. The ‘cut off’ used in various indication is shown in c. Sick* preterm neonate with platelet count <30,000
Tables 4 and 5.
d. Sick* term neonate with platelet count <20,000
e. Preparation for invasive procedure—lumbar puncture
Indications or minor surgery (central line insertion) with platelet
Platelet transfusions are given for thrombocytopenia or for count <50,000
platelet dysfunction. f. Major surgery with platelet count <100,000
1. Decreased platelet production: This is seen when Platelet transfusion in neonate with clinically significant
B
bone marrow failure occurs like in aplastic anemia, bleeding:
Fanconi’s anemia, thrombocytopenia with absent a. Neonate with platelet count <50,000
radius (TAR) syndrome, and other constitutional hypo b. Neonate with condition that increases bleeding, e.g.
plastic anemia. It is also seen when the bone marrow DIC and platelet count <100,000
is infiltrated, e.g. in leukemia and other metastatic c. Neonate with documented platelet function disorder
cancers or in presence of bone marrow suppression irrespective of circulating platelet count
due to chemoradiotherapy or fulminant infections. d. Bleeding neonate meeting the clinical or laboratory
Platelet transfusions have revolutionized the treatment criteria for DIC, at discretion of treating physician
and the outcome of pediatric cancers. The cause of *Definition of a sick neonate:
mortality has shifted from bleeding to infections with a. Cardiovascular instability, HR >180/min or dopamine or
better platelet support available now. ionotropic infusion at >3 micrograms/kg/min
2. Increased consumption of platelets: It is indicated b. Respiratory instability, FiO2 requirements >0.4 or
in disseminated intravascular coagulation (DIC), significant mechanical ventilation MAP>7
c. Central nervous system instability—within 72 hrs of
seizure
Table 4 Indications of using platelets in a >4-month-old child
A specific threshold for transfusion may not be appropriate for
with thrombocytopenia patients with chronic stable thrombocytopenia who are best
• Prophylactic platelets (without bleeding) managed on an individual basis depending on the degree of
– <5–10,000/cumm in a nonsick child hemorrhage
– <20,000/cumm in a sick child with
i. Severe mucositis
ii. Disseminated intravascular coagulation (DIC)
necrotizing enterocolitis (NEC), and Kasabach-
iii. Platelet likely to fall <10,000/cumm before next
Merritt syndrome. In these cases, there is good platelet
evaluation
recovery at one hour after transfusion, but not at 24
iv. Thrombocytopenic bleeding
hours suggesting consumption. In cases with DIC,
– Hyperleukocytosis (Partial exchange preferred) frequent estimation of the platelet count and coagu
i. Bone marrow aspiration/biopsy can be without lation screening tests should be carried out. There
platelet support is no consensus on a target platelet count, but aim is
ii. Lumbar puncture <30,000/cumm to maintain the platelet count > 50,000 as in massive
iii. Other surgeries <50,000/cumm blood loss, would seem to be reasonable practice. In
iv. Surgery at critical sites like CNS, eyes <100,000/ chronic DIC, or in the absence of bleeding, platelet
cumm transfusions should not be given merely to correct a
– <50,000/cumm with acute bleeding, massive low platelet count. Platelets are contraindicated in
hemorrhage, head trauma, multiple trauma thrombotic thrombocytopenic purpura (TTP) and
Chronic stable thrombocytopenia only in presence of hemolytic uremic syndrome (HUS).
•
significant mucosal bleeding 3. Massive transfusions: There is consensus that the
Platelet dysfunction only in presence of significant platelet count should not be allowed to fall below
•
mucosal bleeding 50,000 in patients with acute bleeding. A higher target
Chronic stable DIC only in presence of significant mucosal level of 1,00,000 has been recommended for those with
•
bleeding multiple trauma or CNS injury.
4. Increased platelet destruction: It can occur due to Table 6 Measures to be undertaken in a patient with platelet
immune or nonimmune mechanisms. Non-immune dysfunction with clinical bleeding
destruction can occur following drugs or infections. Avoid/withdraw drugs known to have antiplatelet
Immune destruction can occur in post-trans fusion •
activities
purpura, autoimmune diseases, idiopathic throm Correct baseline condition known to lead to platelet
bocytopenic purpura (ITP), and alloimmune disease •
dysfunction
of newborn. The ITP is the most common scenario • Correct HCT to >33% (with transfusion/EPO)
in this category. Platelet transfusions are generally • Use of DDAVP in a case of storage pool disorder
not effective in this group of diseases, as they will be
• Use of alternate therapy like factor VIIa
immediately destroyed by the antibody present in
Use of platelets when above measures are ineffective or
recipient after transfusion. Platelet transfusions should •
inappropriate
be reserved for patients with life-threatening bleeding
Consider platelet refractoriness after repeated use of
from the gastrointestinal or genitourinary tracts, into •
platelets
the central nervous system or other sites associated with
severe thrombocytopenia. A large number of platelet
concentrates may be required to achieve hemostasis as Clinically one can judge the efficacy by seeing the
a result of reduced survival of the transfused platelets. cessation of bleeding. One can look for the expected
Therapies such as intravenous methylprednisolone increments by calculating corrected count increment
and immunoglobulin should be given at the same time (CCI) (×109/L) as follows by doing platelet count at one
to maximize the chances of stopping the hemorrhage hour and 24 hr after transfusion.
and raising the platelet count.
5. Hypersplenism: Normally 1/3rd of platelets are pooled Post-transfusion platelet count-

CCI = Pretransfusion
in the spleen. This proportion will increase in patients platelet count × BSA m2
_____________________________
with hypersplenism due to any reason. Again platelet Platelets infused × 1011
transfusions may not be effective in such cases, as they Normal CCI is > 7.5 × 109/L at one hour, and > 4.5 × 109/L
will be immediately removed from the circulation into at 20 to 24 hrs. If CCI is normal at one hour, but less at 24
the enlarged spleen. hrs, it suggests consumption coagulopathy. If CCI is less at
6. Dilutional: Dilutional thrombocytopenia can occur 1 hour itself, it suggests immune destruction.
following massive transfusions in patients with massive
hemorrhage or following exchange trans fusions. Side effects: Both RDP and SDP contain platelets, small
Supplemental platelet transfusions may be required in volume of plasma, WBCs and some RBCs or RBC stroma.
such cases to keep platelet counts of > 50,000/cumm. Hence, platelets can lead to similar reactions like other
7. Platelet dysfunction: Various congenital and acquired blood components including febrile reactions, urticaria,
platelet functional disorders may present with signi allosensitization, transfusion associated infections and
ficant bleeding. If local measures fail to control rarely anaphylaxis. In a newborn, antibodies present
bleeding, platelet transfusions will be required. One in the small volume of plasma contained in platelets
should use platelets sparingly in such cases as allo- may be enough to lead to significant hemolysis if
sensitization may prevent good recovery in future there is a mismatch of blood groups between donor
after a number of transfusions are given. One can use and recipient. Hence it may be better to use a donor
HLA matched platelets in such cases. Table 6 shows with low titer anti-A or anti-B antibodies in case of a
the measures to be undertaken in a case of platelet mismatch. If not possible one can use washed platelets
dysfunction with clinical bleeding. as saline washing will remove the antibodies to a large
Platelet transfusion efficacy: One unit of RDP per 10 kg extent. In case of allosensitization patient will become
body weight increases platelet count by 20,000 to 30,000/ refractory to further platelet trans fusions with poor
cumm. SDP is 5 to 7 times more effective than RDP. The recovery of platelet counts in spite of adequate dose.
efficacy of platelet transfusion depends upon various Allosensitization can also increase the chances of graft
factors. Platelet factors like source of platelets, type of rejection in case of future stem cell transplant. Such
platelets, storage, collection and administration will affect patients may benefit by using immune suppression like
the efficacy. Similarly, factors in recipient that affect the low dose steroids, plasmapheresis or using WBC filters
efficacy include pretransfusion count, fever, sepsis, size of while transfusing platelets. Of course one can avoid
liver and spleen, presence of antibodies or consumption many of these complications by using WBC filters with
coagulopathy and drugs taken by the recipient. every platelet transfusion right from 1st transfusion
in patients who are likely to need repeated platelet RDP has 4 to 6 × 107 WBC, SDP has 2 to 4 × 108 WBC and
transfusions. granulocyte pack has 1011 WBC. Ideally all the transfusion
should be leukodepleted especially in patients needing
Granulocytes recurrent transfusions and in immunno compromised
hosts.
Though its use in infections may sound logical, granulocytes
Methods of leukodepletion: There are various ways of
are rarely used in current clinical practice. People have
leukodepletion. Each method has its own merits and
tried giving granulocyte transfusion in patients with
demerits and efficacy.
severe uncontrollable infection in presence of congenital
1. WBC filter: Third generation WBC filters are 99.5
or acquired neutropenia or neutrophil dysfunction. It is
percent efficient in removing the donor lymphocytes.
usually reserved for neutropenic patients with fulminant
Activated lymphocytes can release cytokines like IL2,
sepsis not controlled by antibiotics and antifungal
TNF-α during storage and hence it is best to remove
with ANC < 300 in newborn, ANC < 100 in infants and
the lymphocytes while collecting blood from the donor
ANC < 500 in immune compromised host. It should always
using in-line WBC filter, rather than using the WBC filter
be used along with antibiotics and antifungals. As colony
at bedside while giving the transfusion to the recipient.
stimulating factors are now easily available and affordable,
The advantage of WBC filter is its high efficacy and
use of granulocytes has fallen in to disrepute.
simplicity to use. The disadvantages include its high
Buffy coat preparations are not very satisfactory as the
cost and inability to prevent TAGVHD. Each filter costs
cells tend to become nonfunctional. Packs obtained by
` 400-500/- and is not reusable. Ideally all transfusions
apheresis are the best. They should be used within 24 hr of
should be given using filters especially if patient needs
collection and stored at room temperature. Each pack has
recurrent transfusions and develops NHFTR.
1011 granulocytes in 200 cc of plasma. Dose recommended
2. Washed cells: 90 percent of lymphocytes and 99
is 109 granulocytes/kg each time. It can be repeated every
percent of plasma are removed by washing the PRBC
12-24 hr for 4 to 6 days. It should be given obviously without
with saline or blood processor. This will help reduce
using the WBC filter. It leads to all the side-effected related
NHFTR, allosensitization and other toxicities related to
to plasma and lymphocytes. One should use ABO/Rh
WBC as well as allergic reactions to plasma proteins.
compatible donor.
It is a simple technique and needs cold centrifuge but
is not as effective as the WBC filter for leukodepletion.
Leukodepleted Blood Components One can combine washing and use of WBC filter
where the patient is prone to severe allergic reactions.
Why Leukodepletion? Washing does not prevent TAGVHD. Lastly, washed
Various side effects and toxicities are associated with the platelets from mother are given in a baby suffering
presence of significant number of donor lymphocytes in the from alloimmune thrombocytopenia.
unit of blood component transfused. These include non- 3. Gamma irradiation: TAGVHD can be only prevented
hemolytic febrile transfusion reactions; allosensitization; by gamma irradiating the blood. Dosage of 2500-
increased chances of rejection of graft in candidates for 3500 cGy are used to irradiate the components. The
future transplant; lymphocyte mediated lung toxicity like only disadvantage is need for the sophisticated and
acute respiratory distress syndrome (ARDS); transmission expensive irradiator. There are chances of membrane
of viral infections like HIV, human T-lymphocyte virus leak from the irradiated cells which can result in to
(HTVL), Epstein-Barr virus (EBV), cytomegalovirus increased potassium levels. Hence blood should be
(CMV), etc. which are intracellular pathogens; transfusion irradiated just before infusion or else supernatant
associated graft vs host disease (TAGVHD) in immune plasma should be removed before transfusion.
compromised patients and in transfusion from first Ideally all blood should be irradiated where there is
degree relatives; and immune suppression of the recipient risk of TAGVHD. This includes transfusion given to new-
especially in surgical patients. These donor lymphocytes born especially preterms < 1200 g, intrauterine transfu-
ordinarily do not serve any beneficial effects and hence sions, patient with primary or secondary immunodefi-
should be removed or depleted from the unit transfused to ciency, cancer patients, organ transplant recipients and
eliminate or reduce the chances of these side effects and transfusion given to normal person from a first degree
toxicities. relative donor.
Non-hemolytic febrile transfusion reactions (NHFTR) 4. Frozen cells: This is routinely available in the west but is
occur when > 5 × 106 lymphocytes are present in the unit, rarely available in India. RBC frozen at –70°C has shelf
whereas for TAGVHD it is > 107 cells/kg body weight. life of 5 to 7 years. While freezing, deglycerolization is
One pack of packed red blood cells (PRBC) has 109 WBC, done to prevent intracellular ice formation. It should
be thawed gradually and once thawed should be used patient and can transmit all the plasma borne infections,
within 24 hours. The efficacy for leukodepletion is 90 albumin should be used as a volume expander which
percent and plasma depletion is 99 percent. Hence is much safer. Similarly albumin and not FFP should be
it reduces toxicities related to both lymphocytes and used to replace proteins or albumin. If patient needs both
plasma. Advantage of frozen cells is its availability volume expansion as well as clotting factors like in DIC,
in emergency where one can use O–ve frozen cells sepsis, NEC, etc. one can use FFP. However FFP should
in AB negative plasma. One can collect blood from not be used in a case of DIC without clinical bleeding.
CMV negative donors; HLA matched donor or rare FFP should also not be used prophylactically to prevent
blood group donor and freeze it for future use. Lastly intracranial bleeding in neonate.
autologous blood collected for surgery can be frozen Table 8 summarizes the indications of using FFP in
and used in future if surgery gets postponed for some clinical practice. FFP is mainly used to replace clotting
reasons. Disadvantage of frozen cell is that it needs factors. It can be given when the patient presents
sophisticated instruments to prepare and store it and with bleeding for the first time where the diagnosis is
is extremely costly. It cannot prevent TAGVHD. uncertain as to which factor is deficient. In known cases
of hemophilia, it is better to use factor concentrates,
Fresh Frozen Plasma as they are more efficient and safe. FFP is used for
deficiencies of other factors like factor V, VII, etc. where
Fresh frozen plasma (FFP) is made by freezing the plasma
factor concentrates are not available. It is also used
obtained at the end of centrifugation of the whole blood
where multiple factors need to be replaced as in case of
unit and is stored at <–30° C. The shelf life of FFP is one
hemorrhagic disease of newborn, liver disease, preterm
year when properly stored. It should be thawed at 37°C
with liver dysfunction, DIC, etc. FFP also contains AT
over 30 minutes in the water bath. Thawed FFP should
III, protein C and protein S and hence is useful in the
be used within 4 hours if used for hemophilia A or used
deficiency of these factors too like in the treatment of
within 24 hours if used for other conditions provided it is
purpura fulminans; however in the west activated protein
stored properly. The FFP contains all the plasma proteins
C concentrates are easily available. FFP is used for plasma
including albumin, gamma globulins and most important
exchange in patients with TTP or HUS. It can be used to
clotting factors. As labile factor V and VIII tend to decrease
reconstitute whole blood along with PRBC or to adjust
on storage, freezing of the plasma should be done within 4
HCT of PRBC for exchange transfusion in newborn.
to 6 hours of collection to prevent loss of these factors. One
Lastly, FFP is useful to prevent and treat coagulopathy
unit of FFP has 200 to 250 mL of plasma and 1 mL of plasma
due to L-asperginase in cancer p atients.
contains approximately 1 unit of each clotting factor. The
FFP leads to all the side effects related to plasma like
hemostatic content of a unit of FFP is shown in Table 7. As
allergic reactions like urticaria, anaphylaxis, especially
the maximum tolerated dose of FFP is 10 to 15 cc/kg every
in IgA deficient patient and transmission of plasma
12 hr, one can not achieve very high plasma level of the
borne infections to the recipient. In small babies, it can
missing clotting factors without volume overloading the
lead to hemolysis if it contains high levels of antibodies
patient.
The FFP is often misused as volume expander. As FFP
can lead to allergic reactions, anaphylaxis in Ig A deficient Table 8 Indications of using FFP considered as appropriate
• Inherited factor deficiency
Table 7 Hemostatic content of a unit of FFP a. P
atient with unknown clotting factor deficiency
presenting for the first time
Fibrinogen 2.67 mg/mL
b. Single clotting factor where factor concentrate is not
Factor II 80 IU/mL
available like factor V or XI
Factor V 80 IU/mL
c. Multiple clotting factor deficiency
Factor VII 90 IU/mL
• DIC with clinical bleeding
Factor VIII 92 IU/mL
• Hemorrhagic disease of newborn
Factor IX 100 IU/mL
Liver disease with coagulopathy for prevention and
Factor X 85 IU/mL •
control of bleeding
Factor XI 100 IU/mL Dilutional coagulopathy as seen after massive transfusion
Factor XII 83 IU/mL • (surgical patients) to maintain PT, aPTT to <1.5 time the
Factor XIII 100 IU/mL control
AT III 100 IU/mL • Plasma exchange for TTP/HUS
VWF 80 IU/mL • Sick newborn with coagulopathy and bleeding
against recipient’s blood group antigens. FFP has also 13. WBC filters are an excellent method of leukodepletion
been associated with rare but significant toxicities like and should be used more liberally.
transfusion related acute lung injury (TRALI). 14. One should avoid relatives as donors.
Take Home Messages BIBLIOGRAPHY

1. It is a criminal waste to use whole blood in general as 1. American Association of Blood Banks (2003). Pediatric
one unit of whole blood can satisfy the needs of more Transfusion: A Physician’s Handbook; 1st edition. Edited
than one patient. by Roseff SD.
2. Whole blood is indicated only for massive blood loss 2. Beulter E. Platelet transfusion: the 20000/L trigger. Blood.
and for exchange transfusion. There too one can use 1993;81:1411-3.
reconstituted whole blood. 3. Consensus Conference on Platelet Transfusion. Br J
3. Packed red blood cells can serve both the purposes, Cancer. 1998;78:290-1.
4. Ennio CR. Red cell Transfusion Therapy in Chronic
increasing the oxygen carrying capacity and volume
anemia. Hemat Oncol Clin North Am. 1994;8:1045-52.
expansion in acute blood loss.
5. Guidelines for the use of fresh-frozen plasma, cryopre
4. Transfusions are rarely required if at all for patients
cipitate and cryosupernatant. Brit J Hematol. 2004;126:
with nutritional anemia. 11-28.
5. Transfusions are inappropriate to raise Hb in patient 6. Guidelines for the use of platelet transfusions. Brit J
with nonemergency surgery, as a ‘top-up’ after surgery Hematol. 2003;122:10-23.
and as a ‘panacea’ in a sick malnourished pale child. 7. Indian Academy of Pediatrics transfusion guidelines for
6. Iatrogenic blood loses are the most common cause of neonates and older children (under publication).
anemia in a newborn. Minimize the investigations. 8. Miller JP, Mintz PD. The use of Leucocyte-Reduced blood
7. Thalassemics should ideally receive triple saline components. Hemat Oncol Clin North Am. 1995;9:69-90.
washed, Coombs’ cross matched PRBC using a WBC 9. Rentels PB, Kenney RM, Crowley JP. Therapeutic support
filter. of the patient with thrombocytopenia. Hemat Oncol Clin
8. Platelets are to be stored at 22°C on a constant agitator, North Am. 1994;8:1131-51.
to be used in 20 to 30 minutes and not to be kept in 10. Roseff SD, Luban NL, Manno CS. Guidelines for
fridge at all. Do not use glassware while giving platelets. assessing appropriateness of pediatric transfusion. Trans.
9. Platelets are not indicated in immune causes of throm- 2002;42:1398-413.
bocytopenia (like in ITP); and as also for prophylaxis 11. Rosen NR, Weidner JG, Boltd HD, et al. Prevention of
in a case of chronic stable thrombocytopenia (like in transfusion associated graft-versus-host disease: selection
of sufficient dose of gamma irradiation. Transfusion. 1993;
aplastic anemia).
33:125.
10. FFP is often misused as volume expander (in patient
12. Shah N, Lokeshwar MR. Blood components in pediatric
with shock), as a source of proteins (in preterms), as
practice. Proceedings of South. Pedicon. 2000.pp.55-68.
a source of known clotting factor 9 in a known case of 13. Strauss RG, Levy GJ, Sotelo-Avila C, et al. National survey
Factor VIII deficiency where cryoprecipitate or factor of neonatal transfusion practices: II. Blood Component
concentrates are a better option. therapy. Pediatrics. 1993;91:530-6.
11. FFP is indicated in multiple factor deficiency as seen 14. Transfusion guidelines for neonates and older children.
in liver disease and DIC or as factor replacement in a Brit J Hematol. 2004;124:433-53.
patient with unknown factor deficiency coming for the 15. Voak D, Cann R, Finney RD, et al. Guidelines for
first time, and for plasma exchange for TTP/HUS. administration of blood product transfusion of infants and
12. Leukodepleted blood products are better as the donor neonates. British Committee for Standards in Hematology
lymphocytes present in unit of blood product can lead Blood Transfusion Task Force. Transfusion Medicine.
to unnecessary and avoidable side effects. 1994;4:1411-3.
C H A P T E R 36
Nucleic Acid Amplification Testing
In today’s modern healthcare, blood and blood component transfusions have a very large range of indications and are life-saving for
the patients. However, with the increase in transfusions the risk of transfusion transmitted infections (TTIs ) has also increased. A major
challenge is to use screening assays with maximum sensitivity and specificity to make blood as safe as possible.
In India, the five tests mandatory by Food & Drugs have the highest risk of transfusion transmitted HBV and
Administration (FDA) for the donated blood units are probably would be the most to gain from HBV NAT. In
HBsAg, HIV-Ab, HCV-Ab, VDRL and malarial parasites. addition, worldwide 170 million people are infected with
Currently, testing methods carried out in India are based HCV and 40 million with HIV. These are a serious global
on serological assay detecting either antibodies or antigen. concern.
Naturally, they have a long window period as they basically
detect the host immune response. The ‘window period’ Window Period and its Clinical Significance
is defined as the time period between the start of an
Blood is processed into blood components to enable
infection to the earliest diagnostic detection. Shortening
more than one patient to benefit from a single donation.
this window period has been the focus of attention in
Thus, a single unit of blood collected from a donor in the
transfusion medicine for the last three to four decades.
window period of infection may be transfused to up to four
recipients or may be added to pools of more than 1000
Size of the Problem
units to manufacture blood derived products.
Over two billion people worldwide are infected with The greatest threat to the safety of blood supply is
HBV, which is the leading cause of liver disease. Of these, donation by ‘seronegative’ donors during ‘window period’
more than 350 million are chronically infected, with a of initial infection and detectable seroconversion. Window
higher risk for liver cancer and liver cirrhosis. Currently, period samples have a very low viral load. Detection of very
available screening technologies are designed to detect low viral load samples requires highly sensitive assays.
core antibodies or surface antigens. However, these Hence the Nucleic Acid Amplification Testing
infection indicators do not appear until eight weeks after (NAT)……….
an infection. Thus HBV presents a higher residual risk of With currently used ID-NAT assay the window period
transmission by transfusion than HCV or HIV and the HBV is shortened considerably, as it is a highly sensitive and
infection window period is the real issue in the transfusion specific test that detects very low levels of viral RNA or
setting. Countries like India with a high prevalence of HBV DNA that may be present in donated blood.
Chapter-36 Nucleic Acid Amplification Testing 373
Window Period and Testing Technology: A Schematic Representation
Key for Interpretation

ID-NAT Ultrio Plus
MP-16 NAT Ultrio Plus
Ab or Ag
HIV-1 Detection
4.7
8.1
15.0P24 Antigen
(Window period in days)
HCV Detection
2.2
4.1
58.3-HCV Antibody
HBV Detection
14.9
24.9
38.3 HBsAg
is present. NAT screening can be carried out as a minipool

Testing Options Available and their Principles
testing or individual (ID-NAT) testing. Japan started in
Genomic screening for infectious agents using NAT is 1999 with a minipool of 500. All the countries currently use
performed with several in vitro nucleic acid amplification minipool (6/24/48/96 samples) or ID-NAT testing.
techniques, e.g. transcription–mediated amplification Key to interpretation
(TMA), polymerase chain reaction (PCR), ligase chain • NAT-yield: EIA negative, NAT positive
reaction and nucleic acid sequence-based amplification. • Sero-yield: EIA positive, NAT negative
All these techniques detect the presence of infectious
microorganisms in donor blood by amplifying the nucleic
Impact of NAT: Evidence from Published
acid sequences specific to the microorganism, giving it a
much higher level of sensitivity and specificity than routine Literature
EIA test. Thus the power of NAT lies in its ability to detect A pilot project in India at Apollo Indraprastha Hospital,
viral genomic nucleic acids rather than the presence of showed that out of 12,224 study samples 133 (1.09 %) were
antibodies. NAT screening is characterized by three critical reactive by Ultrio assay. The 84 samples were seroreactive
processes: sample extraction, amplification and detection. but NAT nonreactive. There were 8 NAT yield cases–1 HIV,
NAT is used in addition to the antibody/antigen test since in 1 HIV-HCV coinfection and 6 HBV. Observed NAT yield
some individuals theoretically the amount of virus may have for all three viral TTI’s was 1 in 1528 (0.065 %).
fallen below detectable limits and antibodies could still be However, NAT yield reported from various centers
detectable as in case of HCV. In some cases with HBV again, in India varies from 1 : 300 to approximately 1 : 8000 as
the viral copies may be undetectable but the surface antigen reported at our center.
Implementation of NAT has led to a residual risk of Bacterial screening poses a special challenge in
transfusion transmitted infections of less than 1 : 1 million transfusion medicine. With the introduction of NAT the
in case of HIV and HCV in developed countries. risk of transmission of clinically relevant viral infection is
Blood donor screening by NAT for at least HIV-1 far below the risk of bacterial infections. Many countries
and HCV has been implemented in different countries have implemented culture methods to detect very low
(e.g. USA, Canada, parts of Brazil, Spain, France, the UK, levels of bacterial concentrations. NAT offers a good
Denmark, Germany, the Netherlands, Belgium, Greece, method to detect bacteria.
Slovenia, the Czech Republic, South Africa, Ghana,
Luxembourg, Switzerland, Italy, Japan, parts of China, Practical Considerations
Australia, Poland, Norway, Finland and New Zealand).
One exception in Europe is Sweden. Based on the very low It should kept in mind that a very small number of blood
incidence of HIV-1 and HCV in their donor population, donors may be infected with viral concentrations below
they decided to stop blood donor screening by NAT in the level of analytical sensitivity and therefore NAT can
2008. In India, NAT testing is carried out for HIV, HCV and offer close to 100 percent but not 100 percent safety.
HBV also due to high prevalence of hepatitis B virus in the For proper interpretation of NAT results, in view of very
population. low number of viral copies, based on Poisson distribution
an algorithm was proposed in NAT users meet in India
which is followed by many centers.
Pathogens for which NAT Testing is Available
Proposed algorithm:
a. HIV: 1st case of HIV was reported in 1982. The virus
doubling time is approximately 17 hours and the dia
gnostic window period is reduced to less than 5 days
using ID-NAT.
b. HCV: The virus was first described in 1989 but it was
known since 1970s that a virus other than HAV and
HBV existed, it was called non-A, non-B hepatitis
virus. The virus doubling time is very short (10–12
A
hours), and therefore the diagnostic window period
has been brought down to approximately 4 days using
ID NAT. In Germany, the residual TTI risk for HCV was
estimated to be 1 : 200 and is currently calculated at
1 : 10.8 million.
c. HBV: Compared to HIV and HCV, the virus doubling
time is very low (approx 2.56 days). Hence, with the
current ID NAT assay the diagnostic window period
has been brought down to 15 days compared to 55 days
using EIA. Anti-HBc is used for blood donor screening
in low endemic countries such as USA and Germany.
This is not feasible in high endemic countries such
as India because the percentage of anti-HBc reactive
donors might cause an unacceptable loss of life-saving
blood units.
d. The other viruses which can be detected using NAT
are West Nile virus (WNV) infection (as in USA),
hepatitis A virus infection, hepatitis E virus infection,
parvovirus B19 virus infection, chikungunya virus
infection.
HIV-2 has been found predominantly in West Africa,
and cases have also been reported from India. Since EIA is
always used in conjunction with NAT testing risk of finding
HIV-2 yield is minimum and some NAT systems have
already incorporated HIV-2 in the multiplex screening
procedure. B
Chapter-36 Nucleic Acid Amplification Testing 375
FURTHER READING
1. Kuhns MC, Busch MP. New strategies for blood
donor screening for hepatitis B virus. Mol Diag Ther.
2006;10(2):77-91.
2. Makroo RN, Choudhary N, Jagannathan L, Parihar-
Malhotra M, Raina V, Choudhary RK, et al. Multicenter
evaluation of individual donor nucleic acid testing (NAT)
for simultaneous detection of human immunodeficiency
virus–1 and hepatitis B and C viruses in Indian blood
donors. Indian J Med Res. 2008;127:140-7.
3. Schmidt M, Seifried E. Improving blood donor screening
by nucleic acid technology (NAT), ISBT Science Series.
C 2010;5:219-29.
4. Zanetti AR, Romano L, Zappa A, Velati C. Changing
patterns of hepatitis B infection in Italy and NAT
testing for improving the safety of blood supply.
Journal of Clinical Virology. 2006;36(Suppl):
S51-5.
5. Zou S, Dorsey KA, Notari EP, Foster GA, Krysztof DE,
Musavi F, et al. Prevalence, incidence and residual risk
of human immunodeficiency virus and hepatitis C virus
infections among United States blood donors since
the introduction of nucleic acid testing, Transfusion.
2010;50:1495-504.
D
C H A P T E R 37
Transfusion Transmitted Infections
Most deaths caused by blood transfusion worldwide are due to transfusion transmitted infections. The various agents (viruses,
bacteria or protozoa) responsible share the following features: persistence in the donor’s bloodstream giving rise to carrier states; a
susceptible receptor population; the ability to cause asymptomatic infection; stability in stored blood and in many cases in plasma
fractions. Infectious agents that are only present in blood cells, e.g. malarial parasite can be transmitted by all blood components
except cell-free plasma. On the other hand, those viruses that are present in plasma, e.g. Hepatitis B, can be transmitted by cell-free
plasma and its fractions as well as by cellular components. Screening tests are effective preventive measures but they cannot detect
emerging agents such as HIV in the 1980s or West Nile fever at the beginning of this century. Presently in India, it is mandatory to test
donated blood for hepatitis B and C, HIV 1 and 2, malarial parasites and syphilis.
The following sections review the various transfusion • Yersinia enterocolitica

transmitted infections including their epidemiology, • Other enterobacteriaceae
clinical features, management and preventive measures. • Psychrophilic pseudomonas.
KNOWN TRANSFUSION TRANSMITTED Viral Hepatitis

VIRAL INFECTIONS Despite the dramatic reduction in the risk of viral
transmission during the past three decades, viral hepatitis
• Viral hepatitis
remains a serious complication of transfusions worldwide.
– Hepatitis B
– Hepatitis C
Hepatitis B
– Hepatitis D
– Hepatitis A Hepatitis B virus (HBV) is a major human pathogen
– Hepatitis G that causes acute and chronic hepatitis, cirrhosis and
– Transfusion transmitted virus (TTV) and SEN-V hepatocellular carcinoma.1 The overall prevalence of
• Retroviral infection HBV infection in the United States is about 5.6 percent
– Human T-cell leukemia virus (HTLV) types 1 and 2 as indicated by HBsAg and anti-HBc positivity rates.2 The
– Human immunodeficiency virus (HIV) types 1 estimated prevalence of HBV in India is between 3 and 7
and 2 percent.3 However, the risk of HBV infection in transfusion
• Human herpes virus infection recipients is progressively decreasing with the use of
– Cytomegalovirus (CMV) sensitive screening tests, with an estimated risk of 1 per
– Transfusion transmitted cytomegalovirus (TT-CMV) 205000 units in the USA.4
– Epstein-Barr virus (EBV) HBV is a double shelled DNA virus of the hepadnaviridae
• Human herpes virus (HHV) 6 through 8. family. The virus contains various antigens that may help
• Parvovirus B19 in distinguishing the duration of infection and infectivity
• Bacterial infections of the host.
Chapter-37 Transfusion Transmitted Infections 377
HBV antigens and antibodies in the blood
Fig. 1 Serological markers of HBV and their time of appearance during the course of infection
Hepatitis B virus is spread by transfusing infected Hepatitis B vaccine and hepatitis B immunoglobulin
blood, plasma or coagulation factor concentrates. The (HBIG) are available for prevention of HBV infection.
mean incubation period is 63 days (range 30–150 days) Universal immunization of all children with hepatitis B
in post-transfusion hepatitis cases. Most cases of HBV vaccine is recommended in both pre- and postexposure
infection are asymptomatic as evidenced by high carriage situations and provides long-term immunity. The HBIG is
rate of serum markers in the absence of history of acute recommended in neonates born to HbsAg positive mothers
hepatitis. The prodrome is characterized by lethargy, and children with intimate contact with acute HBV infection.
malaise and anorexia and rise in liver transaminases 6 Interferon-α-2b (IFN-α-2b) and lamivudine are the
to 7 weeks after exposure. Some patients have a serum current two therapies available for chronic HBV infection.
sickness-like illness during the prodrome phase. Jaundice Long-term eradication rates of 25 percent have been
is present in about 25 percent of the patients with onset reported in children. Patients most likely to respond are
about 8 weeks after exposure and lasts for 4 weeks. those with low serum HBV DNA titers, HbeAg, active
Acute fulminant hepatitis with coagulopathy, inflammation and recently acquired disease. Liver
encephalopathy and cerebral edema may occur with a transplantation also has been used to treat patients with
mortality of 0.5 to 1 percent of all HBV infections. end-stage HBV infection.
Five percent develop chronic hepatitis,5 which can
lead to cirrhosis and primary hepatocellular carcinoma. Hepatitis C
The carrier state develops most commonly after Hepatitis C virus (HCV) has now been recognized as the
asymptomatic infection, especially if the infection is cause of almost all parenterally transmitted cases of what
acquired during infancy. was previously called non-A non-B hepatitis. The HCV is
Evaluation of an individual for HBV infection usually globally distributed with a remarkably uniform prevalence
includes testing for serum HBsAg, antibody to HBsAg rate of 1 to 2 percent.7 The epidemiology of HCV in India is
and IgM anti-HBc (antibody to hepatitis B core antigen) not well described, more so in children. The prevalence of
by enzyme immunoassays (EIA). Detection of IgM anti- HCV in blood donors in India (1–1.5%) is higher than that
HBc in serum is helpful in the diagnosis of HBV infection in developed countries (0.3–0.7%).8-10 A high prevalence
during “window period” prior to the appearance of HbsAg of HCV is found in many high-risk groups exposed to
(Fig. 1). Moreover, it can detect recent HBV infection in blood or blood-products like hemophilics (24–90% anti-
rare HBV mutants with altered HBsAg epitopes. Although HCV positive), IV drug users (70–92% anti-HCV positive),
most blood centers perform screening for HBsAg, there patients with pediatric hematological malignancies (55%
is a convincing argument to augment it with anti-HBc HCV-RNA positive) and those with thalassemia (60% anti-
testing.6 HCV positive).8,11,12
Hepatitis C virus (HCV) is a single-stranded RNA virus The clinical outcome of HDV infection depends on the
from the Flaviviridae family. The HCV is transmitted by mode of infection.
blood components and blood products including IVIG, • In coinfection, acute hepatitis, which is much more
anti-D Ig for IV use and factor VIII concentrate. The HCV severe than that caused by HBV alone, is common
has never been transmitted by albumin concentrates or by but risk of developing chronic hepatitis is low. In
anti-D Ig for IM use. superinfection, acute illness is rare and chronic
• Incubation period varies from 7 to 9 weeks. hepatitis is common. However, the risk of fulminant
• Acute infection tends to be mild and insidious in both hepatitis is highest in superinfection.
adults and children. • The diagnosis is made by detecting IgM anti-HDV,
• Only 25 percent cases are icteric. which develop 2 to 4 weeks after coinfection and 10
• Fulminant liver failure rarely occurs. weeks after superinfection.
• About 85 percent cases develop chronic hepatitis. • PCR assays for viral RNA are available only as research
• After about 20 to 30 years 25 percent ultimately tools.
progress to cirrhosis, liver failure and occasionally • There is no vaccine against HDV. However, HDV has
primary hepatocellular carcinoma. little current relevance to transfusion safety, because
Detection of HCV infection is based on EIA for anti- measures used to detect and prevent HBV infection are
HCV antibodies or testing directly for viral RNA or DNA. also effective against HDV.
The recent risk estimate of HCV is 1:103,000 per donor
exposure in the US.13 This was calculated using second Hepatitis A
generation HCV test with window period of 82 days.
Screening by third generation EIA reduces the window Hepatitis A virus (HAV) is a RNA virus belonging to the
period to 66 days and hence further decreases the risk of picornaviridae family. The incidence of HAV varies
transmitting HCV through transfusion to 1:127,000 units significantly with age. Highest incidence rates are seen
transfused.14 With the implementation of nucleic acid in children in the age group of 5 to 15 years accounting
technology-based HCV screening (HCV-NAT) there has for 30 percent of all cases. The HAV is rarely acquired by
been a major decline in the risk of HCV transmission to blood transfusion with a transfusion-associated risk of less
1:3,68,000 units transfused in the US. The NAT testing for than 1 per 1,000,000 units of blood transfused.17 Rarity of
HCV has shown reduction in window period for HCV from parenteral transmission of HAV has been attributed to
66 to 10–30 days.14,15 A recent study in US has shown risk of short duration of viremia, exclusion of infectious potential
HCV infection with mini pool-NAT screening to be as low blood donors on the basis of history and absence of a
as 1 in 2 million.16 Though NAT can significantly improve chronic carrier state. However, rare transmission via blood
the safety of blood supply; its widespread use in developing products18 and clotting factors19 has been reported.
countries like India is unlikely in the near future due to the Currently, no specific laboratory screening of blood
expenditure involved. donations for HAV is performed, as there is no chronic
• No vaccine is available against HCV infection. carrier state. Two inactivated safe and effective vaccines
Immunoglobulin has not been found to be effective in are available with 100 percent immunity after a second
postexposure prophylaxis. dose. IVIG is recommended as pre-exposure prophylaxis
Combination therapy with IFN-α-2b and ribavirin has in susceptible travelers visiting endemic regions and in
resulted in sustained response (defined as normal ALT selected situations for postexposure prophylaxis within 1
levels and negative PCR results 6 months after completion week of exposure.
of therapy) in one-third patients and is now considered
first-line therapy. Monotherapy with IFN-α-2b resulted in Hepatitis G
sustained response in 10 to 15 percent of patients.
Hepatitis G virus (HGV) is a recently discovered RNA virus
Hepatitis D distantly related to HCV (flavivirus). Clinical data derived
from studies of HGV have established its transmission
Hepatitis D virus (HDV) is a small satellite RNA virus, by blood through donor recipient linkages and by the
originally termed the delta virus that can infect only in the recovery of virus in the recipient that was not present prior
presence of concurrent HBV infection: to transfusion.
• Its genome codes a single peptide termed the delta • The HGV is present in 1 to 2 percent of donor
antigen. The infectious form of HDV is coated byHbsAg. population.
• The incubation period in HDV superinfection is 2 to 8 • Detection depends on PCR technology. As yet a
weeks; with coinfection it is the same as HBV infection. causal relationship has not been established between
HGV infection and hepatitis or any other disease transfusion transmitted infection, more than 95 percent
manifestation.20 of HIV infected patients exhibit a wide range of antibodies
• Currently no method is available to prevent HGV to structural env, gag and pol viral proteins.25 As soon as
infection. anti-p24 develops, p24 antigenemia disappears. A long
asymptomatic period follows primary infection, which may
TTV and SEN-V last up to 10 years; however, disease progression continues
relentlessly. Levels of all anti-HIV antibodies are very
These viruses were separately identified among individuals high during the asymptomatic period. Disease develops
with hepatitis and were also shown to be poorly, if at all, when CD4 cells are severely depleted leading to severe
associated with hepatitis. They were readily transmitted immunosuppression and spread of disease to multiple
by transfusion. At this stage, there is little evidence that organs and emergence of opportunistic infections. In the
this virus pair has any pathogenic potential. absence of treatment, disease progression is relentless and
almost invariably fatal.
Retroviral Infection In most countries including India, screening tests for
Prior to the outbreak of the acquired immunodeficiency antiHIV by EIA are now compulsory for blood donation. If
syndrome (AIDS) epidemic in the early 1980s, retroviruses reactive, additional tests are used to confirm the diagnosis
had been identified as a cause of rare malignancies but not of HIV infection.
a threat to transfusion recipients. Presently, the clinically Treatment of HIV infection consists of initiation of
significant transfusion transmitted retroviruses are the antiretroviral medications guided by patient’s CD4 count
human immunodeficiency virus (HIV) types 1 and 2 and and WHO clinical stage and treatment of opportunistic
the human T-cell leukemia virus (HTLV) types 1 and 2. infections.
Risk of transfusion transmitted HIV infection can be
reduced by measures introduced at the blood collection
HIV 1 and 2 centers such as, improved donor screening, education and
Since AIDS was first described in the USA in 1981 in exclusion techniques; enlightened transfusion practice
young, previously healthy, homosexual men, the disease such as, judicious use of allogenic blood components,
has spread worldwide. At the end of 2003, an estimated appropriate use of autologous blood, and alternative to
37.8 million people, 35.7 million adults and 2.1 million transfusion and measures that depend on the development
children younger than 15 years, were living with HIV/AIDS of new technologies, such as viral inactivation of cellular
(UNAIDS 2004). Approximately two-thirds of these people components and safe substitutes for blood. Measures
(25.0 million) live in sub-Saharan Africa and 20 percent (7.4 such as public education, self-deferral of donors engaged
million) in Asia and the Pacific. Between 2002 and 2004, an in high-risk activities and confidential unit exclusion also
estimated 10 million people were infected with HIV and serve a similar purpose.
nearly 6 million died from AIDS.21 The HIV seroprevalence
in Indian scenario has been reported between 0.2 and 1 Human T-cell Leukemia Virus HTLV
percent.22 As per the 2006 NACO surveillance report, 3.8
Types 1 and 2
percent of the total HIV cases are less than 15 years of age.
The HIV is a member of the family Retroviridae and Human T-cell leukemia virus type I (HTLV-I) was the
belongs to the genus Lentiviridae. The genome is a single- first human retrovirus isolated and the first to be causally
stranded RNA. Because HIV is both cell-associated and associated with a malignant disease of humans, the adult
present in the plasma, all blood components are potentially T-cell leukemia.26 It is also associated with myelopathy
infectious. Albumin preparations, immunoglobulins, and tropical spastic paralysis. HTLV-II, which was
antithrombin III and hepatitis B vaccine have not been described later, is known to show 60 percent homology
associated with HIV infection. of genetic sequences to those of HTLV-I.26 The current
For the first few days after infection, no markers of HIV incidence of HTLV infection in the United States is 1 in
can be detected in blood, an interval known as the ‘eclipse’ 6250 individuals being seropositive per year half of these
phase. Viremia follows for a period of several weeks. This being infected with HTLV-I and the rest half with HTLV-
stage is followed by a ‘ramp up’ phase at about day 10 II.27 HTLV-I infection shows geographic clustering with
when HIV viral copy number rises rapidly.23 high endemicity in Japan, sub-Saharan Africa and Central
At about day 17, p24 antigen becomes detectable and South America. HTLV-II shows clustering in Native
in serum and at about day 22, anti-HIV seroconversion American population donors. In these areas, the donors
occurs. During this phase more than 40 percent patients are screened using EIA screening tests.28 The transmission
develop a flu-like illness.24 After 1 to 2 months of rate of HTLV-I or HTLV-II in a recipient of an infective
blood unit is between 20 and 60 percent. The risk of MHC mismatched leukocytes of the donor and
transmission of HTLV from a screened blood unit is low recipients.
(1 in 6,40,000).13 Contact with infected viable lymphocytes 3. Finally, CMV superinfection occurs when a seropositive
can cause infection, as both the viruses are cell-associated. recipient is transfused a new strain of virus.
Transmission is by cellular components and not by cell- The clinical significance of TT-CMV is significantly
free plasma or its derivatives. As refrigeration of blood more than CMV reactivation and superinfection, because
product over 10 days results in degradation of lymphocytes in TT-CMV, the recipient has no immunological memory,
and in decrease in load of infectious viruses, plasma and while the two former conditions are unlikely to cause
plasma derivatives do not transmit the virus.20,27,29,30 The morbidity. Around 1.2 percent of immunocompetent
association of infectivity with fresh cellular components recipients experience TT-CMV.33
raises the possibility that transmission of HTLV by In contrast, TT-CMV causes significant morbidity and
transfusion requires viable T-lymphocytes and that their mortality in immunocompromised recipients with 13 to 37
removal from blood donations may clear the potentially percent acquiring infection from infected and unfiltered
infectious cells. blood.34
With the use of combination of viral lysates from The at-risk groups include premature newborns
HTLV-I and II viruses, there is sensitive detection of born to seronegative mothers, seronegative recipients
both anti-HTLV-I and anti-HTLV-II. Such combination of seronegative bone marrow transplant and solid
HTLV-I/II EIA test is being used in United States for HTLV organ transplant and seronegative patients with AIDS.35
screening as the originally licensed anti-HTLV-I EIA can These patients first have a flu-like illness followed by
miss up to 50 percent of HTLV-II infections.30 HTLV-I disseminated disease including hepatitis, retinitis, ence
and II infections have not been reported in the Indian phalitis, pneumonitis and gastroenteritis.
subcontinent.
CMV Infection can be Detected by Serologic
Assays for AntiCMV Antibodies
Human Herpes Virus Infection
The TT-CMV can be prevented by using seronegative
Of the herpes viruses eight are known to infect humans. blood and filtered blood components. In comparison
Cytomegalovirus (CMV) has the greatest clinical relevance to unscreened blood, the use of seronegative units can
in transfusion medicine. Other herpes viruses that may reduce the incidence of TT-CMV from 13 to 37 percent to
contaminate blood products are Epstein-Barr virus (EBV) 2.5 percent in at-risk individuals.36 No effective vaccine is
and human herpes virus (HHV) 6 through 8. available at present. Ganciclovir and IVIG are used in the
treatment of severe CMV infection immunocompromised
Cytomegalovirus patients.
Cytomegalovirus (CMV) is widely distributed with Epstein-Barr Virus

a seroprevalence of 30 to 80 percent in developed
countries and that approaching 100 percent in developing Epstein-Barr virus (EBV) has been implicated in endemic
nations.31,32 The CMV is transmitted in a latent, particulate Burkitt’s lymphoma, AIDS-related lymphoma, post-trans-
state only by cellular blood components (such as red cells, fusion lymphoproliferative disease and nasopharyngeal
platelets, granulocytes), and the virus reactivates from carcinoma.
donor leukocytes after transfusion. Fresh frozen plasma Transmission of EBV by blood transfusion can manifest
and cryoprecipitate have not been implicated. in a similar manner to classic infectious mononucleosis.
Transfusion can lead to active CMV infection in the Although EBV-seronegative blood components reduce the
recipient by three mechanisms: incidence of TT-EBV, they are difficult to obtain given the
1. The term transfusion transmitted CMV infection (TT- high seroprevalence of EBV.
CMV) is used to describe a primary CMV infection
occurring in a seronegative recipient transfused with Human Herpes Viruses 6 and 8
an infected blood component. With ubiquity of (HHV-6) antibodies and absence of disea-
2. Reactivated CMV infection occurs when a seropositive se associations after transfusion, no recommendations
transfusion recipient experiences reactivation of have been made for protection of seronegative blood
latent CMV infection after a blood transfusion from recipients from transmission by blood components.37
a seronegative donor. The underlying mechanism HHV-8 (Kaposi’s sarcoma associated herpes virus)
involves immunomodulatory interactions between has been found in apparently healthy blood donors
but transfusion transmission of HHV-8 has not been Possible measures to prevent transfusion associated
demonstrated.38 bacterial sepsis include extension of donor screening,
improved donor skin disinfection, removal of first aliquot
Parvovirus B19 of donor blood, limiting storage time, pretransfusion
detection, altered blood processing and chemical and
Parvovirus B19 was discovered incidentally during the
photochemical decontamination.
screening of blood samples for hepatitis B surface antigen.
About 30 to 60 percent of blood donors have antibodies
Syphilis
to parvovirus B19. This is indicative of immunity rather
than chronic persistent infection.39 The virus has been Transfusion transmitted syphilis is not a major hazard of
found regularly in clotting factor concentrates and has modern blood transfusion therapy. Treponema pallidum,
been transmitted to persons with hemophilia. It is also the infectious agent causing syphilis survives at the most
transmitted by cellular blood components and plasma, for 5 days in blood stored at 4°C.43 Only rare cases of
but not intravenous immunoglobulin and albumin.40 transfusion transmitted syphilis have been documented
Parvovirus B19 can infect and lyse red cell progenitors and the causes of this decline are universal donor screening
in the bone marrow41 resulting in sudden and severe and the overall decline in the incidence of syphilis with the
anemia in patients with underlying chronic hemolytic advent of penicillin. The rapid plasma reagin (RPR) test
disorders. Patients with cellular immunodeficiency, is commonly used for screening the blood products for
including those infected with HIV, are at risk for chronic syphilis. Blood donations from individuals who have had
viremia and associated hypoplastic anemia. or been treated for syphilis should be deferred for at least
However, parvovirus B19 screening of whole blood 12 months after successful completion of treatment. As
donations has not been a high priority because of the per the AABB standards, blood donations from any person
benign and/or transient nature of most parvovirus with a positive serological test result for syphilis should
diseases, the availability of effective treatment for chronic be deferred for 12 months.44 It is not the transmission of
hematologic sequelae and the extreme rarity of reports of syphilis that is worrisome. Being a sexually transmitted
parvovirus B19 transmission by individual components. disease, its presence points towards donor’s indulgence
in “high risk” behavior and consequent higher risk of
Bacterial Infections exposure to infections like HIV and hepatitis. However,
the RPR test used for screening is not specific and a large
Septic shock was one of the earliest recognized portion of positive tests in healthy donor population may
complications of blood transfusion. Prospective studies represent a biological false positive reaction.
have indicated that the clinical presentation is wide and
milder reactions are often misdiagnosed as febrile non-
hemolytic transfusion reactions.
Malaria
Transfusion reactions associated with contaminated Malaria can be transmitted by the transfusion of any blood
red cell concentrates are extremely severe with mortality component likely to contain even small number of red blood
of 70 percent.42 Fever, rigors, hypotension, nausea, cells; platelet and granulocyte concentrates, fresh plasma
vomiting and diarrhea are the usual presenting features. and cryoprecipitate have all been implicated. Plasma that
Septic shock, oliguria and disseminated intravascular has been frozen or fractionated does not transmit malaria.
coagulation are frequent complications. Majority of the Malaria parasite of all species can remain viable in stored
reactions are caused by infusion of endotoxins of gram- blood for at least a week and longer in adenine containing
negative organisms such as Yersinia enterocolitica, other solutions. The incubation period of transfusion malaria
Enterobacteriaceae and psychrophilic pseudomonas. depends on the number and strain of plasmodia transfused,
Because platelet concentrates are stored at room on the host and on the use of antimalarial prophylaxis.
temperature, they offer the most favorable media With P. falciparum and P. vivax it is between 1 week and
for bacterial growth thus limiting their permissible 1 month, but with P. malariae it may be many months.45
duration of storage to more than 3 days. Coagulase When blood smear are examined by simple microscopy,
negative staphylococci are the most frequent pathogens. a density of less than 100 parasites per microliter of blood
Pseudomonas species have been isolated from plasma and cannot be detected. Since most apparently healthy donors
cryoprecipitate thawed in contaminated water baths. have very low parasitemia, serological tests are useful in
Possible mechanisms of blood component contami detecting latent malarial infection. In endemic areas, it is
nation include donor bacteremia, inadequate skin recommended that chemoprophylaxis should be given
disinfection and use of contaminated equipment during to all recipients. In nonendemic areas, screening donors
blood collection storage and processing. by travel history can exclude the asymptomatic carriers.
Transfusion transmitted malaria responds to conventional use of sophisticated, sensitive but expensive technologies
anti-malarials. for screening of blood products. The last two decades
have also witnessed surfacing of new and re-emerging
Babesiosis infections. Hence, despite stringent donor eligibility
criteria, improved donor screening and introduction
As with malaria, asymptomatic individuals infected with
of sophisticated technology, transfusion-transmitted
Babesiosis may present as prospective blood donors.
infection continues as a challenge for transfusion experts.
Babesiosis has been transmitted following the transfusion
of infected packed red cells, frozen-thawed-deglycerolized
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17. Dodd RY. Adverse consequences of blood transfusion: 32. Preiksaitis J. Indications for the use of cytomegalovirus-
Quantitative risk estimates. In: Nance ST (ed). Blood seronegative blood products. Transfusion Med Rev.
Supply: Risks, Perceptions and Prospects for the Future. 1991;51:1-17.
Bethesda, MD: Am Assoc Blood Banks. 1994.pp.1-24. 33. Wilhelm JA, Matter L, Schopfer K. The risk of transmitting
18. Giacoia GP, Kasprisin DO. Transfusion acquired hepatitis cytomegalovirus to patients receiving blood transfusion. J
A. South Med J. 1989;82:1357-60. Infect Dis. 1986;154:169-71.
19. Mannucci PM, Gdovin S, Gringeri A, Colombo M, Mele A, 34. Yeager AS. Prevention of transfusion acquired
Schinaia N, et al. Transmission of hepatitis A to patients cytomegalovirus infection in newborn infants. J Pediatr.
with hemophilia by factor VIII concentrates treated with 1981;98:281-7.
organic solvent and detergent to inactivate viruses. Ann 35. Sayers MH. Reducing the risk of transfusion transmitted
Intern Med. 1994;120:1-7. cytomegalovirus infection. Ann Intern Med. 1992;116:55-62.
20. Brecher ME (ed). AABB Technical Manual, 14th edn.
36. Miller WJ, McCullough J, Balfour HH Jr, Haake RJ, Ramsay
Bethesda, MD: Am Assoc Blood Banks. 2002.
NK, Goldman A, et al. Prevention of cytomegalovirus
21. Piot P, Feachem RG, Lee JW. Public health. A global
infection following bone marrow transplantation: A
response to AIDS: Lessons learnt, next steps. Science
randomized trial of blood product screening. Bone
2004;304:1909-10.
Marrow Transplant. 1991;7:227-34.
22. Kaur P, Basu S. Transfusion-transmitted infections:
37. Campadelli-Fiume G, Mirandola P, Menotti L. Human
Existing and emerging pathogens. J Postgrad Med.
2005;51:146-51. herpes virus 6: an emerging pathogen. Emerg Infect Dis.
23. Fiebig EW, Wright DJ, Rawal BD. Dynamics of HIV 1999;5:353-66.
viremia and antibody seroconversion in plasma donors: 38. Chatlynne LG, Lapps W, Handy M, Huang YQ, Masood
implications for diagnosis ans staging of primary HIV R, Hamilton AS, et al. Detection and titration of human
infection. AIDS. 2003;17:1871-9. herpes virus 8 specific antibodies in sera from blood
24. Kahn JO, Walker BD. Acute human immunodeficiency donors, acquired immunodeficiency syndrome patients,
virus type 1 infection. N Eng J Med. 1998;339:33-9. and Kaposi’s sarcoma patients using a whole virus enzyme
25. Horsburgh C, Ou CY, Jason J Jr. Duration of HIV infection linked immunosorbent assay. Blood. 1998;92:53-8.
before detection of antibody. Lancet. 1989.pp.637-40. 39. Luban NL. Human parvoviruses: Implications for
26. Hjelle B. Transfusion-transmitted HTLV-I and HTLV-II. In: transfusion medicine. Transfusion. 1994;34:821-7.
Rossi EC, Simon TL, Moss GL, Gould SA (eds). Principles of 40. Prowse C, Ludlam CA, Yap PL. Human parvovirus B19 and
transfusion medicine, 2nd edn. Baltimore, MD: Williams blood products. Vox Sang. 1997;72:1-10.
and Wilkins. 1995.pp.709-16. 41. Arnold DM, Neame PB, Meyer RM, Soamboonsrup P,
27. Guidelines for counseling persons infected with human Luinstra KE, O’Hoski P, et al. Autologous peripheral blood
T-lymphotropic virus type I (HTLV-I) and type II (HTLV- progenitor cells are a potential source of parvovirus B19
II). Centers for Disease Control and Prevention and the infection. Transfusion. 2005;45:394-8.
U.S.P.H.S. Working Group. Ann Intern Med. 1993;118: 42. Theakston EP, Morris AJ, Streat SJ. Transfusion transmitted
448-54. Yersinia enterocolitica infection in New Zealand. Aus NZ J
28. Okochi K, Sato H, Hinuma Y. A retrospective study on Med. 1997;127:62-7.
transmission of adult T cell leukemia virus by blood 43. Van der Sluis JJ, ten Kate FJ, Vuzevski VD, Kothe FC,
transfusion: seroconversion in recipients. Vox Sang. Aelbers GM, van Eijk RV. Transfusion syphilis, survival
1984;46:245-53. of Treponema pallidum in stored donor blood. II. Dose
29. Lairmore MD, Jason JM, Hartley TM, Khabbaz RF, De B, dependence of experimentally determined survival times.
Evatt BL. Absence of human T-cell lymphotropic virus type Vox Sang. 1985;49:390-9.
I coinfection in human immunodeficiency virus-infected 44. Walker RH (ed). AABB Technical. Manual, 11th edn.
hemophilic men. Blood. 1989;74:2596-9. Bethesda MD: Am Assoc Blood Banks. 1993.
30. Food and Drug Administration. Guidance for industry: 45. Bruce-Cwatt LJ. Transfusion malaria. Bull WHO.
Donor screening for antibodies to HTLV-II (August 15, 1974;50:337.
C H A P T E R 38
Noninfectious Hazards of Blood Transfusion
The risks of blood transfusion should always be borne in mind whilst considering benefits of transfusing red cells, platelets, and
plasma. With the advances in infectious disease testing, noninfectious complications are now many-fold more likely to cause serious
morbidity or death following transfusion. Some of the more common noninfectious hazards of transfusion include transfusion reactions
(hemolytic, febrile, allergic/urticarial/anaphylactic). Other noninfectious hazards include transfusion related acute lung injury, post-
transfusion purpura, transfusion-associated graft versus host disease, transfusion related immunomodulation, alloimmunization,
metabolic derangements, transfusion-associated circulatory overload and iron overload. Individuals who administer blood
transfusions should recognize these complications in order to be able to quickly provide appropriate treatment.
Though transfusion has many benefits it also involves adults, as a single unit of transfused blood forms a greater
risks to the recipient. Any unfavorable event occurring in proportion of their blood volume than that in an adult.
a recipient due to blood transfusion, either during or after Problems may occur due to lack of knowledge of special
transfusion, is considered an adverse effect of transfusion. requirements in the neonatal age group or human error
Transfusion can lead to serious adverse effects such as overtransfusion. It is estimated that the incidence
including infectious and noninfectious complications. of adverse outcome is 18:100,000 red blood cells issued for
With the more stringent donor selection criteria and children aged less than 18 years and 37:100,000 for infants.
use of sensitive screening methods for transfusion The comparable adult incidence is 13:100,000.2 The health
transmissible infectious marker testing, the risk of care personnel involved in the transfusion process should
infectious complications has decreased. The term NISHOT therefore be aware of correct recognition, prevention
(Noninfectious serious hazards of transfusion) was first and appropriate management of the adverse effects of
described in a 2000 AABB bulletin to broadly encompass transfusion.
all noninfectious transfusion complications. Currently, In most instances of transfusion reactions, the
a patient is up to 1000-fold more likely to experience a pathophysiology, clinical diagnosis and management are
NISHOT than an infectious complication of transfusion.1 similar as the transfusion reactions in adults.
The noninfectious complications can be acute (within 24
hours) or delayed. CATEGORIES OF NONINFECTIOUS HAZARDS
Children are a unique patient group and many OF BLOOD TRANSFUSION
have special transfusion requirements. Neonates in
particular are often intensively transfused and are Acute
especially vulnerable to the potential infective and toxic • Allergic
effects of transfusion. They have immature immune • Acute intravascular hemolysis
system and metabolism, and are still undergoing rapid • Febrile nonhemolytic transfusion reaction (FNHTR)
neurodevelopment. The chances for acute side effects • Transfusion related acute lung injury (TRALI)
of transfusion may be greater for small children than for • Metabolic complications.
Chapter-38 Noninfectious Hazards of Blood Transfusion 385
Delayed one or more of the symptoms like urticaria, flushing,

respiratory tract obstruction, cough, chest pain, dyspnea,
• Delayed hemolytic transfusion reaction nausea, vomiting, diarrhea, tachycardia and arrhythmias.
• Alloimmunization Absence of fever helps in differentiating these reactions
• Immunomodulation from hypotension due to hemolytic reaction or bacterial
• Post-transfusion purpura (PTP) contamination.3
• Graft versus host disease (GvHD) Allergic reactions occur due to exposure to soluble
• Circulatory overload substances present in donor plasma which binds with
• Iron overload.
IgE antibodies resulting in release of histamine. This
assumption is based on the facts that these reactions recur
ACUTE HAZARDS OF TRANSFUSION in an affected recipient and can be prevented by removal
Transfusion reactions can occur rapidly after transfusion of plasma. Anaphylactic and anaphylactoid reactions
e.g. hemolytic reaction, or many days or weeks after are sometimes associated with anti-IgA, particularly in
transfusion, e.g. graft versus host disease (GvHD). By IgA deficient recipient. First description of anaphylactic
definition, acute transfusion reactions (ATRs) occur reaction associated with anti-IgA was reported in 1968 and
within 24 hours of transfusion administration3 although approximately 30 additional cases have been reported by
most occur during or within four hours after the end of 1995.7
a transfusion. There are only few studies which have
reported the incidence and type of acute reactions in Treatment and Prevention
pediatric patients. In one prospective study of acute
transfusion reactions in pediatric intensive care units, the Anaphylaxis or anaphylactoid reactions should be
incidence of acute transfusion reactions was 1.6 percent.4 recognized promptly when the recipient shows symp
In that study, however, the only blood product toms and the transfusion should be immediately
transfused was red blood cells (RBCs) and the only stopped. Treatment of transfusion related anaphylaxis
ATR reported was febrile nonhemolytic transfusion or anaphylactoid reaction is same as that of anaphylaxis
reaction (FNHTR). Another prospective study of platelet due to other reasons. Hypotension needs to be managed
transfusions in children by Couban et al5 reported seven by administering fluids and if required administration of
ATRs associated with sixty six leucoreduced platelet epinephrine along with supportive care.
transfusions (11%). ATRs consisted of two FNHTRs, four If the recipient is known to be IgA deficient or there
allergic reactions, and one mixed reaction. According to is a previous history of life-threatening anaphylactic
the Quebec Hemovigilance System (QHS) 2001 report, reaction blood components which lacks IgA, either by
the most frequent immediate transfusion reaction for all washing or obtained from IgA deficient donor, should
components transfused was FNHTR (12:10,000 transfused be administered. Availability of washed red cells is not
units), followed by minor allergic reactions (10:10,000).6 so difficult but washed platelets are not generally easily
available as washing of platelets may result in low platelet
Allergic Reaction recovery.
Treatment of uncomplicated urticaria requires dis
Allergic reactions associated with transfusion may vary continuation of transfusion and administration of anti
from mild uncomplicated allergic reaction to anaphy histaminics. If symptoms are resolved the transfusion may
lactoid and severe anaphylactic reactions. Allergic be restarted.
reactions are the most common cause of adverse
transfusion reactions. Uncomplicated mild allergic Immune Mediated Hemolysis
reactions consists of localized or diffuse urticaria characte
rized by erythematous circumscribe raised lesions present Immune mediated lysis of donor red cells can result
over the upper trunk and neck often associated with in hemolytic transfusion reaction. It is the most severe
itching. Mild urticarial reactions constitute 1 to 3 percent hemolytic transfusion reaction and occurs due to
of adverse transfusion reactions.3 Anaphylaxis is the severe destruction of transfused donor red cells by recipient’s
form of this type of reaction characterized by hypotension, antibodies, e.g. in case of ABO incompatible red blood cell
and is often associated with bronchospasm, dyspnea, and transfusion. Depending on the nature of the antibody the
in rare cases death. These severe reactions may occur after hemolytic transfusion reaction may be acute or delayed
infusion of few mL of blood. and may result in intravascular or extravascular hemolysis.
The anaphylactoid reaction is placed in between Incidence of hemolytic reaction varies from 1:38,000 to
mild urticaria and anaphylaxis and is characterized by 1:70,000.3
Transfusion of an ABO incompatible unit causes rapid half (47%) of the recipients of ABO incompatible red cells
destruction of red cells with the release of free hemoglobin suffered no ill effects even after receiving a full unit. Half
and RBC stroma in the circulation. ABO incompatible (50%) exhibited an acute hemolytic transfusion reaction
transfusion may be life-threatening as it can lead to acute and about 4 percent died as a result.8
renal failure, shock, and disseminated intravascular
coagulation.
In intravascular hemolysis the interaction of red cell Treatment and Prevention
antigen-antibody causes complement activation and Treatment of acute hemolytic reaction depends on its
release of cytokines resulting in clinical manifestations. severity. The aim is to maintain adequate renal perfusion
Complement activation depends on the specificity and in order to prevent renal failure. Adequate renal perfusion
class of antibody, and number of antigen sites. The can be monitored by measurement of urine output
complement activation results in formation of membrane for at least 24 hours. The usual initial step is infusion of
attack complex causing intravascular red cell lysis which intravenous normal saline but clinical monitoring is
leads to hemoglobinemia and, if it exceeds renal threshold, necessary to avoid fluid overload. Furosemide is considered
to hemoglobinuria. The free hemoglobin impairs renal a better diuretic as it improves renal cortical blood flow.
functions. The factors responsible for renal failure in If the intravascular hemolysis is also complicated with
severe cases are hypotension, renal vasoconstriction, coagulopathy, administration of platelets, fresh frozen
antigen-antibody complex deposition, and formation of plasma, and cryoprecipitated antihemophilic factor (AHF)
thrombi in renal vasculature, all of which affect the renal may be necessary.
cortical blood supply. The most common cause of transfusion of incompatible
If the antibody involved in immune reaction does units is clerical error such as incorrect labeling of blood
not fix the complement, there will be acute extravascular sample, blood bag or request form. Failure to follow the
hemolysis. Usually extravascular hemolytic reactions identification procedure during sample collection and just
are not associated with severe clinical symptoms. They prior to transfusion may result in wrong blood transfusion
may present with fever. On investigation the direct anti- and hemolytic episode following transfusion. For bed
globulin test (DAT) may be positive due to binding of side transfusions each institute should develop policies
antibodies to incompatible donor red cells. and procedures to ensure proper patient identification,
Infants less than four months of age generally do not sample collection and labeling and unit identification.
have developed anti-A and anti-B antibodies, and therefore In addition to this continuous monitoring and training of
are not usually susceptible to these reactions. However, the staff responsible for collection and administration of
maternal IgG antibodies can cross the placenta and may blood is also required.
cause hemolysis of transfused red cells. Considering Hemolysis can also occur with the transfusion of
this possibility it is recommended that the compatibility ABO incompatible platelet and plasma products. There
testing should be performed using mothers serum up to have been several reports of severe hemolytic reactions
four months of age. RBCs units lacking corresponding after transfusion of minor ABO mismatched platelet
antigens should be selected for transfusion. transfusion.9
The symptoms of ABO incompatible transfusion are
chills, rigors, fever, abdominal or back pain, pain at the
infusion site, nausea, vomiting, hemoglobinuria, oliguria.
Nonimmune Mediated Hemolysis
These clinical manifestations may be observed after There are chances of in vitro hemolysis due to improper
transfusion of even a few mL of blood. Therefore, the handling during transportation or at the time of
initial period of transfusion is very important, and the rate administration. Malfunctioning of blood warmer or
of transfusion during the initial 15 to 20 minutes should accidental freezing can cause temperature related damage
be slow and can be increased later. Similarly, the recipient to red cells. Addition of drugs or hypotonic solutions may
should be under continuous medical monitoring during result in osmotic lysis of red cells. Transfusion of such
transfusion so that any adverse reaction can be identified units can cause nonimmune mediated hemolysis in the
and managed immediately. Information to the recipient recipient. Treatment of such cases depends on the severity
or attendants of recipient about the symptoms of adverse of the reaction. If the patient develops shock, renal failure
reaction is necessary. intensive medical management is needed.
The severity of hemolytic reaction due to an ABO Such cases are preventable. All the staff involved in
incompatible transfusion varies and depends on the rate processing, issuing and administration of blood should
and the volume of red cells transfused. In a 10 years study be trained for storage and correct handling of blood and
of analysis of transfusion errors it was found that nearly blood components. The equipment used for warming and
for administration of blood should be properly maintained mortality. The incidence of TRALI is estimated to be
and staff should be adequately trained for use of such between 0.08 percent and 15 percent of patients receiving
equipment. a blood transfusion.10 TRALI can occur after transfusion
of whole blood, red blood cells, fresh frozen plasma,
Febrile Nonhemolytic Transfusion Reaction platelet concentrate (random donor and apheresis),
cryoprecipitated antihemophilic factor VIII.
A febrile nonhemolytic transfusion reaction (FNHTR) is
Transfusion related acute lung injury is characterized
defined as temperature rise of more than 1°C associated
by acute noncardiogenic edema and respiratory com
with transfusion without any other demonstrable cause.
promise associated with transfusion. The National Heart,
FNHTRs are caused by antibodies present in recipient’s
Lung and Blood Institute (NHLBI) working group has
plasma that interact with white cells in the transfused
defined TRALI as ‘new acute lung injury occurring during
product and are most frequently HLA antibodies or
or within six hours of transfusion, with a clear temporal
sometimes granulocyte antibodies. This mechanism
relationship to transfusion in the patient without alternate
appears to be the primary cause of FNHTR after transfusion
risk factor for lung injury’.11 Clinically, TRALI should be
of red cells but for FNHTRs consequent to platelet
transfusions the leukocyte derived cytokines, generated considered whenever the recipient experiences acute
during the warmer room temperature storage of platelets, respiratory insufficiency and or chest X-ray shows bilateral
have been implicated. pulmonary edema. It is accompanied by other symptoms
FNHTRs present with fever with or without chills. Most like fever, chills, and hypotension. The severity of TRALI is
FNHTRs are mild and benign but they cause discomfort not related to the volume of blood transfused.
to the recipient. Severe reactions may be accompanied by The exact mechanism of TRALI is not known but it
hypotension, cyanosis, tachycardia, tachypnoea, dyspnea, may result from multiple factors. Antibodies against HLA
cough, transient leukopenia. Febrile reactions should be class I and II or neutrophils antigens present in donor
differentiated from hemolytic transfusion reaction and units are thought to cause increased permeability of
reactions due to a bacterial contaminated product. If the pulmonary microcirculation resulting in collection of fluid
patient has received multiple units it becomes difficult in interstitial and alveolar spaces.
to find out which unit has caused the febrile reaction. In
such instances all units transfused within four hours prior Treatment and Prevention
to transfusion should be included for transfusion reaction The transfusion should be stopped when there is
workup. respiratory distress and the same unit should not be
started even if the symptoms have subsided. Management
Treatment and Prevention of TRALI is mainly supportive with oxygen therapy and
FNHTRs need prompt management and exclusion of ventilation. Most of the patients recover within 48 to 72
other causes of fever. Like any other adverse reactions the hours.
transfusion should be stopped immediately and evaluation No specific precautions are needed if the reaction
done to rule out other causes of fever. Generally, fever has occurred due to antibodies in donor plasma, and
responds to antipyretic agents such as acetaminophen. components from other donors are available. In many
At some centers pretransfusion medication is given centers in the UK the current practice is not to transfuse
specially in recurrent FNHTRs. But this practice is not plasma sourced from female donors.
recommended as it may mask the fever associated with
other types of reactions like hemolytic transfusion and Metabolic Complications
bacterial contamination.
Febrile reactions to red cells can be prevented in most Hypothermia
of the cases by removal of leukocytes by either prestorage
Hypothermia may be caused by rapid infusion of large
or poststorage leukoreduction or administering saline
quantities of cold blood (2–10oC) or RBC units. Due to
washed red cells because leukocyte antibodies are the
their large body surface area-to-weight ratio infants and
primary reason. Cytokines related febrile reactions may be
children are predisposed to hypothermia. Hypothermia
most effectively prevented by prestorage leukoreduction.
during massive transfusion can induce cardiac arrhythmia
and arrest.12 Transfusion of cold blood in neonates has
Transfusion Related Acute Lung Injury been associated with apnea and hypoglycemia.13 Blood
Transfusion related acute lung injury (TRALI) is an does not have to be warmed for transfusions administered
important cause for transfusion related morbidity and at a standard rate. For rapid infusion, generally considered
to be more than 50 mL/kg/hour for an adult and more should be done along with other investigations like direct
than 15 mL/kg/hour for a child, blood should be warmed antiglobulin test (DAT) and serum bilirubin. Detection of
using a monitored blood warming device. red cell alloantibody with the history of recent transfusion
indicates DHTR in such cases. Delayed intravascular
Hyperkalemia hemolysis is uncommon and is often associated with
antibodies to Duffy or Kidd blood group system.18
During storage of red cell products potassium leaks from
the cells, increasing the potassium concentration. Risk
Treatment and Prevention
of hyperkalemia from a blood transfusion depends on
the patient’s size, clinical condition, type and amount of In most of the cases no specific treatment is required.
component transfused, concentration of potassium in the The patient should be monitored for renal function and
plasma. Hyperkalemia resulting from massive transfusion coagulation.
of older RBC units containing elevated amount of For future transfusion requirements the donor unit
extracellular potassium can cause significant cardiac should lack the corresponding antigen for the alloantibody.
complications or possibly death in some patients.14 In one
case of neonatal mortality following transfusion of red Alloimmunization
cells with high plasma potassium levels reported by Hall
et al15 it was observed that the patient’s cardiac arrest was Alloimmunization is one of the clinically significant
probably related to rapid transfusion of RBCs with high adverse effects of blood transfusion. Development of
plasma potassium levels. antibodies against donor antigen present in the blood
In neonatal transfusions, hyperkalemia can be avoided component may pose difficulties in finding compatible
by use of RBC units less than seven days old or older units red cell units or result in platelet refractoriness or adverse
that have been saline washed.16 transfusion reaction. The immune system of the recipient
reacts to donor antigens as they are foreign to recipient.
The first exposure generally sensitizes the immune system
Citrate Toxicity of recipient. With subsequent exposure the secondary
Blood collected for transfusion is anticoagulated with immune response results in rapid production of large
citrate, which chelates calcium ions. Plasma and whole amount of IgG type of antibodies. These antibodies attach
blood are the blood components most likely to cause to the surface of the antigen carrying cells and causes
hypocalcemia because they contain the most citrate destruction of cells by complement system or reticulo-
per unit volume. Rapid blood transfusion can cause a endothelial system. Primary alloimmunization to red cell
transient decrease in ionized calcium and a hypocalcemic or platelet antigens with transfusion is rare in the first
state.17 Symptoms of hypocalcemia include peripheral and few months of life.19 Beyond newborn period, pediatric
perioral paraesthesia, muscle spasm, cardiac arrhythmia patients with clinical conditions like sickle cell disease or
and hypotension. Symptomatic hypocalcemia is rare thalassemia requiring repeated transfusions may develop
and calcium supplementation is usually not required. alloimmunization to red cell antigens.
Mild citrate toxicity is managed by slowing the rate of Alloimmunization to platelet antigens and refracto
transfusion. If severe, parenteral calcium may be required. riness to platelet transfusions may be a problem in
oncology patients or other clinical conditions dependent
on platelets. HLA alloimmunization is the most common
DELAYED HAZARDS OF TRANSFUSION immune cause of platelet refractoriness and can be
Delayed Hemolytic Transfusion Reaction confirmed by demonstration of HLA class I antibodies.20
Clinically the severity varies from mild symptom of
Delayed hemolytic transfusion reaction (DHTR) occurs fever and falling hematocrit to severe effects like platelet
when the antigen on donor red cells elicit immune response refractoriness and bleeding.
in the recipient. Secondary immune response occurs Laboratory work up to detect clinically significant
usually four to seven days after transfusion. Most of the red cell antibodies needs to be done. For HLA antibodies
antibodies associated in DHTR are IgG and the resulting lymphocytic panels and lymphocytotoxic antibody can be
hemolysis is of extravascular type. Secondary immune done using patient’s serum.
response may not result in hemolysis always. In some
patients clinically significant delayed hemolytic reaction
Treatment and Prevention
occurs. Clinically, it is detected by falling hemoglobin,
fever, mild jaundice and positive antibody screen. If DHTR Treatment depends on the severity of reaction. Mild
is suspected, tests for detection of red cell antibodies reaction in case of a single time need of transfusion may
not need any active treatment. Patients requiring multiple Post-transfusion Purpura
transfusions need to undergo antibody identification and
should be transfused preferably with antigen negative Post-transfusion purpura (PTP) manifests as sudden and
blood to prevent further alloimmunization. However, it severe thrombocytopenia occurring one to two weeks
is very difficult to prevent alloimmunization completely. after red cells, plasma or platelet transfusions. The platelet
Selection of phenotype matched blood is recommended count may fall up to below 10 × 109/L. The recipient may
specially in patients requiring multiple transfusions to have hematuria, melena and vaginal bleeding.
prevent alloimmunization. Use of leukoreduced products Post-transfusion purpura is generally thought to be
for transfusion also prevents alloimmunization up to due to the anamnestic development of antibodies against
certain extent. Several strategies have been evaluated donor platelets. These antibodies also start destroying
to prevent alloimmunization to platelets. They include autologous platelets leading to severe thrombocytopenia
reduction in numbers of leukocytes in the platelets and and purpura. Most frequently found antibody is anti-HPA-
use of ultraviolet B irradiation.20 The report of Trial to 1a. Platelet antibodies attach to platelet surface resulting
Reduce Alloimmunization to Platelets (TRAP) study in extravascular destruction of platelets. The exact
group indicated that use of either leukocyte filtered or mechanism of platelet destruction is not fully known.
UVB irradiated blood components reduced the incidence Multiparous female patients are more likely to develop
of HLA antibody generation from 45 percent to 17 and PTP after transfusion because of sensitization during
21 percent respectively.21 previous pregnancies.
Transfusion Related Immunomodulation Treatment and Prevention

Blood transfusion can modulate the immune response of The thrombocytopenia is usually self limiting. This
the recipient. This phenomenon was reported by Opelz condition can be treated by corticosteroids, plasma
and coworkers in 1973, who observed the improved pheresis and exchange transfusions. Intravenous
renal allograft survival in transfused patients.22 However, immunoglobulin can also be given. Platelet transfusions
there may be other adverse effects of transfusion in should be avoided during the treatment of PTP. There are
different clinical situations, such as increased risk of no preventive measures for PTP. History of transfusion
bacterial infection, activation of latent infections and and any adverse reaction should be asked before blood
tumor recurrence. In one controlled study on patients transfusion. Patients with positive history should be tested
with hematologic malignancies, a history of allogeneic for presence of antibodies.
blood transfusion was associated with an increased risk
for lymphoplasmacytic and marginal zone lymphomas.23 Transfusion Associated Graft versus Host
The exact mechanism is not clear but the soluble
Disease
mediators released from WBCs are postulated as a
potential cause for the immunomodulatory effect. Several Transfusion associated graft versus host disease (TA-
studies have been conducted to understand the effect of GvHD) is a severe immunological reaction in a susceptible
blood transfusion on immune response and to develop host resulting from engraftment and proliferation of
preventive strategies. Leukoreduction as a preventive donor lymphocytes present in the transfused product.
strategy remains controversial as the only clinical situation The engrafted lymphocytes elicit immunogenic response
where the findings of randomized clinical trials of adverse against recipient tissues leading to the clinical presentation
transfusion related immunomodulation (TRIM) effects of pancytopenia, bleeding, diarrhea and skin rash.
have been consistent is in cardiac surgery patients. In Transfusion associated graft versus host disease occurs
that setting, the use of leukoreduced allogeneic RBCs has in an immunocompromised host or in immucompetent
been shown to reduce the short-term (up to three months recipient when the donor cells are homozygous for an
post-transfusion) mortality from all causes. Based on the HLA type for which the recipient is heterozygous. The
results of the cardiac surgery RCTs, leukoreduction of all pathophysiology is complex. The donor lymphocytes are
cellular blood components transfused in cardiac surgery not recognized by recipient as foreign and are not cleared
patients should be recommended. In clinical settings by the host immune system resulting in engraftment and
other than cardiac surgery, the available evidence does proliferation in the host. Later these donor cells start
not yet justify implementation of universal leukoreduction attacking host tissue. Clinically, GvHD presents as skin
for the specific prevention of adverse TRIM effects, but rash, blanching and maculopapular erythema of upper
universal leukoreduction may be justified on the basis of trunk, neck, palms and soles that may develop into
other WBC-related adverse effects.24 blistering lesions. Skin biopsy shows infiltration of upper
dermis by mononuclear cells and damage to the basal Iron Overload

layer. Involvement of liver is manifested as hepatitis,
raised bilirubin and enzyme levels. Enterocolitis causes This complication is particularly common in multiple
anorexia, nausea and severe diarrhea. transfusion recipients especially those with hemo
globinopathies since every red blood unit contains
approximately 200 mg of iron. These patients are
Treatment and Prevention multiply transfused at regular intervals to maintain
The clinical effects of TA-GvHD are difficult to treat even hemoglobin levels, resulting in accumulation of
with immunosuppressive drugs. Irradiation of cellular excess iron. During the initial phase iron is stored in
blood components is accepted as method for prevention of reticuloendothelial sites and later in parenchymal cells.
TA-GvHD. The recommended dose is 25 Gy to the center Iron deposition hampers the function of heart, liver,
of the blood container and at least 15 Gy to the periphery of endocrinal glands. Cardiac involvement causes most of
the blood container. This dose renders the T-lymphocyte the morbidity and mortality.
inactive without affecting functional status of red cells,
platelets and granulocytes. These blood components, Treatment
given to recipients from donors homozygous for an HLA
haplotype shared with the recipient, pose a specific risk Treatment is to remove excess iron without affecting
for TA-GvHD. This circumstance can occur when first and hemoglobin levels. Use of iron chelating agents like
second degree relatives serve as directed donors and when desferrioxamine can reduce iron stores.
HLA matched platelet components donated by related or
unrelated individuals are being transfused. Irradiation CONCLUSION
of blood components has been recommended in these Though the list of noninfectious hazards of transfusion is
situations. Other recipients who should receive irradiated long and diverse and although blood transfusion will never
cellular blood component are recipients of hematopoietic be absolutely safe, tremendous progress has been made
progenitor cells transplant and intrauterine transfusions, in the understanding of the complications of transfusion
and patients with congenital immunodeficiency. and their prevention and management. To prevent
Blood transfusion of irradiated blood product in hemolytic reactions, advanced identification systems
pediatric patients requires more attention. Irradiated red that link donor and recipient with greater precision
cells undergo an enhanced efflux of potassium during minimize human error. Febrile non hemolytic transfusion
storage at 2 to 6°C.25 When irradiated red cells are used for reactions (FNHTR) and platelet refractoriness due to HLA
neonatal exchange transfusion or the equivalent of a whole alloimmunization can be prevented by transfusion of
blood exchange is anticipated, red cell washing should be leukoreduced blood. Though TA-GVHD can be prevented
considered to prevent the possible adverse effects caused by selective irradiation concerns about transfusion
by hyperkalemia associated with irradiation and storage.26 related immunomodulation (TRIM) still need to be
resolved. Blood centers and transfusion services should
Circulatory Overload provide education for creating clinical awareness of the
Transfusion therapy sometime may result in circulatory complications of transfusion so that potential transfusion
overload especially in young children and the elderly. risks are identified and managed while they continue to
Rapid increase in blood volume due to transfusion is not occur, until, ultimately they are reduced to a negligible
tolerated by some patients with compromised cardiac concern for transfusing physicians and their patients.
and pulmonary functions. Circulatory overload presents
as dyspnea, cyanosis, severe headache and hypertension. REFERENCES
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transfusion. transfusion. Anesth Analg. 2009;108(3):759-69.
2. Lavoie J. Blood transfusion risks and alternative strategies
Treatment in pediatric patients. Paediatric Anaesthesia. 2011;21(1):
14-24.
The condition can revert if the transfusion is stopped with 3. Technical manual. Noninfectious complications of blood
the onset of symptoms. Diuretics and oxygen support may transfusion. In: Brecher ME, Leger RM, Linden JV, Roseff
be needed in some patients, even if the symptoms are not SD, (eds) Technical Manual. 15th edn. Bethesda MD:
resolved phlebotomy may be required. American Association of Blood Banks. 2005.pp.633-65.
4. Gauvin G, Lacroix J, Robillard P, et al. Acute transfusion red blood cells by washing and by reduction of additive
reactions in the pediatric intensive care unit. Transfusion. solution. Transfusion. 2007;47:248-50.
2006;46:1899-908. 17. Dzik WH, Kirkely SA. Citrate toxicity during massive blood
5. Couban S, Carruthers J, Andreou P, et al. Platelet transfusion. Transfus Med Rev. 1988;2:76-94.
prospective crossover trial of plasma removal and 18. Adverse effects of blood transfusion. In: Roseff SD,
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2002;42:753-8. physician’s handbook. 3rd edn. Bethesda: American
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transfusion reactions. Transfus Med Rev. 1995;9(1):1-8. Transfusion. 2000;40:1463-8.
8. Linden JV, Wagner K, Voytovich AE, et al. Transfusion 20. McFarland JG. Platelet and granulocytes antigens and
errors in New York State: an analysis of 10 years experience. antibodies. In: Roback JD, Grossman BJ, Harris T, Hillyer
Transfusion. 2000;40(10):1207-13. CD (eds). Technical Manual. 17th edn. Bethesda, MD:
9. Lozano M, Cid J. The clinical implications of platelet American Association of Blood Banks. 2011.pp.523-45.
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Transfus Med Rev. 2003;17(1):57-68. fusion Study Group. Leucocyte reduction and ultravio-
10. Vlaar APJ, Juffermans NP. Transfusion related acute lung let B irradiation of platelets to prevent alloimmunization
injury: a clinical review. Lancet. 2013;382:984-94. and refractoriness to platelet transfusions. N Engl J Med.
11. Triulzi DJ. Transfusion related acute lung injury: An 1997;337:1861-9.
Update. Hematology. 2006.pp.497-501. 22. Opelz G, Senger DP, Mickey MR, et al. Effect of blood
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S E C T I O N 6
Hemato-Oncology
CHAPTERS OUTLINE
39. Pediatric Acute Lymphoblastic Leukemia
40. Pediatric Acute Myeloid Leukemia
41. Chronic Myeloid Leukemia
42. Juvenile Myelomonocytic Leukemia
43. Pediatric Hodgkin Lymphoma
44. Non-Hodgkin Lymphoma in Children and Adolescents
45. Langerhans Cell Histiocytosis
46. Hemophagocytic Lymphohistiocytosis: Revisited
47. Bone Marrow Transplantation
C H A P T E R 39
Pediatric Acute
Lymphoblastic Leukemia
Acute lymphoblastic leukemia (ALL) is a neoplasm of precursor hematopoietic cells (B- and T-lymphoblasts) involving bone marrow
(BM) or other tissues like lymph node, thymus with or without peripheral blood involvement. ALL is clinically, morphologically,
immunophenotypically, and genetically a heterogeneous disease. It is the most common childhood malignancy and accounts
for nearly 30 percent of all childhood cancers and approximately 75 percent of all cases of childhood leukemia. The treatment of
childhood ALL is one of the true success stories of modern clinical oncology. Before the advent of effective chemotherapy in the
1960’s, ALL usually was a fatal disease. In the developed countries, with modern intensive protocols approximately 83 to 93 percent
of children with ALL are now long-term survivors.1 However, this is not the case in developing countries where 80 percent of the
children with ALL reside.
INTRODUCTION with signs and symptoms that reflect bone marrow

infiltration and/or extramedullary disease (Table 1).
Though as per the cancer registry data, it has been noted • Bone marrow failure due to infiltration by leukemic
that incidence of ALL is less in India as compared to the blast manifests as anemia, thrombocytopenia, and
developed countries, it is estimated that approximately neutropenia.
8000 new cases of childhood ALL diagnosed each year • History of prior treatment in the form of transfusion or
in India.2 Unfortunately of these only 25 percent receive drugs prescribed especially steroids should be taken as
appropriate treatment. Studies from India have shown that these factors may interfere in diagnosis.
40 to 60 percent of patients treated in a pediatric oncology • Respiratory distress and orthopnea secondary to
center on an affordable protocol, with manageable toxicity a mediastinal mass may be a presenting symptom
can be cured.2,3 There are several reasons for these poor in patients with T-lineage ALL; B-lineage ALL may
results including poverty, lack of awareness, lack of access present with mass in abdomen, head and neck or in
to adequate medical care, lack of adequately trained central nervous system.
personnel and competent oncology units. These factors • Very frequently patients present with life threatening
lead to delayed and sometimes improper diagnosis, delayed complications like congestive cardiac failure, acute
referrals, poor compliance, inadequate or inappropriate bleeding, tumor lysis syndrome (TLS) (hyperuricemia,
therapy and poor outcome. hyperphosphatemia, hyperkalemia hypocalcemia
and/or azotemia)4 and superior vena cava syndrome.
CLINICAL PRESENTATION Clinical examination and investigations (pathological,
Suspected case of acute leukemia should be examined biochemical and imaging) helps to find out need for urgent
meticulously. Evaluation starts with a detailed history and intervention and management of these life threatening
clinical examination. consequences of leukemia.
• Patients commonly present with symptoms including • Differential diagnosis (Table 2): ALL should be differ
weakness, fatigue, fever, bleeding, bone pains, either entiated from benign conditions like juvenile rheu
gradually or abruptly. Children with ALL often present matoid arthritis, infectious mononucleosis, idiopathic
396 Section-6 Hemato-Oncology
Table 1 Symptomatology of acute lymphoblastic leukemia Table 3 Usual cytological features of AML and ALL
• Fever Parameters AML ALL
• Bleeding (e.g. petechiae or purpura) Blast size Large and uniform Small to medium:
• Bone pain variable
• Lymphadenopathy Chromatin Finely dispersed Coarse
• Splenomegaly Nucleoli 1-4 often Absent or 1–2,

prominent indistinct
• Hepatosplenomegaly
Cytoplasm Granules often Granules lacking
• Testicular swelling present
• C
entral nervous system symptoms (cranial nerve palsies, Aur rods 60-70% cases; Absent
intracranial bleed, seizures) diagnostic
Myelodysplasia Often present Absent
Table 2 Differential diagnosis of acute lymphoblastic leukemia

in children
Table 4 Commonly used immunophenotypic markers
• Nonmalignant conditions:
Myeloid or CD13, CD33, CD117, CD15, CD16,
– Juvenile rheumatoid arthritis Monocytic MPO
– Infectious mononucleosis At least 2 of the following:
NSE, CD14, CD64, CD11c, lysozyme
– Idiopathic thrombocytopenic purpura
B-lymphoid CD19, CD20, CD22, Cyto CD79a
– Pertussis; parapertussis
Cyto CD22
– Aplastic anemia
T-lymphoid CD1a, CD2, CD3, CD4, CD5, CD7,
– Acute infectious lymphocytosis CD8, Cyto CD3
• Malignancies: Other markers CD34, Tdt, HLADR, CD41, CD61
– Hematolymphoid malignancies—Acute myeloid leukemia,
Hodgkin lymphoma, non-Hodgkin lymphoma (NHL)
– Neuroblastoma like retinoblastoma, rhabdomyosarcoma, also having
morphology of small round cell tumor, should be kept
– Retinoblastoma
as differential diagnosis though the site of origin and
– Rhabdomyosarcoma immunohistochemistry is helpful to diagnose these solid
tumors.
Laboratory confirmation is mandatory for the diagnosis
thrombocytopenic purpura, pertussis, parapertussis,
and management of ALL.
aplastic anemia, and acute infectious lymphocytosis.
These diseases have overlapping symptomatology.
Hematolymphoid malignancies especially non- LABORATORY DIAGNOSIS OF ACUTE
Hodgkin lymphomas with marrow involvement have a LYMPHOBLASTIC LEUKEMIA
common spectrum of signs and symptoms and should Laboratory investigations should be directed for con
be differentiated with ALL. Clinical symptomatology firming the diagnosis, assessment of organ function, and
like abdominal mass in Burkitt’s lymphoma, bony to rule out need for urgent intervention like tumor lysis
involvement in anaplastic large cell lymphoma syndrome.
helps in making working diagnosis. Clinical features,
morphology and immunohistochemistry are helpful
Confirmation of Diagnosis
tool to distinguish ALL from acute myeloid leukemia
(AML). Some features are given in Tables 3 and 4. Laboratory work up starts with complete blood counts
Solid tumors with advanced stage (i.e. bone marrow (CBC), peripheral blood and bone marrow evaluation.
infiltration) may mimic ALL. Neuroblastoma with bone CBC generally shows anemia and thrombocytopenia.
marrow involvement may have features of anemia, body However patients may have leukopenia, normal leukocyte
pain, abdominal distension, though the early age of count or leukocytosis. Peripheral blood examination is
presentation, ecchymosis, primarily abdominal mass usually followed by bone marrow (BM) examination. BM
may help it to distinguish it from ALL. Other solid tumors aspiration is preferably done from the posterior superior
Chapter-39 Pediatric Acute Lymphoblastic Leukemia 397
Table 5 Immunological markers in B-ALL and T-ALL5

Lineage Immunological subgroup Frequency Immunophenotypic profile Remarks
B-ALL Early pre-B cell 60–70% CD10, CD19, CyCD22, CD34, Good except CD10-ve ALL, especially in <1 year
and Tdt positive. CD24 age group, infantile leukemia associated with
strongly positive, CD45 translocation of 11q23 and have poor prognosis
dim/-ve
Pre-B cell 20% Cyu heavy chain, CD10, Poor compared to early pre-B. Expression of
CD19, cyCD79a, and CD22 t(1:19) is the primary determinant of adverse
+ve prognosis in pre B-ALL
Mature B-Cell 2–5% Surface immunoglobulin Poor with standard ALL therapeutic regimen,
(bright), CD19, CD20, CD22, but dose intensive chemotherapy leads to cure
CD24 +ve CD34. Tdt –ve. rate of 75%.
T-ALL Pro-T-cell CyCD3 and/or CD7 +ve With more intensive chemotherapeutic
approach outcome reaching to the level of non-
T-cell ALL.
Pre-T-cell CyCD3, CD7, CD2 and/or
CD5 +ve
Cortical T-cell CyCD3, CD7, CD2 and/
or CD5 and CD1a +ve, co-
expression of CD4/CD8
Mature T-cell CyCD3, CD7, CD2 and/or

CD5 and CD3 positive,
segregated CD4 or CD8 +ve
iliac bone and sample is aliquoted for morphology, Cytogenetics of Acute Lymphoblastic
flow cytometry, molecular studies (EDTA vacutainer), Leukemia
cytogenetics (Heparin vacutainer) and trephine biopsy
(formalin fixed). The diagnosis of ALL should be Blasts in ALL contain somatically acquired genetic abnor
established by cytomorphological examination of May- malities that help in understanding pathogenesis and
Grunwald Giemsa (MGG) or Wright-stained smears of strongly influence prognosis. This includes changes in
peripheral blood and/or a BM aspirate. chromosome number (hyperdiploid/hypodiploid) and
FAB criteria and scoring system have become chromosomal translocations.
generally accepted for morphological classification. The
myeloperoxidase (MPO) or Sudan-Black (SB) reaction and Numerical Abnormalities
the non-specific esterase (NSE) reactions are recommended
for differentiation from AML. In ALL, the MPO reaction is • High hyperdiploidy: It is defined as 51 to 65 chromo
negative. A positive PAS and/or acid phosphatase reaction somes per cell or a DNA index larger than 1.16.
may support the diagnosis of ALL; these reactions, however, • Hypodiploidy: Patients with fewer than 45 chromo
are not positive in all cases of ALL. somes defined as hypodiploidy.
Flow Cytometry Structural Abnormalities (Chromosomal

Leukemic cells demonstrate particular subsets of surface Translocations)
and intracellular molecules defined as cluster of differen • TEL-AML1, (t [12; 21]): It is seen in 20 to 25 percent
tiation (CD) antigens (Table 5). Identification of which of cases of B-precursor ALL at least in the developed
by flow cytometry (FCM) helps in lineage identification countries. The t (12; 21) occurs most commonly in
and sub-categorization. Diagnosis of acute leukemia by children aged 2 to 9 years.
immunophenotyping is based on the fact that leukemic • Philadelphia chromosome, (t [9; 22] translocation): It is
cells frequently disclose aberrant phenotypes compared present in three percent of children with ALL, and is
to normal hematopoietic cells, known as the leukemia- more common in older patients with precursor B-cell
associated phenotypes (LAP). ALL and high WBC count.
• MLL gene rearrangements: It is seen in five percent of to assess for central nervous system (CNS) involvement
childhood ALL cases. The t(4; 11) is the most common and to administer intrathecal chemotherapy. In males,
translocation involving the MLL gene in children with USG of testis is done to rule out testicular involvement, if
ALL. Patients with t (4; 11) are usually infants with high clinically indicated.
WBC counts; usually have CNS disease.
• E2A-PBX1, (t[1; 19] translocation): It occurs in PROGNOSTIC FACTORS (TABLE 6)
five percent of childhood ALL cases. The t(1;19)
translocation has higher risk of CNS relapses. Outcome of patients with ALL has improved over time
though the prognosis depends on multiple factors, still
Role of Trephine Biopsy type of treatment and its response is the strongest point
for predicting the outcome.
It is especially required in cases wherein the BM Prognosis depends on various factors:
aspirate smears are acellular or dry tap, diluted with • The host genotype (host biology)
peripheral blood, in partially treated cases where blood • The leukemic cell genotype (tumor biology)
transfusion/treatment by steroids and other drugs alter • Response to therapy.
the morphological picture or in cases where the sample
is degenerated during transport to a referral laboratory A. Host Biology
for ancillary investigations. It is recommended to obtain a
trephine biopsy upfront in all new cases of hematolymphoid • Age
malignancies as the paraffin block is an invaluable • Gender.
diagnostic archival material for immunophenotyping.
Age
Organ Function Assessment
The age at diagnosis correlates with clinical outcome.
Renal function test, liver function test, serum electrolyte, In childhood ALL, infants and adolescents have a worse
serum lactate dehydrogenase (LDH) with serology prognosis than patients aged 1 to 10 years6. The improved
especially of hepatitis (B&C), HIV should be done routinely. outcome in patients between 1 to 10 years is due to more
Total leukocyte count, serum LDH, and extramedullary frequent occurrence of favorable cytogenetic features in
involvement (hepatosplenomegaly) are the indicators for the leukemic blasts including hyperdiploidy, or trans
tumor burden and helps to find patients who need urgent location t(12;21).7 Infants with ALL have high-risk of
intervention. treatment failure as they have high presenting leukocyte
counts, increased frequency of central nervous system
Other Tests leukemia at presentation and a very high incidence (~80%)
Lumbar puncture with cytospin morphologic analysis is of rearrangement of the MLL gene on chromosome 11q23.
performed before systemic chemotherapy is administered Amongst infants with MLL gene rearrangements, those
Table 6 Prognostic factors in childhood ALL

Adverse Favorable
Clinical Age <1, >10 1–9.99
WBC >50,000 <50,000
Sex Boy Girl
Laboratory DNA index <1 (Hypodiploidy) >1.16 (Hyperdiploidy)
Immunophenotype T-ALL, EPB CALLA+
Cytogenetics t (4;11) TEL-AML
t (9;22) Trisomy 4,10,17
Treatment Inappropriate Appropriate treatment
treatment
In vivo response Poor ESR* Good ESR*
(Most important) MRD** + MRD** –
*ESR: Early steroid response; **MRD: Minimal residual disease.
presenting at a young age (< 6 months) or with extremely Table 7 Definition for central nervous system
high leukocyte counts (> 300,000/μL) have the worst involvement by leukemia
prognosis.8
• CNS-1 No lymphoblasts
Adolescents (ages 16-21 years) with ALL have a less
favorable outcome than children aged 1 to 10 years, as • C
NS-2 <5 WBCs/mL with definable blasts on cytocentrifuge
examination
they frequently present with T-cell immunophenotype,
high leukocyte counts, and a higher incidence of the • CNS-3 ≥5 WBCs/mL with blasts or cranial palsy
Philadelphia chromosome [t(9;22)].9 Adolescents are also
at higher risk for certain treatment-related complications,
such as hyperglycemia, osteonecrosis, pancreatitis and more intensive intrathecal therapy, especially during
deep vein thromboses, which may also impact prognosis.10 the induction phase.12 Furthermore, a traumatic lumbar
puncture (more than 10 erythrocytes/μL) that includes
Gender blasts at diagnosis appears to be associated with increased
The prognosis for boys with ALL is slightly worse than risk of CNS relapse and requires intensification of therapy.
girls.11 Potential reason for the better prognosis for girls
is the occurrence of testicular relapses among boys. Also, Testicular Involvement at Diagnosis
boys appear to be at increased risk of bone marrow and Overt testicular involvement at the time of diagnosis
CNS relapse for reasons that are not well understood. occurs in approximately two percent of males. Historically,
With current treatment regimens, there is no difference in testicular involvement at diagnosis was identified as an
outcome between males and females.12 adverse prognostic factor, but with aggressive therapy
it has lost its prognostic significance.14 Overt testicular
B. Tumor Biology involvement is not an independent prognostic factor,
• White blood cell (WBC) despite association with high-risk features.15
• CNS status at diagnosis
• Testicular involvement at diagnosis Immunophenotype
• Immunophenotype The World Health Organization (WHO) classifies ALL as
• Cytogenetics either “B-lymphoblastic leukemia”(B-ALL) or “T-lineage
• Numerical abnormalities lymphoblastic leukemia” (T-ALL), based on its cell of origin
• Structural abnormalities (Chromosomal transloca detected by surface or cytoplasmic expression of B or T-cell
tions) antigens. Precursor B-cell ALL is defined by the expression
• Early response to therapy. of cytoplasmic CD79a, CD19, HLA-DR, and other B cell-
associated antigens. It accounts for 80 to 85 percent of
White Blood Cell Count at Diagnosis childhood ALL and has a better prognosis compared to
White blood cell count reflects tumor burden. Although the T-ALL. Precursor B-cell ALL patients are further divided
relationship between WBC count and prognosis is a con into immunologic subtypes, of which Pro-B ALL (CD10
tinuous rather than a step function, the National Cancer negative and no surface or cytoplasmic Ig) is commonly
Institute (NCI) stratifies patients into two subsets based on seen in young infants with a t (4; 11) translocation and has
WBC counts; standard risk (WBC count < 50,000) or high- a poor outcome. T-cell ALL is defined by expression of the
risk (WBC count > 50,000). High WBC count is usually as cytoplasmic CD3, with CD7 plus CD2 or CD5 on leukemic
sociated with unfavourable chromosomal translocations blasts. High-risk features at presentation were significantly
such as t(4; 11), t(9; 22) and T cell immunophenotype.13 more frequent in T-ALL as compared to B-lineage ALL.16
T-ALL is further divided into immunologic subtypes,
of which early T-progenitor (ETP)-ALL (CD1a and CD8
Central Nervous System Status at Diagnosis
negative, CD5 weak, at least one stem-cell-associated or
The presence of CNS disease at diagnosis is an adverse myeloid-associated antigen) has stem-cell-like features
prognostic factor in spite of intensification of therapy. with high-risk of induction failure or relapse.17
Patients are divided into three categories based on the Myeloid antigen expression: Myeloid-associated antigen
number of WBC/μL and the presence/absence of blasts expression is associated with specific ALL subgroups
on cytospin (Table 7). (MLL gene and TEL-AML1 gene rearrangement). No
The adverse prognostic significance associated independent adverse prognostic significance exists for
with CNS2 status can be overcome by the application of myeloid-surface antigen expression.18
Cytogenetics • E2A-PBX1, t(1;19): It is associated with pre-B ALL

immuno phenotype. It was associated with poor
Blasts in ALL contain somatically acquired genetic prognosis in the context of less intensive anti
abnormalities that help in understanding pathogenesis metabolite-based therapy in the past, but with most
and strongly influence prognosis. This includes changes current treatment protocols, the t(1;19) translocation
in chromosome number (hyperdiploid/hypodiploid) and has no adverse prognostic significance except higher
chromosomal translocations risk of CNS relapses.25
Numerical Abnormalities C. Early Response to Therapy

High hyperdiploidy: High hyperdiploidy generally occurs The kinetics of the reduction in tumor burden in response
with clinically favorable prognostic factors (patients aged to treatment has been shown to be highly prognostic
1–9 years with a low WBC count) and is itself an independent of event free survival. Response to therapy is the most
favorable prognostic factor. Hyperdiploid leukemia cells reliable prognostic factor, as it reflects leukemic cell drug
are particularly susceptible to undergoing apoptosis and sensitivity, intensity of therapy, and pharmacogenomic as
accumulate higher levels of methotrexate and its active well as pharmacodynamic features of the host.
polyglutamate metabolites which may explain the favorable
outcome commonly observed for these cases19. Among
specific trisomies, patients with triple/trisomies (4, 10, and
Peripheral Blood Response to Steroid
17) have been shown to have an improved outcome.19 Prophase
Hypodiploidy: Patients with fewer than 44 chromosomes Patients with a reduction in peripheral blast count to
have a worse outcome than patients with 44 or more less than 1,000/μL after a 7-day induction prophase with
chromosomes in their leukemic cells. Cases with 24 to 28 prednisone and one dose of intrathecal methotrexate
chromosomes (near haploidy) have the worst outcome.20 (good prednisone response) have a more favourable
prognosis than patients whose peripheral blast counts
remain above 1,000/μL (a poor prednisone response).26
Structural Abnormalities
German Berlin-Frankfurt-Munster (BFM) clinical trials
(Chromosomal Translocations) group stratifies its treatment based on early response to
• TEL-AML1, (t[12; 21]): The t(12; 21) occurs most the 7-day prednisone prophase.
commonly in children aged 2 to 9 years.21 It has good
prognosis. However, its impact may be modified by Day 7 and Day 14 Bone Marrow Responses
factors such as early response to treatment, NCI risk
Patients who have a rapid reduction in leukemia cells to
category, and treatment regimen. There is a higher
less than 5% (M1 marrow) in their bone marrow within 7
frequency of late relapses in patients with TEL-AML1
or 14 days following initiation of multiagent chemotherapy
fusion compared with other B-precursor ALL.22
have a more favorable prognosis than do patients who
• Philadelphia chromosome, (t[9; 22] translocation): It is
have slower clearance of leukemia cells from the bone
associated with poor prognosis especially in those who
marrow.27
present with a high WBC count or have a slow early
response to initial therapy. However, its prognosis
seems to have improved by incorporation of tyrosine Peripheral Blood Response to Multiagent
kinase inhibitors, such as imatinib, in the treatment. Induction Therapy
A COG study, using intensive chemotherapy and Patients with persistent circulating leukemic cells at 7 to
concurrent imatinib given daily, demonstrated a 10 days after the initiation of multiagent chemotherapy
3-year EFS rate of 80.5 percent.23 are at increased risk of relapse compared with patients
• MLL gene rearrangements: The t(4; 11) is the most who have clearance of peripheral blasts within 1 week of
common translocation involving the MLL gene in therapy initiation.28
children with ALL. Patients with t(4; 11) are usually
infants with high WBC counts; usually have CNS disease
and respond poorly to initial therapy. Children with MLL
Induction Failure
rearrangement have a better prognosis than infants.24 Five percent of patients do not achieve complete mor
The t(11; 19) occurs in one percent of cases and phologic remission by the end of induction therapy. A
occurs in both early B-lineage and T-cell ALL. Outcome cut-off of five percent blasts in the bone marrow is used
for infants with t(11; 19) is poor, but outcome appears to determine the remission status. Patients at highest risk
favorable in older children with T-cell ALL. of induction failure include T-cell phenotype and patients
with B-precursor ALL with very high presenting leukocyte respectively predicted by use of MRD or IKZF1 alone. The
counts or the Philadelphia chromosome. Induction failure above findings signify that the use of combined parameters
portends a very poor outcome.29 enhances the risk stratification, particularly for patients
originally classified as nonhigh-risk.34
Minimal Residual Disease
Pharmacogenetics
Bone marrow morphology cannot discriminate well
between patients at high-risk of relapse and patients with It is the study of genetic variations in drug-processing
excellent prognosis. Therefore, more sensitive techniques genes and individual responses to drugs which enables
have been developed for detection of submicroscopic improved identification of patients at higher risk for
levels (<5%) of malignant cells during and after treatment, either disease relapse or chemotherapy-associated side
i.e. MRD. Minimal residual disease (MRD) is defined effects. Patients with ALL who are homozygous for TMPT
as the detection of the clones of cells resistant to the mutant alleles experience severe or fatal myelotoxicity and
chemotherapy given. increased relapse because of long delays in therapy.35
Three types of techniques allow detection of MRD Studies from St Jude Children’s Research Hospital
of 10-3 to 10-6 (1 leukemic cell in 1000 to 1 million cells): (SJCRH) have shown that when patients are treated
Multiparametric flow cytometry (MPFC) for surface pharmacologically according to phenotype or genotype,
phenotype of leukemia cell (sensitivity of 10-3–10-4) can be carriers of variant TMPT alleles experience outcomes as
used in up to 80 to 90 percent patients with ALL; PCR for good as, or better than, those with wild-type TPMT.36
T-cell receptor or immunoglobulin gene rearrangement or The reduced folate carrier (RFC) is the primary
fusion transcripts (sensitivity 10-3– 10-5) can be performed transporter of MTX into cells. RFC expression in leukemic
in 90 to 95 percent patients with ALL; PCR Analysis of blasts is linked to MTX sensitivity, while defective
breakpoint fusion regions of chromosomal aberrations can transport associated with reduced RFC expression is a
be performed in 30 to 45 percent patients MRD is the most common mechanism of acquired methotrexate resistance.
robust and strongest independent predictor of outcome in Increased copies of RFC are present in hyperdiploid
children and adolescents with ALL, which is independent blasts and MTX-polyglutamate accumulation in blasts
of age, sex, immunophenotype, WBC count and treatment correlates with better outcome in hyperdiploid ALL. The
group. null genotype of glutathione S-transferases, enzymes that
MRD discriminates outcome even in subsets of patients catalyze the inactivation of many antileukemic agents, has
defined by cytogenetic abnormalities and other prognostic been associated with a reduced risk of relapse.
factors. Patients with higher levels of end-induction MRD
(>0.01%) have a poorer prognosis than those with lower RISK STRATIFICATION OF CHILDHOOD ALL
or undetectable levels. Therefore, post-induction MRD
is utilized as a factor determining the intensity of post- NCI Risk-grouping
induction treatment. MRD levels at earlier (e.g. day 8 and Age and WBC count at diagnosis strongly correlates with
day 15 of induction) and later time points (e.g. week 12 of outcome in B-Precursor ALL. The high predictive value
therapy) also predict outcome.30 of age and WBC among all studies, and the fact that
these variables can be easily and reliably measured by
Newer Factors all investigators worldwide, make them one of the most
important prognostic factors based on which the patients
Molecular Genetic Abnormalities with ALL are divided into two risk groups:
IKAROS/IKZF1 deletions, JAK mutations and kinase
expression signatures have been associated with poor 1. Standard risk Age 1–9.99 years
prognosis in B cell acute lymphoblastic leukemia.31-33 WBC <50000/cumm
Based on combination of gene expression profile and flow 2. High-risk Age >10 years
cytometric measures of minimal residual disease (MRD),
WBC >50000/cumm
children with high-risk B-precursor ALL can be classified
as low, intermediate, and high-risk and thus allow
prospective identification of children who respond or fail
Children’s Oncology Group Risk Stratification
current treatment regimens. Furthermore, integrated use
of both MRD and IKZF1 status allows prediction of 79% of In the current children’s oncology group (COG)
all the relapses with 93% specificity in MRD-medium risk classification system and treatment algorithm, patients
group as compared to 46 and 54 percent of the relapses with precursor B-cell ALL are initially assigned to a
standard-risk or high-risk group based on NCI grouping. Table 9 Key components of treatment in ALL
All children with T-cell phenotype are considered
• Protocol based therapy
high-risk regardless of age and initial WBC count. Early
treatment response, assessed by day 7 or day 14 marrow • Risk-directed therapy
morphology along with end-induction MRD assessment • Empiric multiagent induction therapy
and cytogenetics is subsequently used to determine the • Presymptomatic CNS therapy
intensity of post-induction therapy. Patients are classified
• Early intensification of chemotherapy
as very high-risk if they have very high-risk cytogenetics
with poor response or induction failure as detailed here. • Systemic and IT
In developing countries outcome of disease is also • Consolidation/intensification
affected adversely by inadequate supportive care, delay in • Early reinduction
diagnosis, and poor access to acute care.2
• Augmented therapy only in high-risk patients
Late intensification where indicated:
MANAGEMENT OF ACUTE LYMPHOBLASTIC
• Delayed intensification
LEUKEMIA IN CHILDREN
• Double delayed intensification
• Induction chemotherapy for ALL
Extended continuation of treatment:
• Consolidation/Intensification therapy
• Maintenance therapy • Dose intensity of antimetabolites
• Central nervous system (CNS) therapy • Dexa/VCR pulses
The treatment of childhood acute lymphoblastic leukemia
(ALL) has advanced significantly over the past 3 decades,
with overall survival rates progressing from 20 to 95 adopted. The NCI criteria risk stratified in to standard and
percent.1,37,38 The standard “backbone” of treatment for ALL high-risk based on: age, initial white blood cell (WBC)
has remained unchanged for over 25 years and includes count, and the presence of extramedullary disease at
remission induction, consolidation, treatment to prevent diagnosis.39
overt leukemic infiltration of the central nervous system Currently most cooperative groups use additional risk
(CNS directed therapy), and continuing (maintenance) factors that have been shown to have an impact on patient
therapy. The steady improvement in survival of children outcomes (e.g. ploidy, blast karyotype/cytogenetics, and
with ALL is a result of a number of modifications of this early morphologic response). The resulting classification
treatment, the value of which have been confirmed by system thus incorporates the strongest prognostic
randomized clinical trials (Tables 8 and 9). indicators predictive of outcome, and stratifies the treat
In an effort to appropriately balance the risks and ment based on their risk of relapse.
benefits of therapy, “risk-adapted therapy” has been
Induction Chemotherapy for ALL
Table 8 Principles of childhood ALL therapy Three-drug induction therapy using vincristine,
• T reat patients on cooperative group based clinical trials, if corticosteroid (prednisone or dexamethasone), and
possible L-asparaginase in conjunction with intrathecal (IT)
• A
dopt effective treatment components of successful clinical therapy, results in complete remission (CR) rates of
trials: greater than 95%. For patients who are at standard risk
– Reinduction therapy: BFM, CCG or low risk of treatment failure, four-drug induction
– Intensive asparaginase: DFCI therapy does not appear necessary for favorable outcome
– Augmented therapy: BFM provided that adequate post remission intensification
– Intensive intrathecal therapy: SJCRH therapy is administered. Because of the likelihood of
• Individualized therapy: increased toxicity with four-drug induction therapy, many
– Risk assessment based mainly on MRD studies co-operative groups including the Children’s Oncology
– Targeted HD-MTX dose Group (COG), National Cancer Institute (NCI), protocols
– Mercaptopurine dose based on TPMT, 6TGN and ANC for standard-risk precursor B-cell acute lymphoblastic
• Risk-adapted therapy to decrease late complications: leukemia (ALL) utilize a three-drug induction consisting
– Omit cranial irradiation in all patients of dexamethasone, vincristine, and PEG-L-asparaginase.
– Decrease dose of anthracyclines to bare minimum For patients presenting with high-risk features, a
– Avoid use of etoposide more intensive induction regimen (four or five agents)
may result in improved event-free survival (EFS), and CCG developed an augmented BFM treatment regimen
such patients generally receive induction therapy featuring repeated courses of escalating-dose intravenous
that includes an anthracycline (e.g. daunorubicin) in methotrexate (without leucovorin rescue) given with
addition to vincristine, prednisone/dexamethasone, plus vincristine and asparaginase during interim maintenance
L-asparaginase. and additional vincristine/L-asparaginase pulses during
initial consolidation and delayed intensification. Augmen
Consolidation/Intensification Therapy ted therapy also included a second interim maintenance
and delayed intensification phase. Of note, there is a
Once remission has been achieved, systemic treatment in significant incidence of osteonecrosis of bone in teenaged
conjunction with central nervous system (CNS) sanctuary patients who receive the augmented BFM regimen.
therapy follows. The intensity of the post induction The augmented BFM regimen has also been evaluated
chemotherapy varies considerably depending on risk in children with high-risk ALL and a rapid early response
group assignment, but all patients receive some form of to induction therapy. For these children, augmented
intensification following achievement of remission and intensity during consolidation, interim maintenance,
before beginning maintenance therapy. Intensification and delayed intensification resulted in a higher EFS rate
may involve use of the following: than that achieved with standard-intensity treatment.
Intermediate-dose or high-dose methotrexate with Increased duration of intensive therapy was not beneficial,
leucovorin rescue or escalating-dose methotrexate without and a single application of delayed intensification was as
rescue;40 drugs similar to those used to achieve remission effective as two applications.46
(re-induction or delayed intensification);41 different drug For children with Ph+ ALL, imatinib mesylate in
combinations with little known cross-resistance to the conjunction with chemotherapy during post induction
induction therapy drug combination; L-asparaginase for therapy has produced a 3-year EFS of 87.7 ± 10.9 percent.
an extended period of time; or combinations of the above.42 These patients fared better than historic controls treated
In children with standard-risk acute lymphoblastic with chemotherapy alone (without imatinib), and at least
leukemia (ALL), regimens utilizing a limited number of as well as the other patients on the trial who underwent
courses of intermediate-dose or high-dose methotrexate as allogeneic transplantation. Longer follow-up is necessary
consolidation followed by maintenance therapy (without to determine if this novel treatment improves cure rate or
a re-induction phase) have been used with good results. merely prolongs DFS.
Similarly favorable results for standard-risk patients have Infant ALL is uncommon, representing approximately
been achieved with regimens utilizing multiple doses of 2 to 4 percent of cases of childhood ALL. Despite the
L-asparaginase (20–30 weeks) as consolidation, without inclusion of post induction intensification courses with
any post induction exposure to alkylation agents or high doses of cytarabine and methotrexate. Long-term
anthracyclines.43 EFS rates remain below 50 percent, and for those infants
Post induction consolidation for regimens using a with MLL gene rearrangement, the EFS rates continue to
German Berlin-Frankfurt-Munster “BFM-backbone,” such be in the 17 to 40 percent range.47
as those of the Children’s Oncology Group (COG), include Factors predicting poor outcome for MLL-rearranged
a delayed intensification phase, during which patients infants include a very young age (<6 months), extremely
receive a 4-week re-induction (including anthracycline) and high presenting leukocyte count (300,000/mL), and high
reconsolidation containing cyclophosphamide, cytarabine, levels of MRD at the end of induction and consolidation
and 6-thioguanine given approximately 3 months after phases of treatment.48
remission is achieved. In a Children’s Cancer Group (CCG) The role of bone marrow transplantation in infants
study, which included a three-drug induction and utilized with MLL-rearranged ALL remains controversial.
prednisone as the corticosteroid throughout all treatment
phases, two blocks of delayed intensification produced
Maintenance Therapy
a small event-free survival (EFS) benefit compared with
one block of delayed intensification in intermediate-risk (Standard risk and high-risk ALL) The backbone of
patients.44,45 maintenance therapy in most protocols includes daily
In high-risk patients, a number of different approaches oral mercaptopurine and weekly oral methotrexate.
have been used with comparable efficacy. Treatment On many protocols, intrathecal chemotherapy for CNS
for high-risk patients generally is more intensive than sanctuary therapy is continued during maintenance
that for standard-risk patients, and typically includes therapy. The use of continuous 6-thioguanine (6-
higher cumulative doses of multiple agents, including TG) instead of 6-mercaptopurine (6-MP) during the
anthracyclines and/or alkylating agents. The former maintenance phase is associated with an increased risk of
hepatic complications, including veno occlusive disease – Postremission therapy

and portal hypertension. Because of the risk of hepatic – Continuation chemotherapy
complications, 6-TG is no longer utilized in maintenance – Hematopoietic stem cell transplantation (HSCT)
therapy in current protocols. • Treatment of central nervous system relapse
Pulses of vincristine and corticosteroid are often • Isolated testicular relapse.
added to the standard maintenance backbone, A CCG
randomized trial demonstrated improved outcome Diagnosing Relapse of ALL
in patients receiving monthly vincristine/prednisone
pulses,49 and a meta-analysis combining data from six Suspected patients should undergo bone marrow aspira
clinical trials showed an EFS advantage for vincristine/ tion with morphology, surface marker studies and
prednisone pulses. Maintenance chemotherapy generally cytogenetic studies. Examination of extramedullary sites
continues until 2 to 3 years of continuous complete like CNS, testis, eyes, and skin should be done at the time
remission. On some studies, boys are treated longer than of diagnosis of relapse.
girls; on others, there is no difference in the duration of
treatment based on gender. Extending the duration of Risk Stratification of Relapsed ALL
maintenance therapy beyond 3 years does not improve
outcome.50 It is important to have risk stratification of relapse for
counseling and tailoring the salvage treatment.
Central Nervous System Therapy • Length of 1st CR: Patients with very early (< 18 months
from start of therapy) BM relapse have < 10 percent
Options for central nervous system (CNS)-directed long-term survival (LTS) compared to approximately
therapy include IT chemo therapy, high dose systemic 50 percent LTS for patients who have late BM relapse.
chemotherapy, and cranial radiation. The type of CNS- • Immunophenotype: Childhood ALL patients with
therapy, i.e. used, is based on a patient’s risk of CNS- T-cell ALL fare more poorly than those with CALLA+
relapse, with higher-risk patients receiving more intensive ALL.
treatments. The proportion of patients receiving cranial • Site of relapse: Historically, isolated marrow relapse
radiation has decreased significantly over time, with those has had the worst prognosis; isolated CNS, testicular,
receiving cranial radiation, the dose has been significantly or other extramedullary relapse carried a significantly
reduced. IT chemotherapy is usually started at the better prognosis, and combined marrow and extra-
beginning of induction, intensified during consolidation medullary relapse, an intermediate prognosis
and, in certain protocols, continued throughout the • The nature and intensity of previous therapy: Patients
maintenance phase. IT chemotherapy typically consists of previously treated with lower intensity primary therapy
either methotrexate alone or methotrexate with cytarabine have a higher reinduction rate.
and hydrocortisone. Unlike IT cytarabine, IT methotrexate • Minimal residual disease (MRD) studies: MRD is an
has a significant systemic effect, which may contribute to important tool even for patients with ALL at time of
prevention of marrow relapse. relapse. The level of minimal residual disease after
Systemically administered drugs, such as dexame achieving second remission or before transplant may
thasone, L-asparaginase, high-dose methotrexate with predict outcomes.52
leucovorin rescue, and high-dose cytarabine, provide
some degree of CNS protection. For example, in a
randomized CCG study of standard-risk patients who all Treatment of Marrow Relapse
received the same dose and schedule of IT methotrexate Reinduction Therapies after Marrow Relapse
without cranial irradiation, oral dexamethasone was
associated with a 50 percent decrease in the rate of CNS Typical treatment of first relapse involves a combination
relapse compared with oral prednisone.51 of vincristine, a glucocorticoid (prednisone, prednisolone,
or dexamethasone), and asparaginase, plus an anthra
cycline, methotrexate, or cytarabine in varying doses and
RELAPSE OF DISEASE AND
schedules. Only a few randomised trials have investigated
ITS MANAGEMENT reinduction therapy.53
• Diagnosing relapse of ALL In the absence of randomized trials and consistent risk-
• Risk stratification of relapsed ALL stratified reporting of patient’s outcomes, with possibly
• Treatment of marrow relapse exception of mitoxantrone, no reinduction combination is
– Reinduction therapies after marrow relapse significantly superior to the others.
Postremission Therapy relapse is around 2 to 5 percent with current protocols.

Effective treatment includes systemic chemotherapy and
Therapeutic options after CR2 include further chemo administration of local radiotherapy. Doses of 2400 cGy to
therapy and HSCT. Most pursue HSCT options for patients both testes is optimal.
with early relapses, although outcomes remain poor for
most patients with measurable MRD after reinduction.
Similar outcomes are reported for matched related donor
LATE EFFECTS OF THERAPY
and matched unrelated donor transplants. Prolonged consequences of ALL therapy should be kept in
For late marrow relapse, outcomes are similar with mind while following these patients and need special after
chemotherapy and HSCT options. Some recommend therapy clinic to diagnose and manage these effects.
HSCT options for patients with late marrow relapse and
an MRD-positive CR2. Chemotherapy options may be • Central nervous system:
pursued for isolated extramedullary relapse with success – Cortical atrophy
in most patients. – Necrotizing leukoencephalopathy
– Subacute leukoencephalopathy
– Mineralizing microangiopathy
Continuation Chemotherapy
• Neuroendocrine abnormalities:
All patients who achieve a second remission receive – Growth hormone deficiency
additional chemotherapy, even if hematopoietic stem cell – Obesity
transplantation is planned. To maintain control of disease,
• Cardiac abnormalities:
higher dose intensity is used, and higher regimen-related
toxicity is tolerated than in first-line treatment. – Cardiomyopathy
Most reports describe single-arm studies with combi – Late onset congestive heart failure
nations of vincristine, glucocorticoids, metho trexate, • Others toxicities:
cytarabine, etoposide, cyclophosphamide or ifosfamide, – Avascular necrosis
and thiopurines, with or without maintenance therapy for – Primary gonadal failure
up to 2 years. – Second malignancies (SMN)
CNS prophylaxis includes high-dose methotrexate or – Post-traumatic stress disorder (PTSD)
cytarabine, intrathecal chemotherapy, and in more recent
BFM group trials, 1200–1800 cGy cranial irradiation.54
SUMMARY
Hematopoietic Stem Cell Transplantation Acute lymphoblastic leukemia is the most common
The only strategy that has shown to improve outcome of malignancy in pediatric age group. With current chemo
patients with relapsed ALL with high-risk features is to therapy agents high cure rate can be achieved. Detailed
give intensified consolidation chemotherapy regimen, work-up and sophisticated management is required
followed by hematopoietic stem cell transplantation while dealing with child of acute lymphoblastic leukemia.
(HSCT). Recent data has shown that outcome of HSCT Type of treatment and minimal residual disease (MRD)
is better than chemotherapy alone even in patients with measurement are promising tool to predict the prognosis.
intermediate risk relapsed ALL and HSCT is currently Treatment is based on risk stratification or response
being offered to these patients if they have an HLA evaluation. Though the treatment is prolonged with
matched sibling donor. significant toxicities, still outcome is quite satisfactory.
Unlike relapse of other malignant condition pediatric
Treatment of Central Nervous System Relapse acute lymphoblastic leukemia can be treated with
intensive chemotherapy and hematopoietic stem cell
A common approach is first to induce a CSF remission transplant with reasonable outcome. Major worry after
with IT chemotherapy, reinstitute systemic therapy, and completion of treatment is post treatment consequence
then later administer craniospinal irradiation, at doses of of chemotherapy and radiotherapy which needs special
2,400 to 3,000 cGy to the cranial vault and 1,200 to 1,800 attention.
cGy to the spinal axis.55
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C H A P T E R 40
Pediatric Acute Myeloid
Leukemia
Acute myeloid leukemia (AML) is a heterogeneous disease. AML accounts for 15 to 20 percent of all acute leukemias in children.1
Five-year survival rates for children in the US younger than age 15 years with AML increased from < 20 percent in 1975 to 1978 to 58
percent in 1999-2002.2 Although there are no systematic statistics for pediatric AML from India, survival outcomes range from 23 to
53.8 percent.3 The understanding of disease biology has resulted in changes in classification and risk stratification. This, along with
improvements in supportive care which allow for more intensive treatment, risk-adapted treatment approaches, and the selective
use of hematopoietic stem cell transplant (HSCT) have all contributed to improvements in outcome. However, Pediatric AML has
unfortunately not replicated the success story of pediatric acute lymphoblastic leukemia (ALL), and still remains a challenging disease
to treat, especially so in resource-poor settings.
BIOLOGY AND PATHOGENESIS Class 2 mutations impair cell differentiation and confer
self-renewal properties and are gene fusions commonly
A number of inherited and acquired disorders have generated by chromosomal translocations, such as t(8;21)
been associated with development of AML in children, (q22;q22)/RUNX1-RUNX1T1 and inv(16)(p13.1;q22)/
although the cause is unknown in the vast majority of CBFB-MYH11.6 Data suggest that class 1 and 2 mutations
patients. Inherited conditions like Down syndrome, co-operate to induce leukemia. Currently, more than 90
Fanconi anemia, congenital neutropenia, inherited bone percent of pediatric AML cases are identified to have at
marrow failure syndromes; acquired conditions like least one known genomic alteration.7
aplastic anemia, paroxysmal nocturnal hemoglobinuria,
myelodysplastic syndrome as well as environmental CLASSIFICATION
exposures to drugs (alkylating agents and topoisomerase
inhibitors) and ionizing radiation have been found to have Although, the French-American-British (FAB) classi
an increased association. fication8 (based on morphological features) continues to be
The cancer stem cell model is increasingly being widely used, the most comprehensive classification of AML
accepted in the pathogenesis of AML—it proposes that is the WHO 20089 classification, which includes clinical
AML cells, like normal hematopoietic progenitors, features, morphological findings, immunophenotyping
are hierarchically organized into compartments that and cytogenetics, to define specific disease entities.
contain leukemic stem cells (LSCs) that have unlimited (Table 1). Although, the present classification is for adult
self-renewal capacity and are capable of propagating population, it is being incorporated slowly in the practice
leukemia.4,5 Multiple genetic mutations have also been of pediatric oncology.
implicated in the pathogenesis of AML. These have been
broadly classified as type/class 1 and 2 mutations. Class
CLINICAL FEATURES AND DIAGNOSIS
1 mutations include lesions that confer a proliferative Pediatric AML can have a varied presentation. As in
and/or survival advantage by aberrant activation of other acute leukemias, patients can present with fever,
protein kinases, such as FLT3, cKIT, and RAS, whereas pallor, bleeding, hepatosplenomegaly and infections.
Chapter-40 Pediatric Acute Myeloid Leukemia 409
Children presenting with high leukocyte counts may • Other laboratory features may include electrolyte
have symptoms due to CNS or pulmonary leukostasis. abnormalities suggestive of tumor lysis (hyperuricemia,
Approximately 10 to 20 percent of AML may present with hyperkalemia, hyperphosphatemia), although less
extramedullary myeloid tumors (EMMT, granulocytic commonly than in acute lymphoblastic leukemia
sarcoma/chloroma) due to infiltration of tissues with (ALL). A small proportion of patients also may present
leukemic blasts.5 Common sites include lymph nodes, with coagulopathies—a DIC like picture may be seen
skin (leukemia cutis), gums and orbits. These are usually most commonly in acute promyelocytic leukemia
associated with t (8; 21), inv (16) or 11q23 translocation. (APML) but also in AML M4/5 and in the presence of
Central nervous system (CNS) involvement in the infection.
form of CSF involvement or CNS chloromas can occur in • The definitive diagnosis can be made by bone marrow
approximately 11 percent10 of children with AML, and is aspirate and trephine biopsy. The differential count is
more common in subtypes M4/5, and hyperleukocytosis. to be done on at 500 bone marrow cells (or 200 cells
Children with AML have a lower number of functional in peripheral blood). For the diagnosis of AML, there
neutrophils and thus may present with infections should be 20 percent or more myeloblasts in the
(including florid), even at diagnosis. peripheral blood or bone marrow. Certain types of
An initial step in diagnosis is examination of complete translocations, such as t (8;21),t(15;17) and inv 16, are
blood count and peripheral smear, which typically show also diagnostic of AML irrespective of blast percentage.9
pancytopenia with circulating blasts. Twenty percent On morphology, myeloblasts are classically described
of children may have a WBC count > 100,000/mm3 at as being large and uniform with finely dispersed
diagnosis.5 chromatin, with 1 to 4 nucleoli, granular cytoplasm
and the presence of Auer Rods in 60 to 70 percent of
Table 1 Current classification of myeloid neoplasms cases. Myelodysplastic changes may be seen in cases
(WHO 2008)9 of secondary AML.
1. AML with recurrent genetic abnormalities: • A bone marrow trephine biopsy is especially required
• AML with t (8; 21) (q22; q22), RUNX1-RUNX1T1 (CBFA/ETO). in cases wherein the BM aspirate smears are acellular
• AML with inv (16) (p13; q22) or t (16; 16) (p13; q22), CBFB- or dry tap, diluted with peripheral blood, in partially
MYH11. treated cases where blood transfusion/treatment
• Acute promyelocytic leukemia with t (15; 17) (q22; q11-12), by steroids and other drugs alter the morphological
PML-RARA. picture or in cases where the sample is degenerated.
• AML with t (9; 11) (p22; q23), MLLT3-MLL. The paraffin block is an invaluable diagnostic archival
• AML with t (6; 9) (p23; q34); DEK-NUP214. material for immunophenotyping and other studies.
• AML (megakaryoblastic) with t (1; 22) (p13; q13), RBM15- • Myeloperoxidase (MPO) stain is specific for the
MKL1.
diagnosis of AML and positivity in > 3 percent of
• AML with mutated NPM1.
blasts is considered diagnostic. Blasts of AML-M0,
• AML with mutated CEBPA.
2. AML with myelodysplasia-related features.
3. Therapy-related myeloid neoplasms. Table 2 Initial evaluation of a patient with suspected acute
4. AML, not otherwise specified: myeloid leukemia
• AML with minimal differentiation. Diagnostic tests/procedures

• AML without maturation. • Complete blood count with differential count
• AML with maturation. • Bone marrow aspirate and trephine biopsy
• Acute myelomonocytic leukemia.
• Acute monoblastic and monocytic leukemia. • Lumbar puncture
• Acute erythroid leukemia. • Immunophenotyping
• Acute megakaryoblastic leukemia. • Cytogenetics
• Acute basophilic leukemia.
• Acute panmyelosis with myelofibrosis • Molecular genetics/translocations/mutations
5. Myeloid sarcoma Additional evaluation
6. Myeloid proliferations related to Down syndrome: • Physical examination
• Transient abnormal myelopoiesis. • Syndromes/constitutional anomalies

• Myeloid leukemia associated with Down syndrome • Biochemistry, coagulation
7. Blastic plasmacytoid dendritic cell neoplasm. • Chest X-ray, echocardiography
M6 (erythroblasts), M7 (megakaryoblasts) and M5 Table 3 Risk status based on validated cytogenetic and
(monoblasts) are MPO negative. Nonspecific esterase molecular abnormalities
(NSE), alpha naphthyl butyrate (ANB) and alpha Risk status Cytogenetics Molecular abnormalities
naphthyl acetate (ANA), show diffuse cytoplasmic
Better risk Inv (16) or t (16;16), Normal cytogenetics—
activity in monoblasts and monocytes.
t (8;21) with NPM1 mutation
• Immunophenotyping utilizes various lineage-specific t 915;17 or isolated CEBPA
monoclonal antibodies that detect antigens on mutation in the absence
AML cells, and should be used at the time of initial of FLT3-ITD
diagnostic workup. The common myeloid markers Intermediate Normal cytogenetics t (8;21), inv (16),
are CD13, CD33, CD117, CD15, CD16, and MPO. For risk +8 t (16;16); with c-KIT
the diagnosis of monocytic lineage, at least 2 of the t(9;11) mutation
following are required: NSE, CD14, CD64, CD11c, and Other nondefined
lysozyme Table 2. Poor risk Complex (>= 3 Normal cytogenetics:
The basic work-up of a patient with suspected clonal chromosomal with FLT3–ITD mutation
acute myeloid leukemia is given in Table 2. abnormalities)
-5, del 5q, -7, del 7q
RISK STRATIFICATION AND PROGNOSTIC 11q23-non t (9;11)
Inv(3), t (3;3),
FACTORS t(6;9).t(9;22)
As with ALL, multiple variables have been associated Adapted from the National Comprehensive Clinical Network
with outcomes in pediatric AML. Host factors such as (NCCN) Clinical Practice Guidelines in Oncology Acute Myeloid
younger age11 and constitutional abnormalities (Down Leukaemia v 2.201328
syndrome)12,13 have been found to be favorable. Other
factors include disease related features—FAB morphology
(M3-favorable; M0 and M7-unfavorable), cytogenetics – Nucleophosfomin (NPM1) mutations: These are
and molecular markers. The presence of CNS disease has usually seen associated with normal karyotypes,
not been found to affect overall survival.10 and carry a favourable prognosis,20-22 with OS > 80
The current focus is on genetic/molecular abnor percent.
malities and Minimal residual disease. – FMS-like tyrosine kinase (FLT3)-ITD mutations:
• Cytogenetic abnormalities: Multiple recurrent There is strong evidence that these mutations
cytogenetic abnormalities are described in AML, confer a higher risk of relapse in children with
some of which have a consistent clinical and AML.23,24 The ratio of FLT3-ITD to wild type allele >
immunophenotypic profile. These are summarized in 0.4 is an independent predictor of adverse outcome
Table 3. with a PFS of <10 percent.27 Outcomes might be
• Favorable cytogenetic Abnormalities include t(8; 21), improved by HSCT.25
inv(16), and t(16;16). This group is classically described – c-KIT mutations: Usually seen in CBF leukemias.
as core binding factor (CBF) leukemias, and found to Although they confer inferior prognosis in adults,
have a favorable prognosis in many studies13-18 with their role in children is not clear.
overall survival > 90 percent. – CEBPA mutations: Like NPM mutations, they are
• Intermediate risk cytogenetic abnormalities have associated with a normal karyotype and favorable
classically included those with normal cytogenetics, outcome.26
trisomy 8 and a few abnormalities associated with Other molecular markers with adverse prognostic
11q23 translocations – t (1; 11) and t (10; 11). significance include WT1 mutations and high expression
• Poor risk cytogenetics include monosomy 5 and of BAALC.23
7, deletion 5q and 7q, and other less common • Minimal residual disease (MRD): A recent advance in
abnormalities like inv(3), t(3;3), t(6;9) and t(9;22). risk adapted approach to AML has been monitoring of
t(9;11) has been found to be favorable19 in some studies MRD, and is currently the most important predictor of
and unfavorable16,18 in others. outcome. The presence of MRD (with varying cut-offs)
• Molecular genetics: The widespread use of molecular at the end of induction has been found to consistently
markers has helped further refine the risk stratification predict a higher rate of relapse, and have been found
of AML. The commonly used mutations/abnormalities to be a better predictor of outcome compared to a risk
include: stratification schema based on FAB, cytogenetics and
Day 15 blast percentage.14,27 MRD levels at later time • Dose intensification: The classic example of dose
points do not appear to have any significance. intensification by compression of timing was
demonstrated by the CCG-2891 study36 where 4-day
TREATMENT OF PEDIATRIC AML treatment courses were separated by only 6 days
rather than the standard timing of 2 weeks or longer;
Although treatment approaches to pediatric AML have EFS in the former group was better. Similarly, in the
evolved over the past few decades, the outcomes have MRC-10 trial,37 the duration of cytarabine infusion was
not paralleled that of pediatric ALL. Improvements in prolonged to 10 days in order to intensify dose with
risk stratification (including monitoring of MRD) and improved outcomes.
supportive care, as well as intensive treatment regimens The choice of anthracycline in the treatment of AML
and the selective use of hematopoietic stem cell transplant has been a topic of debate.38-40 Certain anthracyclines
(HSCT), have brought up the 5-year event-free survival (idarubcin and mitoxantrone) are favored for their
(EFS) of children with AML to only 40 to 55 percent perceived greater antileukemic effect and/or their lower
(Table 4).29 cardiotoxicity, but no anthracycline agent has been
demonstrated to be superior.
Induction Therapy
The primary goal of induction therapy is to achieve a Postremission Therapy
significant reduction of leukemia burden, i.e. achievement Most protocols for pediatric AML (including CCG,33
of remission defined as a normal peripheral blood cell
BFM,41 POG42 and MRC37 groups) consolidate therapy
count (absolute neutrophil count >1,000/mm3 and
with a backbone of high-dose cytarabine ), although the
platelet count >100,000/mm3), normocellular marrow
timing, dose and accompanying agents vary considerably.
with less than 5 percent blasts in the marrow and no signs
Other strategies used in consolidation include continuous
or symptoms of the disease.30
delivery of multiagent low-dose chemotherapy (thioguan
Most treatment regimens use an intensive combination
ine, vincristine, cyclophosphamide, fludarabine), delivery
of anthracycline and cytarabine. The classic ‘3 + 7’ regimen
of repeated cycles of myelosuppressive therapy with
(daunorubicin 45 mg/m2 per day for 3 d; and cytarabine
or without stem cell rescue and allogeneic stem cell
100 mg/m2 per day for as continuous infusion for 7 d) was
transplantation.1 The optimum number of cycles for post-
demonstrated to induce remission in 60 to 70 percent
remission chemotherapy has yet to be determined and
of AML patients and became the standard of care for
probably depends on the therapy used for induction. In
induction therapy in the 1980s.1
the MRC AML 12 trial,39 children randomly assigned to
There have been several attempts to improve on this
receive four cycles vs. five had the same EFS and OS rates.
regimen—these include:
• Higher doses: Intensification of cytarabine dose has
been most recently studied in the AML02 trial, where Maintenance Therapy
the introduction of high-dose (18 g/m2) versus low- In the background of more intensive induction and post-
dose cytarabine (2 g/m2) did not significantly lower the remission therapies, most studies33,43 have shown no
rate of MRD-positivity after induction 1.14 Similarly, additional benefit of additional maintenance therapy in
POG 9421 study which compared different doses—high non-M3 AML. However, some groups44,45 continue to use
dose cytarabine (1 g/m2) and standard dose cytarabine either oral or parenteral maintenance chemotherapy to
in 3+7 regimen failed to show any improvement in improve outcome.
remission rates; however 3 y EFS was higher in those
who received two courses of high-dose cytarabine.31 Current Status of Stem Cell Transplantation in
Randomized trials in adults comparing high and Pediatric AML
standard doses of daunorubicin, have had conflicting
results, and there is no clear data in pediatric Although allogeneic SCT has improved outcomes in some
population. Also, dose intensification of anthracyclines subsets of pediatric AML, the role in other subsets as well
has an increased risk of cardiotoxicity, especially in as the timing, remain a controversial issue. Studies have
children. varied in recommending HSCT in first complete remission
• Addition of other agents: Various groups have tried to (CR1), 2nd complete remission (CR2) or at relapse.
add other agents in an attempt to improve outcomes, but Studies from the North American CCG trials (251, 213
there has been no convincing effect of benefit. Examples and 2891)46,47 demonstrated the superiority of allogeneic
include 6-thioguanine,32 etoposide,33 cladribine,34 SCT over autologous SCT, and also that there was no
fludarabine and gemtuzumab ozogamicin.35 benefit of HSCT in favorable risk cytogenetics. However,
European studies32,40 have not demonstrated the survival risk cytogenetics. There is some evidence of benefit for
advantage of allogeneic HSCT on outcomes as compared those with intermediate risk disease. However, outcomes
to intensive chemotherapy. This variation in outcomes at remain dismal in high risk disease, and HSCT is of unknown
different centers has led to varying recommendations. In benefit in these patients.5 The potential benefits of SCT
general, study groups in the United States recommend need to be weighed against the risk of transplant related
HSCT for a larger proportion of patients than do the mortality and morbidity. Autologous transplantation is
European groups. A meta analysis of co-operative trial not recommended in the management of pediatric AML.
groups48 (POG, CCG and MRC) indicated that HLA
matched related donor HSCT is an effective treatment of SUPPORTIVE CARE
intermediate risk AML in first CR, but that patients with
high risk AML fare poorly even with HSCT. Infectious complications remain a major cause of morbid
There is emerging data to suggest that among children ity and mortality in children with AML, both at presentation,
with high risk AML, the 5-year OS does not differ according as well as following intensive chemotherapy/SCT.50,51 Both
to donor source.49 bacterial and fungal infections can be life-threatening.
In summary, most pediatric co-operative groups agree Randomized, controlled trials which demonstrate the
that there is no role for HSCT in patients with favorable role of prophylactic antibiotics in reducing the rates of
Table 4 Result from recent pediatric AML trials (Source: Reference 2)

Study Years of Eligible age Number of CR rate* Outcome Reference
enrollment (years) patients (%)
MRC AML 10 1988–1995 ≤14 341 92 7-year EFS: 48% Stevens et al (1998)
7-year OS: 56%
LAME 89/91 1988–1996 <20 268 90 6-year EFS: 48% Perel et al (2002)
6-year OS: 56% Perel et al (2005)
TCCSG M91-13, M96-14 1991–1998 NA 192 89 5-year EFS: 54% Tomizawa et al (2007)
5-year OS: 60%
AML-BFM93 1933–1998 <18 471 82 5-year EFS: 50% Creutzig et al (2001a)
5-year OS: 58% Creutzig et al (2001b)
NOPHO-AML93 1993–2000 <18 219 91 7-year EFS: 49% Lie et al (2003)
7-year OS: 64%
POG9421 1995–1999 ≤21 565 89 3-year EFS: 36% Gale et al (2005)
3-year OS: 54%
MRC AML12 1995–2002 <16 529 92 10-year EFS: 54% Gibson et al (2011)
10-year OS: 64%
CCG2961 1996–2002 ≤21 901 88 5-year EFS: 42% Lange et al (2008)
5-year OS: 52%
AML-BFM 98 1998–2003 <18 473 88 5-year EFS: 49% Creutzig et al (2006)
5-year OS: 62% Lehrnbecher et al (2007)
AML99 2000–2002 ≤18 240 95 5-year EFS: 62% Tsukimoto et al (2009)
5-year OS: 76%
SJCRH AML02 2002–2008 ≤21 216 94 3-year EFS: 63% Rubnitz et al (2010a)
3-year OS: 71%
COG AAML0391 2003–2005 ≤21 350 87 3-year EFS: 53% Cooper et al (2012)
3-year OS: 66%
NOPHO-AML 2004 2004–2009 ≤18 151 92 3-year EFS: 57% Abrahamsson et al (2011)
3-year OS: 69%
Abbreviations: BFM: Berlin-Frankfurt-Münster study group; CCG: Children’s cancer group; LAME: Leucamie aique myeloide enfant (The
French Cooperative AML Group); MRC: Medical research council; NOPHO: Nordic society of paediatric haematology and oncology;
POG: Pediatric oncology group; SJCRH: St Jude children’s research hospital; TCCSG: Tokyo children’ cancer study group; CR: Complete
remission; EFS: Even-free survival; OS: Overall survival.
*CR rate after two courses of induction therapy.
bacterial infection in children are lacking.50 Prophylaxis • Epigenetic agents: Histone deacetylase inhibitors
with intravenous cefepime or a vancomycin regimen, and (HDAC) inhibitors
voriconazole, reduced morbidity in children with AML, HDAC inhibitors are a class of agents that modulate
and resulted in dramatic decreases in the incidence of the expression of genes by causing an increase in
septicemia and hospitalization days in a retrospective histone acetylation, thereby regulating chromatin
single center analysis.52 A current COG open-label, structure and transcription. There is increasing evide
randomized, controlled trial (ACCL0934) is designed to nce to show that HDAC inhibitors and demethylating
evaluate whether prophylactic therapy with levofloxacin agents may have a prominent role in the treatment of
will decrease the incidence of bacteremia in children AML.55 HDAC inhibitors evaluated in clinical trials in
undergoing intensive chemotherapy/HSCT. On the basis adults with AML/MDS are depsipeptide, vorinostat
of randomized, controlled trials of prophylactic antifungal and valproic acid.56 A drug which holds promise in
therapy in adults with cancer studies, many pediatric resource poor settings is sodium valproate (VPA),
oncologists recommend antifungal prophylaxis with which has recently been identified as an antitumor
voriconazole, posaconazole, micafungin, or caspofungin, agent with HDAC inhibition. Pediatric phase I studies
which have a broad-spectrum of activity and good of decitabine, valproic acid, and SAHA are underway.57
tolerability. A recent survey conducted by COG53 found that
Antibacterial and G-CSF prophylaxis reduced infection TREATMENT OF RELAPSED ACUTE
rates while mandatory hospitalization did not reduce MYELOID LEUKEMIA
infection or significantly affect nonrelapse mortality.
Acute myeloid leukemia recurs in 30 to 40 percent of
Novel Therapeutic Agents patients and is associated with a poor prognosis; less
than one-third of children with relapsed AML survive.
With outcomes remaining poor in many subtypes of AML, The length of first remission (CR1) is an important factor
there is a constant search for newer therapeutic agents. affecting both remission rates, as well as survival; those
Many new agents (Table 5) with diverse mechanisms of with CR1 < 1 year have substantially lower rates of both
action are currently being studied in relapsed/refractory than those with CR1 >1 year.58 Other prognostic factors
disease, but only a few are being used in children. following relapse include not having undergone HSCT
• Gemtuzumab ozagamicin (GO): GO is a humanized in CR1, and favorable cytogenetics (t(8;21), t(15;17),
anti-CD33 antibody conjugated to cytotoxin and inv(16).58 The standard approach is to use intensive
calicheamicin. It has been evaluated both singly,54 chemotherapy regimens to achieve remission, followed
as well as in combination with chemotherapy, (in by consolidation with allogeneic SCT. It is often necessary
previously untreated as well as relapsed/refractory to administer non-anthracycline based chemotherapy as
patients) with encouraging results.14,35 Although, it most patients are heavily pretreated. Various remission
was approved for use in AML by the US Federal Drug induction regimens, including fludarabine plus cytarabine
Administration in 2000, it was withdrawn in 2010 and mitoxantrone plus etoposide,58,59 have been used
when an interim analysis of a randomized trial [SWOG in children. The targeted immunotherapy agents such
(South Western Oncology Group) 106] in adults as gemtuzumab ozogamicin and clofarabine along with
demonstrated an increased early death rate in the GO newer chemotherapeutics as mentioned earlier in this
arm of the trial. Judicious use of GO in combination chapter have shown some activity in clinical trials.
with chemotherapy may improve clinical outcome in
selected subgroups of newly diagnosed patients and ACUTE MYELOID LEUKEMIA IN CHILDREN
may also be a useful agent in the treatment of relapsed
WITH DOWN SYNDROME
AML.
• FLT3 inhibitors: FLT3 is a trans-membrane enzyme Children with Down syndrome (DS) have a ten-fold
that promotes proliferation after activation by ligand to twenty-fold increased risk of leukemia compared to
binding and plays an important role in the survival and children without DS. The ratio of ALL to AML incidence is
proliferation of AML blasts. The signaling inhibitors similar to that in normal children, except in the first 3 years
lestaurtinib, midostaurin, quizartinib, and sorafenib of life, where AML, particularly the M7 (megakaryoblastic
are being tested in adult and pediatric trials. subtype dominate. Typically, AML in DS exhibits a
• Farnesyltransferase inhibitors (FTI): Farnesylated distinctive biology characterized by GATA1 mutations.
protein products of the Ras gene family are frequently Ten percent of neonates with Down syndrome may also
activated in MDS and AML. Two farnesyltransferase develop a transient myeloproliferative disorder (TMD),
inhibitors, tipifarnib and lonafarnib, are currently which typically improves spontaneously within the first 3
under development for the treatment of AML. months of life. Although, TMD is usually a self-resolving
Table 5 Novel therapeutic agents in the treatment of AML

Class Agent(s) Target
Deoxyadenosine analog Clofarabine Ribonucleotide reductase, DNA
polymerase, mitochondria
Tyrosine kinase inhibitor Sorafenib, quizartinib, lestaurtinib, Tyrosine kinase (e.g. FLT3-ITD)
midostaurin, sunitinib
Demethylating agent Azacitidine, decitabine DNA methyltransferase
Proteosome inhibitor Bortezomib Proteasome
Histone deacetylase inhibitor Valproic acid, vorinostat, panobinostat, Histone deacetylase
depsipeptide
Farnesyltransferase inhibitor Tipifarnib, lonafarnib Ras, lamin A
Janus kinase (JAK) inhibitor Ruxolitinib, TG101348 JAK
Chemokine receptor (CXCR4) antagonist Plerixafor CXCL12/CXCR4 axis
Apoptosis inducer Obatoclax, oblimersen BCL2
Angiogenesis inhibitor Bevacizumab Vascular endothelial growth factor
(VEGF)
Multidrug resistance inhibitor Cyclosporine P-glycoprotein
Lineage-specific antibody Anti-CD33 (mylotarg), -CD45, -CD66 Lineage-specific antigen
Immune therapy NK cells, T cells Leukemia cells
condition, it can be associated with significant morbidity t-MDS) resulting from epipodophyllotoxins and other
and may be fatal in 10 to 20 percent of affected infants. topoisomerase II inhibitors (e.g. anthracyclines) usually
DS children with leukemia frequently experience higher occur within 2 years of exposure and are commonly
levels of treatment-related toxicity, including cardiac and associated with chromosome 11q23 abnormalities
infectious complications; with an increased sensitivity to whereas t-AML following exposure to alkylating agents
cytarabine and daunorubicin. The outcome is generally or ionizing radiation often occurs 5 to 7 years later and is
favourable (EFS > 80%), especially in children aged 4 years commonly associated with monosomies or deletions of
or younger at diagnosis. Appropriate therapy for these chromosomes 5 and 7.
children is less intensive than current AML therapy, and Treatment of t-AML is challenging due to the
hematopoietic stem cell transplant is not indicated in first following due to previous treatment, and poor remission
remission. Since, this is an exhaustive topic beyond the rates resulting from adverse cytogenetics. HSCT is
scope of this chapter, interested readers may refer to these the only curative option, although rates of transplant
publications.12,13,60-62 related morbidity and mortality are high. Studies
Treatment of AML in children with Down syndrome describing treatment outcomes in t-AML have not been
is a large topic in itself, which is why we have given the encouraging.63,64
references of standard review articles on this topic.
ACUTE PROMYELOCYTIC LEUKEMIA
Therapy-related Acute Myeloid Leukemia Although acute promyelocytic leukemia (APML) was
The development of AML following treatment with conventionally classified as AML FAB M3, it is considered
ionizing radiation or chemotherapy, particularly alky to be a separate entity in view of its unique morphology,
lating agents and topoisomerase inhibitors, is termed cytogenetics and molecular characteristics. Cytogene
therapy-related AML (t-AML). The risk of t-AML/t-MDS tically, it is characterized by a balanced translocation
is related to the cumulative doses of chemotherapy between the PML gene on chromosome 15 and the RARA
agents received, as well as the dose and field of radiation gene on chromosome 17, i.e. t(15;17). This translocation
administered. High cumulative doses of either epipodo leads to the PML-RARA transcript which leads to matura
phyllotoxins (e.g. etoposide or teniposide) or alkylating tion arrest at the promyelocyte stage. This translocation
agents (e.g. mechlorethamine, melphalan, busulfan, is also a target for therapy, and the discovery of all
and cyclophosphamide) are implicated. T-AML (and transretinoic acid (ATRA, which induces differentiation of
leukemic blasts into mature granulocytes) has dramatically care measures, along with a risk-adapted approach, will
improved outcomes in APML.65 hopefully improve outcomes in children with AML.74
Acute promyelocytic leukemia in children behaves
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of a suitable donor. acute myeloid leukemia does not affect survival: a
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11. Creutziüg U, Büchner T, Sauerland MC, et al. Significance
CONCLUSION AND FUTURE DIRECTIONS of age in acute myeloid leukemia patients younger
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understanding of the biology of disease will allow more demography, biology, and outcome of acute myeloid
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existing infrastructure, as well as scrupulous supportive of 1,612 patients entered into the MRC AML 10 trial.
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C H A P T E R 41
Chronic Myeloid Leukemia
Chronic myeloid leukemia (CML) is clonal hematopoietic stem cell disorder involving the entire myeloid lineage and at least some
of the lymphoid lines. It is characterized by myeloid hyperplasia of the bone marrow, extramedullary hematopoiesis, leukocytosis
(presence of complete range of granulocyte precursors in peripheral blood) and a specific cytogenetic signature.
CML was a model disease from its discovery: the word “leukemia” (white blood) was coined to describe the neoplastic nature
of colorless corpuscles or leukocythemia seen in the blood of these patients by Virchow in 1845.1 CML usually progresses from a
chronic phase through an accelerated phase to a myeloid/lymphoid blast crisis (BC), which depicts the molecular “multi-hit” theory of
oncogenesis. CML was the first neoplasm associated with a chromosomal aberration, known as the Philadelphia chromosome (Ph).2
The elucidation of molecular pathogenesis of this disease led to development of specific therapy, making CML a model of targeted
therapy for human malignancies.
EPIDEMIOLOGY results from t(9:22) (q34;q11) balanced reciprocal trans

location. This translocation leads to juxtaposition of
CML is primarily a disease of middle age; peak incidence the ABL1 gene from chromosome 9 and BCR gene from
being in the 4th and 5th decade of life with annual chromosome 22, resulting in formation of the BCR-ABL
incidence of 1–2 cases per 100,000 population/year. In fusion oncogene that encodes a chimeric protein with a
children, CML comprises 3 percent of newly diagnosed constitutive tyrosine kinase activity (Fig. 1).1
leukemia. It is even rarer in children younger than 4 years ABL1: The wild type of ABL1 gene encodes a 145-kd
with 80 percent of cases of pediatric CML diagnosed after protein that is predominantly located in the nucleus and
4 years and 60 percent after 6 years of age.3 As per the is universally active in hematopoietic cells at all stages of
population-based registry of Mumbai, proportion of CML differentiation. The ABL1 protein participates in signal
is 5.1 percent of all newly diagnosed leukemias in females transduction and regulation of gene transcription, also
and 2.5 percent in males.4 acting as a tyrosine kinase.
There is no ethnic or genetic predisposition. Ionizing BCR: The BCR region is a part of a much larger gene
radiation is a risk factor for development of the disease with known as BCR, which encodes for a 160-kd protein.
an increased incidence in radiologists, survivors of atomic BCR-ABL1: This fusion gene encodes tumor specific
explosions and in persons exposed to therapeutic radiation. 210-kd hybrid protein that differs from normal ABL1
However, radiation and other environmental exposures kinase in following aspects:
have not been demonstrated to be causal in children. • It has higher and constitutive TK activity
• It has ability to autophosphorylate
PATHOPHYSIOLOGY • It is translocated to cytoplasm, thereby exposing it to
CML is disease of hematopoietic stem cells, having new spectrum of substrates
cytogenetic hallmark in form of Ph chromosome, which • It binds to F-actin.
Table 1 BCR-ABL1: Alternate splicing patterns

Fusion gene Region Clinical phenotype
p190 kd* Minor BCR Ph positive ALL
p210 kd Major BCR Positive CML
p230 kd μ-BCR Chronic neutrophilic leukemia
*Has 5-fold higher TK activity than p 210
hemic cell lines and in some instances lymphoid lineages.

An initial triggering event (possibly rearrangement of
C-ABL) may occur prior to the formation of BCR-ABL
Fig. 1 The Philadelphia chromosome fusion protein, which is acquired at some point in the
disease evolution. The cytogenetic changes in CML
precursor cells are expressed in its progeny as multiple
Formation of BCR-ABL1 fusion protein in CML cytologic abnormalities that confer a distinct survival
activates numerous downstream signaling pathways advantage, in turn producing a neoplastic clone (Table 2).
including RAS pathway leading to increased transcription Overtime approximately 80 percent of patients with CML
of MYC and BCL-X genes, activation of BCL-2 by abroga develop additional nonrandom cytogenetic abnormalities
tion of inhibitory effect of interferon consensus sequence- in Ph cell, i.e. “clonal evolution”, which reflects the genetic
binding protein and activation of RAC family kinases such instability and transformation to advanced-phase CML.5
as CRKL, ERK as well as c-Jun N-terminal kinase, which The most common of these are trisomy 8, isochromosome
leads to increased proliferation and survival of myeloid 17, and duplicate Ph chromosome. At the molecular level,
progenitors. most common mutations associated with CML progression
There are 3 main breakpoint cluster regions (m-bcr, are p53 (25%–30% of patients with myeloid BC) and INK4A/
M-bcr, and μ-bcr) in BCR and ABL1 contains 2 alternative ARF exon 2 (50% of cases of lymphoid BC).6
first exons (1b and 1a) with different breakpoints. The
combination of breakpoints within BCR and ABL1 genes Clinical Features
generates at least 3 different fusion transcripts encoding
proteins with distinct molecular weights (p210, p190, p230) The natural history of CML is triphasic progressing through
that are associated with different neoplastic phenotypes 3 phases, from a predominantly mature hyperproliferative
(Table 1). p190 is found to be associated with Ph+ acute phase called ‘chronic phase’ (CP) through an advanced
leukemia in almost 80 percent of pediatric patients and 50 ‘accelerated phase’ (AP) to a predominantly immature
percent of adults; and has got five times higher tyrosine ‘blast crisis’ (BC), as defined in Table 3. Approximately,
kinase activity as compared to p210 protein. 95 percent of children with CML present in CP and only
5 percent in advanced phases.7 Chronic phase usually
lasts for 3 years. Sudden onset of blastic phase, i.e.
Biology of CML
within 3 months of previously documented complete
CML is an acquired clonal disorder of unicellular hematological response can rarely occur at a rate of 0.4 to
origin, where the target of neoplastic transformation is 2.6 percent in first 3 years with interferon8 and 0.7 percent
multilineage stem cell with potential for generating all on imatinib.
Table 2 Cytologic abnormalities in CML

Abnormality Consequence
Distorted adhesive kinetics Reduced stromal/stem cell interaction and abrogation of normal cell surface signal
maturation
Discordant nucleocytoplasmic maturation Extension of late progenitor proliferative phase
Resistance to apoptosis Increased survival and accumulation
Abnormalities in feedback regulation
• Production of inhibitory molecules Suppression of normal HSC
• Insensitivity to feedback inhibition Selective growth advantage
Altered cell kinetics Prolonged survival/increased accumulation
Chapter-41 Chronic Myeloid Leukemia 421
Table 3 WHO criteria for chronic, accelerated and blast phase9

CML–CP CML–AP CML–BC
Documentation of Blasts 10–19% of peripheral blood WBC or bone marrow Blasts >20% of WBC or BM cells
t( 9,22) or BCR-ABL fusion gene (BM)
Bone marrow Peripheral blood basophils at least 20% Extramedullary blast proliferation
blast <10%
Does not meet criteria for AP Persistent thrombocytopenia unrelated to therapy Large foci/clusters of blasts in BM biopsy
or BC
Increasing spleen, or WBC count or high platelets
unresponsive to therapy
Cytogenetic evidence of clonal evolution
Megakaryocytic in sheets/clusters, with marked fibrosis,
and/or severe granulocytic dysplasia
Symptoms and Signs normocytic normochromic anemia, hyperleukocytosis

(mean WBC count of 2.5 lac/mm3 which is usually
Patients in CML-CP usually present with complains higher than in adults) with predominant neutrophilia
of fever, night sweats, fatigue, asthenia, and left upper with or without thrombocytosis (mean: 5,00,000/
quadrant pain. Complications in form of priapism, cumm). Peripheral smear shows myeloid cells in all
neurologic dysfunction or respiratory distress can be seen stages of differentiation with predominant neutrophils,
with hyperleukocytosis. increased absolute basophil and eosinophil count;
On examination, patient would usually have pallor, however, myeloblasts and promyelocytes account for
massive splenomegaly, and hepatomegaly. Papilledema, less than 15 percent of differential count. Accelerated
retinal hemorrhages, visual loss, and tachypnea can be seen phase manifests as increasing leukocyte/platelet
in patients presenting with hyperleukocytosis. The onset counts with higher proportion of blasts between 10 to
of accelerated phase is marked by progressive systemic 19 percent and more than 20 percent basophils. Blast
symptoms and increasing splenomegaly despite therapy. crisis usually manifests as pancytopenia and more
Occasionally, the first manifestation of progression could than 20 percent blasts. The characteristic biochemical
be extramedullary, i.e. meningeal leukemia or a chloroma. abnormality of the granulocytes in CML is reduced
In patients with extreme basophilia, histaminemic symp leukocyte alkaline phosphatase (LAP) score.
toms would be present in form of cold urticaria, pruritis • Bone marrow examination (Fig. 3): In CML-CP,
and gastric ulceration. Blast crisis usually presents with bone marrow is hypercellular with granulocytic and
signs and symptoms of acute leukemia. megakaryocytic hyperplasia and less than 10 percent
blasts. Accelerated phase has 10 to 19 percent blasts
Investigations and blast crisis is characterized by >20 percent blasts.
• Complete blood counts and peripheral smear (Fig. 2): Myelofibrosis may be seen in 30 to 40 percent of cases
Chronic phase CML is generally characterized by a during the course of disease.
Fig. 2 Peripheral blood picture of CML-CP Fig. 3 Bone marrow picture of CML-CP
DIFFERENTIAL DIAGNOSIS
Leukemoid reaction, juvenile myelomonocytic leukemia
(JMML) and other myeloproliferative disorders may
mimic CML.
• Leukemoid reaction usually has an obvious inflamma
tory focus, a high LAP score, a less marked splenomegaly
and absence of Ph chromosome. JMML is generally
characterized by greater involvement of skin, lymphoid
tissue and monocytic cell line as compared to CML, with
lesser leukocytosis and splenomegaly, and absence of
A B Ph chromosome, though the LAP score may be low.
• CML may be differentiated from other myelopro
Figs 4A and B Interphase FISH (A) 2 Red (BCR) and 2 green (ABL)
signals of normal nucleus; (B) Two yellow-white fusion signal liferative disorders by more pronounced involvement
corresponds to Ph chromosome of the granulocytic cells and the presence of Ph
chromosome.
• Blast crisis mostly follows chronic phase and does
not pose diagnostic dilemma. However, children who
• Cytogenetics: The “gold standard” for the diagnosis of
CML is either the demonstration of the Philadelphia initially present in BC need to be differentiated from
chromosome by conventional cytogenetic techniques, de novo acute leukemia and de novo acute Ph positive
or the demonstration of the products of the underlying leukemia. A combination of massive splenomegaly,
t(9;22) translocation, namely the BCR-ABL fusion basophilia and Ph chromosome helps distinguish BC
mRNA or the BCR-ABL protein. of CML with de novo leukemias. However, BC of CML
• Conventional cytogenetics: Conventional bone marrow and de novo Ph positive acute leukemias need to be
cytogenetics should be done for initial workup, as it not differentiated at molecular and cytogenetic level. Ph-
only confirms presence of Ph chromosome but also positive acute leukemia will produce a 190 kd protein,
detects other clonal abnormalities that may indicate while CML-BC produces a 210 kd protein and has
advanced phase CML. specific nonrandom cytogenetic aberrations.
• Fluorescence in situ hybridization technique (FISH):
FISH employs large DNA probes linked to fluorophores; Prognostic Factors
it permits direct detection of the chromosomal position
of the BCR and ABL1 genes when employed with The major predictors of survival are phase of disease
metaphase chromosome preparations. Its advantage and duration of chronic phase. Unlike adults; spleen
is that it can also be utilized on interphase cells from size, burden of white cells, platelets, basophils or blast
bone marrow or peripheral blood, in which physical co- percentage in peripheral blood have not been found to
localization of BCR and ABL probes is indicative of the be prognostically useful in children and hence prognostic
presence of the BCR-ABL fusion gene (Figs 4A and B). score like Sokal, Hasford or EUTOS are not applicable in
• Reverse transcription polymerase chain reaction children. Early response to imatinib has been found to be
(RT-PCR): Reverse-transcription polymerase chain prognostic in recent studies.
reaction (RT-PCR) is a highly sensitive technique that
employs specific primers to amplify a DNA fragment Treatment (Fig. 5)
from BCR-ABL mRNA transcripts. Depending upon the
combination of primers used, the method can detect The therapy for CML is has evolved significantly over
the e1a2, e13a2 (b2a2), e14a2 (b3a2) and e19a2 fusion time from earlier (Phase-1) palliative approaches such
genes. The use of nested primers and sequential PCR as arsenic, splenic RT, busulphan and hydroxyurea (late
reactions makes the technique extremely sensitive, 1860s–1970) to phase-II of aggressive nontargeted curative
capable of routine detection of one Ph positive cell approaches such as allogenic stem cell transplant,
in 105 to 106 normal cells. Quantitative RT-PCR is interferon with or without cytarabine (1970s–2000) finally
recommended before starting therapy as well as for to the current phase of targeted molecular therapies such
monitoring response to therapy. as imatinib, dasatinib, and other tyrosine kinase inhibitors.
preferentially expanded in CML. In addition, it also has an

immunomodulatory action.10,11 In the scant pediatric data
available, nearly 58 percent pediatric patients treated with
interferon achieved complete hematological response,
50 percent achieved major cytogenetic response and 14
percent showed complete cytogenetic responses, with an
overall survival of 60 percent at 8 years.12,13 In one study
combination of IFN and AraC was found to be superior to
IFN alone.14
Tyrosine Kinase Inhibitors (TKI)

The unique disease biology of CML paved way for the
development of first molecular targeted therapy in human
cancers in the form of imatinib mesylate which has changed
the entire outlook towards management of CML, receiving
an accelerated FDA approval for pediatric use in 2003.
Imatinib, a small 2-aminopyridine molecule, competitively
inhibits the inactive configuration of the BCR-ABL protein
tyrosine kinase by blocking the ATP binding site and
Fig. 5 Evolution of treatment for CML thereby preventing a conformational change to the active
form. It inhibits cellular proliferation without inducing
apoptosis, producing a 92 to 98 percent decrease in CML
Medical Management colony growth in vitro without inhibiting normal colony
growth. Imatinib also inhibits platelet-derived growth factor
The initial goal of therapy in CML is to reduce the
(PDGFR) and c-kit, but not the SRC family kinases.15,16 The
leukocytosis and organomegaly. Early agents like busu
second generation TKIs inhibit both BCR-ABL and other
lphan and hydroxyurea did achieve this goal but could
signaling pathways. Dasatinib and bosutunib inhibit both
not achieve cytogenetic remission. Interferon was the first
BCR-ABL and SRC kinases. Nilotinib, like imatinib, is an
drug to achieve a significant cytogenetic remission. Since
inhibitor of BCR-ABL, c-kit, and PDGFR. Both nilotinib and
its introduction, tyrosine kinase inhibitors have become
dasatinib are >100-fold more potent than imatinib in vitro.
the frontline therapy and helped achieve rapid and
durable complete cytogenetic and molecular remissions.
Dose of TKI
Hydroxyurea The recommended standard dose of imatinib in children
is 260 to 340 mg/m2 (maximum absolute dose 400 mg/
It is an inhibitor of ribonucleoside diphosphate reductase,
day) which gives drug exposures similar to the 400 to
preventing conversion of ribonucleotides to deoxyribo
600 mg adult dosage levels. The recommended pediatric
nucleotides and interferes with DNA synthesis during
dose for CML-AP is 400 mg/m2 daily (maximum absolute
the S phase. Recommended starting dose is 25 to 50 mg/
dose, 600 mg) and for CML-BC is 500 mg/m2 daily (maximum
kg, which is further adjusted according to hematological
absolute dose, 800 mg). The anticipated decrease in the
response.
white cell count may be observed not earlier than 2 weeks
after the start of treatment and complete hematological
Interferon (IFN) response is usually achieved after a median of 4 weeks.
In 1980s, interferon with or without cytarabine (AraC) Pediatric experience with second generation TKI is
constituted standard management of patient awaiting limited though dasatinib is being evaluated in pediatric
transplant or not eligible for transplant. IFN exerts anti phase II trial (NCT00777036) with a dose of 60 mg/m2
proliferative effect on myeloid precursors, in parti daily for CML-CP and 80 mg/m2 daily for more advanced
cular on those in the late progenitor phase which is phases. Dosage recommendations for children concerning
nilotinib cannot yet be made. The dose approved for cytogenetic and molecular responses as defined in Table 5.
treating adults is 300 to 400 mg twice daily. Table 4 details The response achieved in relation to time, i.e. milestones
the starting dose and administration guidelines for TKI. is used to grade response as optimal, suboptimal and
failure, providing basis for timely alteration of therapy as
Response Rates and Response detailed in Table 6.17,18
The pediatric TKI treatment results are comparable
Assessment to TKI to the results achieved in adults. Data from pediatric
Effectiveness of therapy with TKI is assessed based on the trials have shown that with use of imatinib in CML-CP,
achievement of landmark responses, i.e. hematologic, 96 percent achieve CHR and 69 percent achieve CCyR after
Table 4 Starting doses and instruction for administration of TKI25

TKI Dose (mg/m )2
Instructions
Imatinib 340, OD To be dispersed in water or apple juice, 50 mL for 100 mg tablet. Take with water or food to avoid
esophageal irritation
Dasatinib 60–80, OD Dissolve in 30 mL lemonade/preservative-free apple or orange juice
Nilotinib* 170–230, BD Capsules may be dispersed in 5 mL of apple juice/sauce and ingested immediately on an empty
stomach, abstain from eating for at least 1 hour
* Pediatric dosing has not been established but is based on 300 mg or 400 mg twice daily, if less than or greater than 40 kg,
respectively.
Table 5 Landmark responses to treatment17,18

Hematological response (CBC and Clinical) Cytogenetic response (Ph+metaphases) Molecular response (BCR-ABL transcripts)
Complete: Complete: 0% Complete: Transcripts not detectable,
1. WBC <10 ×103/L Partial: 1–35% or >3-log reduction in transcript from
2. Platelets <450×109/L Minor 36–65% diagnosis
3. Differential with no immature Minimal 66–95% Major: <0.1%
granulocyte and <5% basophils
4. Spleen not palpable
Table 6 Milestones of response in CML-CP17,18 with therapeutic option

Time (Months) Optimal Suboptimal Failure
Diagnosis NA NA NA
3 CHR and minor CyR CHR and no CyR No CHR
6 Partial CyR Minor/Minimal CyR No CyR
12 Complete CyR Partial CyR Less than partial CyR
18 MMoIR <MMoIR <Complete CyR
Anytime Stable or improving MMoIR Loss of MMolR, BCR-ABL kinase Loss of CHR, CCyR, new
domain mutations still sensitive chromosome abnormalities in
to imatinib, >0.05% increase in presence of Ph+, BCR-ABL kinase
transcript levels domain mutations insensitive
to TKI
Management Continue same with monitoring Increase dose of imatinib Introduce second generation TKI
Consideration13-15 Introduce second generation TKI Consider HSCT
Table 7 Response rates to imatinib in pediatric CML trials

Study Dose CHR (%) CCyR (%) MMR (%) OS
At months (m) At months (m) At months (m)
COG-phase I (N = 20)19 260–570 100 83 NA 78.5% (24 m)
European phase II (N = 30) 26
260–340 80^ 60^ 50 95^ (12 m)
75* 29* 0* 75* (12 m)
French national (N = 44)20+ 260 98 62 (12 m) 31 (12 m) 98 (36 m)
AAML0123 (N = 50) 23+
340 78 91 (9 m) 98 (12 m)
CML-PAED II ( N = 51) 21+
300** 95 93 (12 m) 85 (18 m) –
I-CML-Ped (N = 150)27+ – – 63 (12 m) awaited 97 (42 m)
^ : In CP, *: advanced phase, ** : 400 for AP, 500 for BC
+ : Patients treated upfront with imatinib
1 year 19-23 as depicted in Table 7. Imatinib is more effective Resistance

in CML-CP with overall survival of 95 percent at 1 year as
It is divided into two categories:
compared to 75 percent in advanced phases.26
1. Primary resistance: It is defined as failure to achieve a
Dasision and ENESTnd Trial in treatment naïve CML
timely response (Table 6). It has been seen in 10 to 20
adults have demonstrated a significantly faster as well as
percent of CML-CP pediatric trials of imatinib. It could
deeper responses and better transformation free survival
be due to inadequate inhibition of tyrosine kinase or
with dasatinib and nilotinib as compared to imatinib,
BCR-ABL mutations.
however, not showing a difference in the PFS and OS at 3
2. Secondary resistance: Loss of a previously achieved
and 4 years respectively.28-31 There is very scant data and
response constitutes secondary resistance. It occurs
little experience with second generation TKI in pediatric
in 80, 50, and 15 percent of patients in blast crisis,
population, with two phase 1 trials confirming effectiveness
accelerated phase, or chronic phase, respectively
of dasatinib in pediatric age group.24,33 In the COG phase
after 2 years of imatinib therapy. It is due to reactiva
1 trial in the dose range of 60 to 110 mg/m2 once daily,
tion of BCR-ABL signaling (mutation of BCR-ABL,
dasatinib could achieve CCyR in 3 and partial CyR in 3 of
imatinib excretion, overexpression of BCR-ABL)
8 evaluable patients with resistant CML-CP.24 In another
or activation of other signaling pathways including
CA180-018 phase I study of 17 children with resistant CML-
SRC kinases.
CP, 14 (82%) achieved complete cytogenetic response
(CCyR) and eight (47%) achieved major molecular res
Managing Resistance
ponse. Of 17 patients with advanced-phase CML or
Ph-positive ALL, six (35%) achieved CHR and 11 (65%) Once resistance is suspected, the disease status should
achieved CCyR.33 be re-evaluated. Compliance and tolerance to therapy
should be confirmed in patients suspected to have
resistance, as none of the patients with compliance ≤80
Intolerance and Resistance to TKI percent to imatinib could achieve a MMR in past studies.
Up to one-fourth of patients either may not tolerate or may The trough plasma imatinib levels should be obtained
show resistance to TKI. since level <1000 ng/mL is associated with higher rate of
progression of disease. In addition, mutational analysis
Intolerance of BCR-ABL should be performed. Primary resistance
due to inadequate inhibition of tyrosine kinase can be
A patient is considered to be intolerant to therapy overcome by increasing the dose of imatinib. Resistance
when a nonhematologic toxicity of at least grade three due to mutation can be overcome by changing the TKI
recurs despite appropriate dose reductions and optimal known to be active for the particular mutation keeping
symptomatic management. In general <5 percent of the side effect profile and comorbidities in the child in
patients are intolerant to imatinib therapy. mind.
Toxicity of TKI Allogenic Stem Cell Transplant (Allo-SCT)

Imatinib is generally well tolerated in children. Table 8 Prior to imatinib, allogenic stem cell transplant was the
enlists the major acute side effects with TKI as well as their treatment of choice for CML and was usually performed
management. Generally, the acute side effects of TKI tend during the early chronic phase since outcome of Allo-
to be manageable and decrease over time. SCT in BC is dismal with less than 20 percent long-term
However, the developing organs and potentially survival.32 Allo-SCT is the only known curative treatment
prolonged use of TKI, known to act on off target kinases available for children with CML. In most studies of
involved in normal function of organs, in pediatric pediatric patients, the survival ranges between 60 to 80
patients makes them more particularly vulnerable to percent with better results in matched sibling donors
long-term side effects of TKI, many of which may not compared to voluntary unrelated donors. However, owing
be apparent for years (Table 9). Among these, most to the complications of high early mortality, growth failure,
concerning long-term side effects are growth retardation, infertility, graft versus host disease, metabolic syndrome,
impact on bone health, and cardiac dysfunction. Growth and secondary malignancies associated witth Allo-SCT,
delay has been demonstrated in many recent studies due it has become a form of rescue treatment for patients in
to disturbance in GH:IGF-1 axis. Similarly, altered bone advanced CML (AP/BC), treatment failure after receiving
mineralization has been shown in few studies. The cardiac second generation TKI, and patients with T315I tyrosine
dysfunction is still a topic of debate, however, definite in- kinase mutation (till proven role of ponatinib).
vitro evidence definitely warrants more evaluation. Thus, However, in developing countries, where one time
TKI administration in children requires close monitoring cost of transplant is significantly lesser as compared
of side effects.25 Hence, the risk and benefits of TKI to potentially lifelong therapy with TKI and good
therapy should be weighed against the alternative options contemporary outcome and potential cure with matched
available, including allogeneic SCT. sibling donor transplant, it remains an attractive option in
Table 8 Major acute side effects of TKI25

TKI side effects Recommended intervention
Hematologic toxicity: Hold TKI until count recovery for up to 2 weeks; G-CSF may be administered to treat neutropenia;
Neutropenia, anemia, or restart at full dose if cytopenia persists < 2 weeks; reduce dose by 20% if longer than 2 weeks
thrombocytopenia
Rash Observation; consider topical steroids
Elevated liver function tests Observation, avoid ibuprofen
Muscle cramps Check CK and electrolytes, consider electrolyte repletion
Nausea, vomiting Supportive care, consider ondansetron
Headache Supportive care
Table 9 Chronic side effects of TKI25

TKI side effects Recommended intervention
Cardiac toxicity Consider ECG, echocardiogram, troponin, electrolytes, if there is clinical concern
Imatinib: Possible, not proven Do not use nilotinib with history of cardiac or electrolyte problems
Dasatinib: Possible, not proven
Nilotinib: QT prolongation
and sudden death have
been reported
Effusions Hold TKI; if multiple sites of edema, give diuretics; and if severe, thoracocentesis and brief
Dasatinib: Pleural effusions course of steroids
Decreased height/growth retardation Closely monitor height, GH stimulation tests and IGF-1 levels
Poor bone health Closely monitor calcium and phosphorus; vitamin D repletion as necessary
Teratogen Avoid TKIs during pregnancy
Table 10 Laboratory monitoring on TKI therapy

Test Recommendation
Bone marrow cytogenetics/FISH At diagnosis to establish disease phase and additional mutations, if any
Every 3 months till complete CyR
Rising levels of BCR-ABL(1 log) without MMR
Quantitative RT-PCR (peripheral blood) At diagnosis to determine baseline transcript type and levels
Every 3 month after cytogenetic CR till MMR, then 6–12 monthly
Rising levels of BCR-ABL (1 log) with MMR then 1–3 monthly
TKD mutations Failure to reach (suboptimal response) or maintain CHR, CCyR, or MMR; rise in quantitative
PCR after MMR; cytogenetic relapse or increase in Ph chromosomes, if CCyR not obtained
CML-CP in presence of a matched sibling donor. Recent Flow chart 1 Management of CML-CP in children
studies have shown promising early results with reduced
intensity conditioning transplants in children with CML
with significantly lower long-term morbidity. Also,
preliminary data using imatinib for cytoreduction pre-
transplant has demonstrated a significantly lower risk of
death attributable to the lower disease burden at the time
of transplant making it an attractive strategy. Also, imatinib
has shown to be effective in relapse post-transplant as well
as maintenance post-transplant, with most centers using
1 year of imatinib post-transplant to reduce the risk of
disease relapse.
Guidelines for Management of

CML in Children
Chronic Phase CML (Flow chart 1)
Initiate treatment with hydration, hydroxyurea, allopu
rinol and start imatinib at 340 mg/m2 per day after
confirmation of diagnosis. Monitor disease status through
CBC, differential count and peripheral blood FISH every 3
months till achievement of cytogenetic remission followed
by quantitative RQ-PCR every 3 month till MMR, then 6
to 12 monthly (Table 10). If child fails to achieve optimal
response (Table 6), check for compliance, imatinib levels,
BCR-ABL mutations and switch to dasatinib 60 mg/m2 per
day if none or sensitive mutations are present and initiate
screening for related and unrelated allogeneic stem cell
donors. If second line-TKI is not affordable/available, or
child is noncompliant with low-serum levels consider
increasing the dose of imatinib and follow for response. In
children with resistant mutations (T315I), or progression
or relapse on dasatinib, allo-SCT should be done with the
myeloablative allogeneic SCT if sibling donor is available
best available donor as soon as possible.
after achievement of remission. If patient fails or has
a suboptimal response at any time (Table 6), proceed
Accelerated Phase CML (Flow chart 2) immediately to myeloablative allogeneic SCT with best
Start imatinib 400 mg/m2 or dasatinib at 80 mg/m2 per available donor. These patients may benefit from post-
day in 2 divided doses (preferred). Initiate search for transplant maintenance with TKI in view of high risk of
HLA-matched sibling or unrelated donors and proceed to relapse.
Flow chart 2 Management of CML-AP in children REFERENCES

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C H A P T E R 42
Juvenile Myelomonocytic Leukemia
Juvenile myelomonocytic leukemia (JMML) is a clonal panmyelopathy and a unique kind of chronic myeloproliferative disorder seen
almost exclusively in infants and young children. It is characterized by myeloid proliferation, especially in the monocytic lineage
and occurs due to well-characterized molecular defects at the genetic level which directly affect granulocyte macrophage-colony
stimulating factor (GM-CSF) invoked downstream signaling pathways involved in cellular proliferation—especially the RAS pathways.
This results in a progressive organ infiltration of the spleen, liver and finally lungs in a progressive, relentless march to fatality if
untreated. Allogeneic Hematopoietic Stem Cell Transplantation (Allo-HSCT) is the only known cure resulting in long-term remission.
The unique leukemogenesis model of JMML has provided in depth understanding of cancer cytogentics and offers a wide array of
potential targeted therapies that may have applications across several malignancies.
Juvenile myelomonocytic leukemia (JMML) is a clonal leading to much confusion over its classification and its
panmyelopathy, which presents with leukocytosis, spleno persistent identification as the “ph negative” chronic
megaly and organ infiltration due to excessive proliferation myeloid disorder. Subsequently, the finding of elevated
of cells of the monocytic and granulocytic lineages. It is one HbF levels,6 and the identification of monosomy 7 as a
of those enigmatic diseases of childhood that has evaded unique chromosomal anomaly in many infants and young
categorization and generated considerable confusion over children with recalcitrant myeloid proliferations and acute
its classification over the years until recently. It was first leukemias7 led to further disorders being broadly clubbed
broadly categorized under “myeloproliferative” disorders, under this entity.
a term coined by Dameshek in 1951 when he observed that After the French-American-British (FAB) group had
different chronic proliferative disorders sharing similar successfully classified MDS in adults, there was a move
clinical and hematologic features often developed a more to extend the same for the pediatric entity and called
aggressive form over the course of their illness,1 and has it chronic myelomonocytic leukemia (CMML) instead
jumped categories till its molecular basis has been better of jCML. In 1997, Niemeyer reported one of the largest
understood in recent times. series to date and described in painstaking of CMML in
childhood, jCML and the infant monosomy 7 syndrome
highlighting their common biological features.8 Later
HISTORY AND CLASSIFICATION
leading an international group she proposed the term
Earlier termed a chronic myeloproliferative disorder, it was JMML to include all these entities and established its
only after the discovery of the Philadelphia chromosome diagnostic criteria.9 The 2001 classification system of the
by Nowell and Hungerford in 19602 which characterized World Health Organization (WHO) grouped these into a
adult-type chronic myeloid leukemia (CML), that it was separate category of myelodysplastic/myeloproliferative
shown that this unique anomaly was absent in children disorders as it reflects both elements, i.e. dysplastic and
and infants with a similar clinical picture.3 Hardisty in proliferative features of the myeloid cells.10 With the recent
1964 had coined the term “juvenile CML” (jCML), which identification of certain genetic mutations in JMML,
stayed in popular parlance for several decades since,4,5 a revised set of diagnostic criteria was proposed by the
Chapter-42 Juvenile Myelomonocytic Leukemia 431
International JMML Working Group in 2006.11 The existing higher risk of developing JMML.8,19 While about 11 percent
criteria and the proposed ones are given in Tables 1 and 2 of patients with a clinical diagnosis of NF1 develop JMML,
respectively. another 15 percent of JMML patients without a clinical
diagnosis of NF1 have mutations in the NF1 gene.14,20,21
EPIDEMIOLOGY A few cases of JMML have been associated with Noonan
syndrome (NS), which is diagnosed in about 2 percent of
From the few population-based studies, it can be derived JMML patients registered. In all such cases a heterozygous
that JMML represents about 2 to 3 percent of leukemias PTPN11 mutation, a gene that encodes the nonreceptor
in children, and has an annual incidence of 1.2/million protein tyrosine phosphatase SHP-2 was observed.22
children per year.12,13 JMML predominates in infants
and toddlers with a median age at diagnosis of 1.8 years,
and close to 85 percent of all cases are diagnosed from 4
PATHOPHYSIOLOGY AND GENETICS
months to 6 years of age. A male preponderance is seen As previously noted, JMML came to be defined among the
through all age groups at with a sex ratio of about 2:1.8 Few group of disorders primarily identified by the absence of
reports exist of a familial occurrence in twins pairs and the Philadelphia chromosome. In contrast monosomy
other siblings.14,15 7 was identified in about 25 percent of patients, other
JMML is closely associated with neurofibromatosis abnormalities in 10 percent and a normal karyotype
type 1 (NF1).16-19 These patients have a 200- to 350-fold in 65 percent.8,14,23 When monosomy 7 is present, it is
generally the sole abnormality. Among the chromosomal
Table 1 Current JMML diagnostic criteria (2nd International abnormalities other than monosomy 7, loss of material on
JMML Working Group11) the long arm of chromosome 7 is the most frequent.8
While JMML involves the myeloid, erythroid and
All of the following At least 2 of the following
megakaryocytic lineages and transforms into myeloid
Absence of the t(9;22) BCR/ Circulating myeloid precursors leukemias, clonal involvement of the B-lymphoid lineage
ABL fusion gene is also seen, including lymphoid blast crisis.24,25 It may
Absolute monocyte count White blood cell be gathered that malignant transformation takes place
>1000/micL > 10,000/micL at a stage of a committed stem cell that has the ability
<20% blasts in the bone Elevated fetal hemoglobin for myeloid, as well as early B-lymphoid differentiation.
marrow (HbF) Involvement of T-lymphoid precursors has only been
GM-CSF hypersensitivity reported in one child with JMML and T-cell lymphoma.26
Molecular Aspects
Table 2 Proposed criteria of the 2nd International Molecular pathways in JMML have been extensively
JMML Working Group11 studied and still draw interest as they serve as models
Category 1 Category 2 Category 3 for studying leukemogenesis. Several molecular events
All of the following At least 1 of the At least 2 of the that activate RAS dependent signaling pathways and
following following deregulate growth and survival of leukemic cells, have
Splenomegaly Somatic Circulating myeloid been described in JMML27,28 (Fig. 1), primarily including
mutations in RAS precursors mutations in the genes encoding RAS, NF1 and SHP-2. In
or PTPN11 addition Casitas B Lymphoma (CBL) is also increasingly
Absolute monocyte Clinical diagnosis WBC >10,000/μL
recognized as a causative gene.29 Figure 1 also highlights
count >1000/μL of NF1 or NF1 the targets of JMML therapy covered later in this text.
gene mutation
Blasts in PB/BM Monosomy 7 Elevated fetal ONCOGENIC RAS MUTATIONS
<20% hemoglobin (HbF) A hypersensitivity of JMML cells to GM-CSF has been
for age
well-established and was used as a diagnostic test for
Absence of the Clonal cytogenetic its confirmation long before the molecular pathways
t(9;22) BCR/ABL abnormalities responsible became apparent. A specific defect in the
fusion gene excluding GM-CSF signal transduction pathway was postulated
monosomy 7
to drive the pathogenesis of this disease. However, no
Age less than 13 abnormalities in the GM-CSF receptor (GM-CSFR)
years could be found, but a host of abnormalities in the
Fig. 1 Molecular activation pathways in JMML and the potential therapeutic targets28
downstream pathways were soon identified in the RAS/ Table 3 Summary of gene mutations in JMML28
MAPK pathway.27 The RAS family of signaling proteins
Gene Sites of mutation Frequency
regulate cellular proliferation by cycling between an active
guanosine triphosphate (GTP)-bound state (RAS-GTP) PTPN11 E76K, D61Y, D61V, E69K, 35%
and an inactive guanosine diphosphate (RAS-GDP)-bound A72T, A72V, E76V/G/A
state. Mutant RAS alleles encode proteins that accumulate RAS 25%
in the GTP-bound conformation because of defective NRAS Codons 12 and 13
GPT hydrolysis. Common sites of these mutations along KRAS Codon 13
with other candidate genes are summarized in Table 3.27,30 HRAS No mutation in codons 12,
13, and 61 was found
Oncogenic point mutations of NRAS and KRAS are seen in
15 to 20 percent of children with JMML.27,30 NF1 Loss of wild-type NF1 11–15%

Other genetic pathways include NF1 in neurofibro allele
matosis and PTPN11 in Noonan syndrome. A group CBL Codons 371, 380, 381, 384, 17%
of GTPase- activating proteins (GAPs) facilitate the 396, 398, 404, and 408.
conversion from active RAS-GTP to the inactive RAS GDP Splice sites 1227, 1228,
state. Neurofibromin, the protein encoded by the gene for and 1096
NF1, functions as GAP and negatively regulates RAS.27,28,30
As described above, 25 to 30 percent of JMML cases carry
the clinical diagnosis of NF114 or are known to harbor gene (TSG) that functions by negatively regulating RAS
NF1 gene mutations.20,21 Extensive studies in the field signaling.
by Shannon and co-workers who demonstrated loss of
heterozygosity (LOH) by loss of the normal NF1 allele from Noonan syndrome (NS): It is a heterogeneous disorder
the bone marrow of children with type 1 neurofibromatosis defined by short stature, facial dysmorphia, cardiac defects
and malignant myeloid disorders,31 were instrumental in (most commonly pulmonic stenosis and hypertrophic
proving that NF1 works as a myeloid tumor suppressor cardiomyopathy), skeletal defects, mental retardation
and bleeding diathesis, in which JMML is an occasional of NF1.17 Abdominal distension and discomfort are
association, NS is caused by germline mutations in generally due to hepatosplenomegaly. Gut infiltrates may
PTPN11, the gene encoding the nonreceptor protein predispose to diarrhea and gastrointestinal infections.
tyrosine phosphatase (PTP) SHP-2, 130 a member of a small Unlike acute monoblastic leukemia, JMML rarely involves
subfamily of cytoplasmic src-homology 2 (SH-2) domain the central nervous system (CNS). A small number of
containing PTPases.32 It is required for hematopoietic cell patients with CNS chloroma8 and with ocular infiltrates37
development and participates in signal transduction of have been described. Pituitary infiltration and diabetes
a number of cytokines, including GM-CSF and IL-3.30,32 insipidus, responsive to antileukemic therapy, has also
Binding of the SH2 domain to phosphorylated tyrosine been described.38 In addition, clinical features of NF1
residues induces a conformational shift that relieves the with multiple cafe au lait spots, and the characteristic
inhibitory interaction between the SH-2 domain and the dysmorphisms of NS may be seen in 11 percent and 7
catalytic PTP domain. Heterozygous germline mutations percent of JMML cases respectively.8
of PTPN11 are present in children with NS and JMML.22
PTPN11 mutations also represent a major molecular event HEMATOLOGICAL AND
in nonsyndromic JMML. About 35 percent of patients with LABORATORY FEATURES
JMML harbor somatic mutations in PTPN11.28,30
The Casitas B-cell lymphoma (CBL, c-CBL) protein, is • The hematologic profile is characterized by leuko
also now increasingly recognized to have a causative role cytosis, anemia and thrombocytopenia. Unlike CML,
in JMML.29 First reported by Loh, et al. c-CBL mutations the median white blood cell (WBC) count is 33 × 109/L
have been detected in up to 17 percent of cases.29,30 The and rarely exceeds 100 × 109/L.12,25,47 Rarely the counts
germ line mutation represents the first hit, with somatic may be less than 10 × 109/L, especially in children with
loss of heterozygosity being the second hit positively monosomy 7.8,14
selected in JMML cells. Individuals with germ line CBL • Both mature and immature myeloid lineage cells are
mutations are at increased risk of developing JMML, seen along with a characteristic monocytosis, often
which might follow an aggressive clinical course or resolve with dysplastic cell forms. An absolute monocyte
without treatment.33,34 count of more than 1 × 109/L has been retained as a
diagnostic criteria (Table 2).9,11
CLINICAL FEATURES • Occasionally, eosinophilia and basophilia may be
seen.
The typical presentation is that of an infant with • The median blast cell percentage in PB smears is less
progressive pallor, fever, infection, petechiae, cough and than 2 percent8 and rarely exceeds 20 percent.
progressive abdominal distention due to splenomegaly • In about 14 percent of children, the platelet count at
and hepatomegaly.8 Occasionally, the spleen may be diagnosis is below 20 × 109/L.
normal at diagnosis, but will rapidly increase thereafter. • Most patients have a hemoglobin level between 7
However, most patients presenting in the author’s and 11 g/dL. The reticulocyte count and the number
experience have had large spleens with varying degree of of normoblasts vary over a wide range. Red cells are
hypersplenism (personal experience, unpublished data). generally normocytic, while macrocytosis is noted in
Lymphadenopathy is fairly common too, a feature which some patients with monosomy 7. Microcytosis may be
distinguishes it from CML.8,23 The tonsils may be markedly due to iron deficiency, but can often be noted even in
enlarged due to infiltration. Dry cough, tachypnea the absence of laboratory detected deficiency.8
and interstitial infiltrates on chest X-ray are signs of Bone marrow (BM) aspirate shows increased
peribronchial and interstitial pulmonary infiltrates. cellularity with predominance of granulocytic cells in
Patients with advanced disease frequently have cachexia. all stages of maturation. Monocytosis in BM is usually
Skin is usually involved by eczematous eruptions or less than in PB, with a median of 10 percent cells.8 The
erythematous maculopapules on the face, trunk, and BM blast count is moderately elevated, but is more than
hands.23,35 Indurated raised lesions with central clearing,35 10 percent in only 10 percent of patients.8,10 Dysplasia of
and petechiae may also be seen. granulocytes is usually minimal, with hypogranulation
In addition to these often nonspecific lesions, juvenile of neutrophil cytoplasm and pseudo-Pelger-Huët
xanthogranulomas composed of numerous foamy cells forms.10 Besides macrocytic differentiation in a few
may be seen in JMML. They are present by the end of cases, erythroid cells mature normally. Megakaryocytes
the second year of life and are often multiple.36 In some are reduced or absent in about 75 percent of children.8
but not all children, xanthogranulomas are associated Cytochemical and immunophenotypic studies are
with multiple cafe au lait spots and the clinical diagnosis not specific, but might be helpful in identifying the
monocytic population.10 Because smears of PB and These include fusing of diphtheria toxin to GM-CSF,45 the
BM provide sufficient information, BM biopsy may use of GM-CSF receptor antagonist E21R,46 and inhibition
be omitted in most cases. Reticulin fibrosis has been of production of GM-CSF, TNF-α and IL-1β by IL-10.47
noted in biopsies of some patients.8,39 “Spontaneous” growth of JMML myeloid progenitors in
• HbF synthesis is increased especially in those with vitro can also be inhibited by 13-cis or all-trans retinoic
a normal karyotype resulting from a high number acid48-50 possibly due to their antagonistic effect of retinoic
of circulating F cells. In addition, other fetal red cell acid on the transcription factor AP-1, which is activated by
characteristics, such as increased expression of the I Jun/Fos oncoproteins shown to be upregulated in JMML.51
antigen and decreased carbonic anhydrase levels, are Interferon-α (IFN-α)52 and farnesyltransferase inhibitors
present.40 Despite these changes, maturation of red (FTIs)53 have also been shown to inhibit colony formation.
cells does not seem to be compromised.
• While clinical features of patients with monosomy Natural Course and Prognostic Factors
7 and those with a normal karyotype are similar,8
hematological differences are often seen. Monosomy 7 The JMML is a relentlessly aggressive disorder and
patients have a lower median WBC but similar absolute uniformly fatal in untreated patients. Allogeneic hema
monocyte count, red blood cells are often macrocytic, topoietic stem cell transplantation (HSCT) offers the
and erythropoiesis in BM is more pronounced. In only hope of a permanent cure. Thrombocytopenia, age
addition, they have a normal or only moderately above 2 years and high HbF at diagnosis predict a poor
elevated HbF, which is often elevated in patients with outcome.8,14 A scoring system was devised in which HbF
normal karyotype.8 of 10 percent or higher and a platelet count of 33 × 109/L
• Immunological abnormalities are frequently seen or less had an adverse impact on outcome.14 JMML rarely
in JMML. Serum IgG, IgM and IgA levels are often transforms to a blastic stage and infiltration of the lungs
increased in a polyclonal fashion. Autoantibodies, such leading to respiratory failure is the usual cause of death in
as antinuclear antibodies, antibodies against red cells progressive disease. Watchful observation may be of use in
causing a positive Coomb’s test and anti-thyroglobulin cases of Noonan’s syndrome as some of them are known
antibodies may be present.8 to spontaneously remit.
DIFFERENTIAL DIAGNOSIS MANAGEMENT OF JMML

Several viruses have been implicated in creating a clinical It has now long been recognized that Allo-HSCT offers the
and hematological picture resembling JMML. These only real chance of cure in JMML. However, historically
include cytomegalovirus (CMV), Epstein-Barr virus in the developed countries and in the current reality
(EBV), human herpesvirus 6 (HHV-6) and parvovirus of developing countries, few have reached the stage of
B19.41-43 Concomitant viral infections must therefore transplant. In the absence of this possibility, either from
always be carefully excluded, especially in those with a lack of donor availability or socioeconomic constraints,
normal karyotype or when a molecular analysis has not a host of alternative options have long been practiced. In
been feasible. Other disorders like leukocyte adhesion addition there is now a growing list of molecular directed
deficiency (LAD) variant and some metabolic disorders therapies as the JMML model of leukemogenesis has
can also mimic JMML. Strict adherence to the diagnostic become increasingly well-defined and newer targets
criteria can usually avoid misdiagnosis. identified.27,28,30 Comparative evaluation of the efficacy of
these different approaches has been hampered to a large
Hematopoiesis in Cell Culture Studies extent by the lack of adequate response criteria. This was
addressed to an extent by the 2nd International JMML
When JMML cells are cultured in semisolid media, an Working Group which evolved the new response criteria
increased number of monocyte-macrophage colonies reflected in Table 4.11
are formed even in the absence of added growth factors.44
This hypersensitivity of JMML cells to GM-CSF, since its
Low-dose Conventional Chemotherapy
first identification, has become the hallmark of the disease
and an essential element of its diagnosis.9,11,44 The shift to Historically 6-mercaptopurine (6-MP) has been used
left in colony assays of JMML cells in absence of GM-CSF either as a single agent54 or in combination with low-
stimulation, as compared to controls is characteristic. dose cytarabine or etoposide,54,55 and was shown to be of
GM-CSF appears to be obligatory for survival of JMML benefit. In a later update in 2006 of the original 110 cases
cells. This has led to several novel approaches in blocking reported by Neimeyer, sixty-three patients who did not
colony formation of JMML cells by different strategies. receive transplant were analyzed and all the above agents
Table 4 Response criteria of the 2nd International of hypersplenism. Its role is largely limited currently
JMML Working Group11 to symptomatic relief or to tackle hypersplenism due
to massive spleens or prior to Allo-HSCT to accelerate
Complete clinical Partial clinical
hematologic recovery and reduce the risk of hemorrhagic
response response
complications post-transplant. 8,54,56
White blood cell <20000/μL <50% of initial WBC
count count but total
still greater than Experimental Therapeutic Approaches
20000/micL The recent focus in JMML has been on designing targeted
Splenomegaly Normalization of 25% decrease from drug therapy to the many recognized molecular defects.
spleen size initial size Most of these therapies have been tested and developed
on mouse models, while some have undergone early phase
human trials. Since RAS hyperactivation remains a crucial
used either singly or in combination in a maintenance element in the pathogenesis, suppression of its activity has
like therapy showed some efficacy in producing partial been one of the prime targets, and various methods have
response or stable disease three months from start of been used to achieve this end (Fig. 1).
therapy. However, the variability in response evaluation • One such method has been targeting RAF1—a MAP
made it unreliable and the setting up of uniform criteria kinase which functions downstream of the RAS
was the main thrust of this paper.56 This came to fruition subfamily of proteins and plays an important role in
with the efforts of the 2nd International JMML Working the signal transduction. A DNA enzyme designed to
Group recommendations in 2009.11 At best, this therapy specifically cleave mRNA for RAF1, has been found
may be used currently only in the palliative setting. to be very specific for JMML cell lines while sparing
normal marrow cells, which indicates a high level of
Intensive Chemotherapy safety.64 Another means of achieving the same purpose
is the use of a RAF1 inhibitor. BAY 43-9006—a low
This has usually involved AML type protocols and has been
molecular-weight agent binds at the active site of the
far more controversial. A few small series have reported
RAF1 kinase65 and has undergone further trials.11
benefit,57,58 with one CCG study reported remission in seven
• Another target has been stopping RAS activation at
of 12 patients who received intensive chemotherapy.59 This
the cell membrane level where addition of a farnesyl
success has allowed some groups to use a combination of
group to the newly translated protein is one of the first
intensive cytoreduction with cis-retinoic acid as a bridge to
steps of RAS activation. Farnesyltransferase inhibitors
Allo-HSCT. However, these results have not been widely
(FTIs) are able to prevent RAS translocation to the
replicated and many other studies have reported prolonged
plasma membrane, leading to downregulation of RAS-
aplasia which is often fatal.60,61 In addition, there may be no
activated cellular pathways.53 L-744,832 is one such
difference in outcome between those who receive intensive
FTI, which can abolish the in vitro growth of myeloid
therapy and those who do not, as in the absence of Allo-
progenitor colonies in response to GM-CSF.66 Other
HSCT, both groups do poorly with overall survival (OS) of 6
FTIs too, have reported some in vitro activity, however,
percent at 10 years.
they have shown only modest to little activity in clinical
trials when used as a single agent.
Other Measures • SHP-2 phosphatase has presented a very tempting
In vitro sensitivity of JMML cells to IFN-α led to its initial target for inhibition due to the direct correlation
use in JMML.52 However, apart from isolated case reports, between the driving mutation of PTPN11 and JMML,
no benefit has ever been proven. A POG study using an much like the tyrosine kinase inhibition of imatinib
IFN-α dose of 30,000 units/m2 was stopped for excessive in CML with a direct driving gene at the bcr-abl locus.
toxicity.62 None of the evaluable patients had either a However in JMML, this has been plagued with issues
partial or complete response. A similar basis was found related to the highly selective SHP-2 inhibition required
to justify the use of 13-cis retinoic acid (isotretinoin).48,49 due to a shared homology in catalytic pathways with
However, the earlier promise was not borne out a phase II SHP-1 which has a negative role in cytokine signaling67,68
POG study 63 or by other investigators.54 resulting in neutralized end results of SHP-2 inhibition.
The role of splenectomy has remained undecided. It is The key role of GM-CSF stimulation and proliferation
often offered to reduce respiratory distress or abdominal of JMML cell lines has also led to interest in the role of
discomfort due to massive spleen size, and sometimes GM-CSF antagonists for its cure, which has already been
also to reduce transfusion requirements in the presence covered in this text elsewhere.
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60. Festa RS, Shende A, Lanzkowsky P. Juvenile chronic 74. Urban C, Schwinger W, Slavc I, et al. Busulfan/
myelocytic leukemia: experience with intensive combina cyclophosphamide plus bone marrow transplantation is
tion chemotherapy. Med Pediatr Oncol. 1990;18:311-6. not sufficient to eradicate the malignant clone in juvenile
61. Hasle H, Kerndrup G, Yssing M, et al. Intensive chronic myelogenous leukemia. Bone Marrow Transplant.
chemotherapy in childhood myelodysplastic syndrome. 1990;5:353-6.
A comparison with results in acute myeloid leukemia. 75. Sanders JE, Buckner CD, Thomas ED, et al. Allogeneic
Leukemia. 1996;10:1269-73. marrow transplantation for children with juvenile chronic
62. Maybee D, Dubowy R, Krischer J, et al. Unusual toxicity myelogenous leukemia. Blood. 1988;71:1144-6.
of high dose alpha interferon (aIFN) in the treatment of 76. Donadieu J, Stephan JL, Blanche S, et al. Treatment of
juvenile chronic myelogenous leukemia (JCML) [abstract]. juvenile chronic myelomonocytic leukemia by allogeneic
Proc Am Soc Clin Oncol. 1992;1:950a. bone marrow transplantation. Bone Marrow Transplant.
63. Castelberry RP, Chang M, Maybee D, et al. A phase II 1994;13:777-82.
study of 13-cis retinoic acid in juvenile myelomonocytic 77. Locatelli F, Noellke P, Zecca M, et al. Hematopoietic stem
leukemia [abstract]. Blood. 1997;90:346a. cell transplantation (HSCT) in children with juvenile
64. Iversen PO, Emanuel PD, Sioud M. Targeting Raf-1 gene myelomonocytic leukemia (JMML): results of the EWOG
expression by a DNA enzyme inhibits juvenile myelomono MDS/ EBMT trial. Blood. 2005;105:410-9.
cytic leukemia cell growth. Blood. 2002;99(11):4147-53. 78. Yoshimi A, Locatelli F, Trebo M, et al. Treatment for
65. Thompson N, Lyons J. Recent progress in targeting the Raf/ patients relapsing with juvenile myelomonocytic leukemia
MEK/ERK pathway with inhibitors in cancer drug discovery. after allogeneic stem cell transplantation: the EWOG-MDS
Current Opinion in Pharmacology. 2005;5(4):350-6. study. Leuk Res. 2005;(Suppl 1):516 [abstract].
C H A P T E R 43
Pediatric Hodgkin Lymphoma
Hodgkin lymphoma (formerly called Hodgkin’s disease) is a malignant lymphoma, first described by Hodgkin, in 1832 as some morbid
appearances of the absorbent glands and spleen that accounts for approximately 5 to 7 percent of childhood cancers and 1 percent
of all deaths.1 Hodgkin lymphoma (HL) is characterized by a progressive painless enlargement of lymph nodes and defined by
specific histopathological features. Pediatric HL is one of the few pediatric malignancies that shares aspects of its biology and natural
history with its adult counterpart. The odyssey of treatment in HL, which began with radiotherapy and then got revolutionized with
multiagent chemotherapy, has continued to grow better in terms of cure rates. With the currently available treatment modalities
(multiagent chemotherapy either alone or in conjunction with low-dose involved-field radiation therapy) and the use of risk-adapted
therapy, over 90 percent of children diagnosed with HL are long-term survivors. Currently, management designed to achieve maximal
cure rates with the fewest late-effects of therapy continues to be the paradigm for pediatric oncologists across the world.
EPIDEMIOLOGY appears to decrease the risk of Hodgkin lymphoma,

most likely by maturation of cellular immunity.2
Variation in the incidence, age, and gender distribution of 2. Young adult form (Ages 15–34 years): This is associated
HL occurs in different populations according to geographic
with a higher socioeconomic status in industrialized
location, socioeconomic status, and immunologic status.
countries, increased number of siblings, and earlier
In Industrialized countries, HL presents with a bimodal
birth order. Delayed EBV infection, particularly when
distribution with regard to age, with a rise in incidence
associated with infectious mononucleosis, is a risk
in young adults (20–34 years) and in the elderly (55–74
factor for the young adult form. It has been proposed
years).2 In contrast, in low-income countries, there is a
that delayed exposure to a common infectious agent
trimodal distribution. There is an inverse relationship
may also play a role in EBV-negative young-adult
between the incidence of the HL in children and young
adults within countries according to their economic cases, although the identity of the agent has not been
development.3 Such patterns of occurrence being similar established.6
to Epstein-Barr virus (EBV), tuberculosis and poliomyelitis 3. Older adult form: Most commonly presents in indi
infections; the role of an environmental exposure has been viduals aged 55 to 70 years.
suggested as a possible etiology of HL. As many as 20 to In India, lymphomas are the second most common
30 percent of childhood HL cases in developing countries malignancies in children ahead of CNS tumors especially in
occur before 5 years of age4,5 against some 5 percent in males unlike west where brain tumors are more common.
industrialized countries. Overall, there are three distinct Importantly, HL exceeds non-Hodgkin lymphoma in India
forms of Hodgkin lymphoma: in contrast to west due to significantly higher incidence of
1. Childhood form (Ages 14 years and younger): This HL in males in India. The age standardized incidence of
type increases in prevalence in association with larger HL in Indian children ranges from 8.2 to 19.6 per million
family size and lower socioeconomic status. Early children per year compared to 5.7 in USA and 6.4 in
exposure to common infections in early childhood Britain. Furthermore, mixed cellularity is most common
phenotype in India (likely related to early childhood EBV node contains a variable cellular infiltrate consisting of
exposure) leading to much younger age peak (median lymphocytes, eosinophils, macrophages, plasma cells,
age 8–9 years) compared to 16–30 years in west where and fibroblasts. These infiltrating cells secrete an array of
nodular sclerosis is most common.7 Pediatric HL shows a cytokines and chemokines, which are important for HRS
significant male predominance in low-income countries cell survival and maintenance of the characteristic cellular
including India (male : female ratios being 2.5:1 to 8:1) infiltrate. These malignant cells have three distinct origins;
compared to ratio of about 1.5:1 in west.8,9 in nodular lymphocyte predominant HL, the tumors cells
(L&H) derive from germinal centre (GC) or postgerminal
ETIOLOGY centre B-cells and retain expression of all B-cell specific
antigens. In classical HL, HRS cells are GC B-cells but have
The etiology of Hodgkin lymphoma is believed to be
crippling mutations that destroy the coding capacity of
multifactorial and may include the following;
their functional IgV gene rearrangements. In a minority
• Infectious agents: Several studies have documented
(2%) of classical HL cases, HRS cells display a cytotoxic
a link between Hodgkin lymphoma and EBV. EBV
T-lymphoid phenotype.
DNA can be identified in tumor cells in approximately
In EBV-positive Hodgkin lymphoma, EBV-encoding
50 percent of patients in the United States as well
genes play a role in preventing apoptosis. Latent membrane
as Western Europe and in more than 90 percent of
protein-1 (LMP-1) expressed in EBV-positive HRS cells
patients in developing countries.10 EBV positivity is
mimics an activated CD40 receptor, activating the anti-
most commonly observed in tumors with mixed-
apoptotic nuclear factor–kappa-B (NF-κB) pathway. A
cellularity histology and is almost never seen in
paracrine activation of NF-κB in Hodgkin lymphoma is
patients with lymphocyte-predominant histology. EBV
observed; both HRS cells and the surrounding supporting
positivity is more common in children younger than
cells produce cytokines that upregulate several members
10 years compared with adolescents and young adults.
of the TNF receptor superfamily, including CD30, CD40, or
Patients with a prior history of serologically confirmed
EBV latent membrane protein-1 (LMP-1). The production
infectious mononucleosis have a four-fold increased
of the ligand for these receptors is responsible for the
risk of developing EBV-positive HL but are not at
phosphorylation and translocation to the nucleus of
increased risk for EBV-negative HL.11
NF-κB. The constitutive translocation of NF-κB to
• Genetic predisposition: Clustering in families suggests
the nucleus of HRS cells is essential for the malignant
a genetic predisposition, with an increased incidence
transformation of HRS cells. It leads to inhibition of
observed among same-gender siblings, monozygotic
apoptosis, proliferation, and secretion of proinflammatory
twins, and parent-child pairs. Familial Hodgkin
cytokines.12
lymphoma has been associated with specific human
leukocyte antigens. Familial cases account for 4.5
percent of all cases. PATHOLOGIC CLASSIFICATION
• Immune dysregulation: The increased susceptibility to The current World Health Organization classification
HL in patients with T-cell immunodeficiency, human classifies HL in two broad types based on both morphologic
immunodeficiency virus (HIV) infection, or congenital
appearance as well as immunophenotypic characterization
immunodeficiency syndromes suggest a role for
including type of neoplastic cells, inflammatory mileu and
immune dysregulation in its development.
overall growth pattern as detailed here.13
• Environment: Clustering of cases in families or racial
groups supports the idea of a common environmental
link. At present, no conclusive association is recognized Classical Hodgkin Lymphoma
with common environmental factors other than EBV The hallmark of classic HL is the Reed-Sternberg cells
infection. (R-S) cells and their mononuclear (Hodgkin cells) and
multinucleate variants which lack the immunophenotypic
BIOLOGY evidence of B-cell differentiation. R-S cells almost always
HL is a B-lineage lymphoma. The malignant cells of HL are express CD30, and approximately 70 percent of patients
clonal Hodgkin/Reed-Sternberg (HRS) cells or lymphocytic express CD15. CD20 is expressed in approximately 6 to
and histiocytic (L&H) cells or their morphologic variants, 10 percent of cases, and generally RS cells do not express
which usually constitute less than 1 percent of the cells B-cell antigens such as CD45, CD19, and CD79A.
in involved lymph nodes. Characteristic RS cells are The classical HL is subclassified into four subtypes
binucleate or multinucleate giant cells with prominent according to the number of R-S cells, characteristics of
nucleoli and abundant cytoplasm. The rest of the lymph the inflammatory milieu, and the presence or absence of
Chapter-43 Pediatric Hodgkin Lymphoma 441
Table 1 Histopathological classification of classical Hodgkin lymphoma

REAL subgroups Distinctive features Relative frequency (%)
Lymphocyte rich (LR) Benign appearing lymphocytes with or without histiocytes. Few Reed- 10–15
Sternberg (RS) cells. No fibrosis
Nodular sclerosis (NS) Thickened capsule with proliferation of orderly collagenous bands that 20–50
divide lymphoid tissue in nodules: Lacunar variant of RS cells
Mixed cellularity (MC) 5–15 RS cells per high power field. Fine fibrosis in interstitium. Focal 20–40
necrosis may be present
Lymphocyte depletion (LD) Abnormal cells with relative paucity of lymphocytes. Fibrosis and necro- 5–16
sis common but diffuse
fibrosis. The histologic features and clinical symptoms Hepatosplenomegaly

of HL have been attributed to the numerous cytokines
Hepatic and/or splenic enlargement may be present in
secreted by the R-S cells, which include interleukin-1,
patients with advanced stage HL. Overall, children are
interleukin-6, and tumor necrosis factor. Classical HL
more likely than adults to present with stage I/II disease
subtypes are detailed in the Table 1.
and less likely to present with stage IV disease.
Nodular Lymphocyte—Predominant Hodgkin Mediastinal Mass

Lymphoma
Unlike adolescents and young adults, only few young
This pathologic class of Hodgkin lymphoma is children with HL have mediastinal disease at presentation
characterized by large cells with multilobed nuclei, (approximately 75 vs 33 percent, respectively), in part
referred to as popcorn cells. These cells express B-cell reflecting the tendency of these patients to have mixed
antigens, such as CD19, CD20, CD22, and CD79A, and are cellularity histology. Mediastinal masses are almost always
negative for CD15 and may or may not express CD30. The present in association with low cervical or supraclavicular
OCT-2 and BOB.1 oncogenes are both expressed unlike adenopathy. Such bulky mediastinal disease may cause
classical HL. Nodular lymphocyte-predominant Hodgkin dysphagia, dyspnea, cough, stridor and the superior vena
lymphoma (NLPHL) is most common in males younger cava syndrome.
than 10 years and constitutes 5 to 10 percent of all HL
cases. Patients with NLPHL generally present with slow
Systemic Symptoms
growing, localized, non-bulky disease usually in axilla and
inguinal regions without any B symptoms. Patients with HL may present with nonspecific systemic
symptoms including fatigue, anorexia, and weight
loss. Fewer than 20 percent of children with HL have B
PRESENTING SYMPTOMS AND SIGNS
symptoms which include unexplained persistent fever
Presenting symptoms and signs of HL in children include (above 38°C or 100.4°F), drenching night sweats and weight
lymphadenopathy, systemic symptoms, and mediastinal loss (more than 10 percent of body weight) in the previous
mass. HL almost always presents at a site above the six months. These symptoms have important implications
diaphragm, with only 3 percent of cases presenting in a for staging and prognosis. As in adults, pruritus, which
purely subdiaphragmatic location.14 typically resolves with treatment, has been described.
Rarely, patients present with autoimmune disorders such
Lymphadenopathy as autoimmune hemolytic anemia, thrombocytopenia, or
neutropenia.15–18
Most common presenting sign is painless lymphadenop-
athy. Approximately, 80 percent of young children pre-
sent with cervical lymphadenopathy. The affected lymph
DIFFERENTIAL DIAGNOSIS
nodes typically feel rubbery and more firm than inflam- The presenting symptoms and signs of HL in children and
matory adenopathy; they may be sensitive to palpation, if adolescents may be caused by a variety of diseases and the
they have grown rapidly. differential diagnosis includes other malignant, infectious,
and inflammatory diseases. In India, mycobacterial history of immune deficiency should undergo a detailed
infections are the most common differential diagnosis. immunologic evaluation.
Others include EBV infection, Non-Hodgkin lymphoma,
metastatic adenopathy from other primary tumors (e.g. IMAGING STUDIES
nasopharyngeal carcinoma, soft tissue sarcoma), toxo
plasmosis, systemic lupus erythematosus, and other The goal of imaging is to define the accurate extent and
disorders causing reactive hyperplasia of lymph nodes.19 stage of the disease. The following studies should be
obtained:
DIAGNOSTIC EVALUATION
Anatomical Imaging
A complete evaluation of patients with suspected HL is
mandatory before beginning treatment. The goal is to CT of neck, thorax, abdomen, and pelvis (with and without
confirm the diagnosis, stage the disease, document other intravenous contrast) should be obtained. Establishment
prognostic factors, and to evaluate organ function that of lymphomatous involvement on CT-scan is complicated
may influence the selection of therapy. by great variability of normal nodal size with body region
and age as well as the frequent occurrence of reactive
hyperplasia. However, contiguous nodal clustering or
ESTABLISHING THE DIAGNOSIS matting, focal mass lesion in a visceral organ, size on long
After a careful physiological and radiographical evaluation axis of 2 cm or greater or between 1 cm and 2 cm with
of the patient, the least invasive procedure should be used other suggestive clinical features should be considered
to establish the diagnosis of lymphoma by biopsy of one significant. Currently, definitions of bulky disease are not
or more peripheral lymph nodes. Fine-needle aspiration uniform and often depend on the clinical protocol with
cytology alone is not recommended because of the lack bulky peripheral (non-mediastinal) lymphadenopathy
of stromal tissue, the small number of cells present in varying from aggregate nodal masses exceeding 4 to 6 cm
the specimen, and the difficulty of classifying Hodgkin and bulky mediastinal mass as a transverse mediastinal
lymphoma into one of the subtypes. An image-guided diameter over one-third of the maximum intrathoracic
biopsy may be used to obtain diagnostic tissue from intra- diameter on an upright posterior-anterior (PA) chest
radiograph. However, the Cotswolds modification of the
thoracic or intra-abdominal lymph nodes. Patients with
Ann-Arbor classification has defined lymph nodes more
large mediastinal masses are at risk of cardiac or respiratory
than 10 cm in greatest dimension on CT imaging as bulky.
arrest during general anesthesia or heavy sedation. In
these patients, peripheral lymph node biopsy or image-
guided core-needle biopsy of mediastinal lymph nodes Combined Anatomical and Metabolic Imaging
may be feasible using light sedation and local anesthesia PET-CT, which integrates functional and anatomic tumor
before proceeding to more invasive procedures. Supine characteristics, is being increasingly used for staging and
position should be avoided and procedures should be monitoring of pediatric patients with HL. In PET scan,
done with the patient on his or her side or prone. If airway uptake of the radioactive glucose analog, 18-fluoro-2-
compromise precludes biopsy, immediate treatment deoxyglucose (FDG) correlates with proliferative activity
with steroids or localized radiation therapy should be in tumors undergoing anaerobic glycolysis and adds to the
considered and biopsy performed as soon as feasible anatomical information from CT scan. In recent studies,
preferably within 48 hours. PET findings resulted in a change in staging in more than
50 percent of patients and subsequent adjustments in
involved-field radiation therapy treatment volumes in 70
LABORATORY STUDIES
percent of patients. Concordance between PET and CT is
Hematological and chemical blood parameters show generally high for nodal regions, but lower for extranodal
nonspecific changes that may correlate with disease sites such as spleen, lung nodules, bone/bone marrow, and
extent. Abnormalities of peripheral blood counts may pleural and pericardial effusions. Generally, a suspected
include neutrophilic leukocytosis, lymphopenia, eosino anatomic lesion which is PET-negative should not be
philia, and monocytosis. Acute-phase reactants such as considered involved unless proven by biopsy and areas
the erythrocyte sedimentation rate and C-reactive protein, of PET positivity that do not correspond to an anatomic
if abnormal at diagnosis, may be useful in follow-up lesion should be disregarded in staging. PET scan is
evaluation. Patients with history of recurrent infections, more accurate in detecting viable HL in post-therapy
autoimmune and inflammatory disorders, or a family residual masses. Residual or persistent FDG avidity has
Table 2 Cotswold’s revision of Ann Arbor staging classification

Stage Definitions
I Involvement of a single lymph node (LN) region (I) or of a single extranodal organ or site (IE)
II Involvement of two or more LN regions, on the same side of the diaphragm (II) or localized involvement of an
extralymphatic organ or site and one or more LN region on the same side of the diaphragm (IIE)
III Involvement of LN regions on both sides of the diaphragm (III), which may be accompanied by involvement of the
spleen (III S) or by localized involvement of an extralymphatic organ (III E) or both (IIISE)
IV Noncontiguous involvement of one or more extralymphatic site with or without LN involvement
Annotations Definitions
A No B symptoms
B At least one of the following within the last 6 months:
a. Weight loss >10%
b. Unexplained persistent or recurrent fever
c. Drenching night sweats
X Bulky disease (>6 cm in diameter or mass >1/3 of mediastinal) diameter
E Extension to a single extralymphatic organ adjacent to a known involved site
been correlated with prognosis. Rapid early response pediatric HL. Different clinical trial groups have established
documented by significant reduction in disease volume various risk categorizations as outlined in Table 3. In
and PET negativity at an early stage (after one or two cycles general, these incorporate stage of disease, disease bulk, and
of chemotherapy) is associated with a favorable outcome. systemic (B) symptoms.23-26
PET scanning should be performed at baseline and a not
earlier than 3 weeks postchemotherapy completion and 8 TREATMENT
to 12 weeks post-radiation.20–22
The treatment of Hodgkin lymphoma (HL) in children
Staging and Prognostic Factors requires a careful balance between providing enough
therapy to eradicate the tumor and avoiding unnecessary
Current Hodgkin’s lymphoma staging is based on treatment that could result in excessive long-term
Cotswold’s modification of Ann Arbor staging proposed treatment-related side effects. Hence, focus is on
in 1998 (Table 2). The assessment to determine stage of maximizing treatment efficacy and minimizing risks for
disease involves scrupulous history for B symptoms and late toxicity associated with both RT and chemotherapy.
laboratory studies including a blood count, LDH, ESR, and The treatment paradigm for childhood HL is risk,
liver function tests apart from imaging studies detailed gender and response adapted use of non-crossresistant
here. Bone marrow biopsy is indicated in those with combination chemotherapy with or without low-dose
advanced disease (stage III/IV), B symptoms, elevated involved-field radiation therapy. This approach is
alkaline phosphatase or hematologic abnormalities. supported by information obtained from clinical trials
Recently, with the advent of PET-CT scan, which is highly and meta-analyses of randomized trials evaluating the
sensitive for bone marrow involvement, bone marrow influence of radiation field size, dose and the role of
biopsy is considered mandatory only in ambiguous cases. chemotherapy in children with HL.
Sites of bulk disease, disease infiltration into extranodal
tissues, presence of B-symptoms, ESR, hemoglobin, WBC Combined Modality Therapy in Children
count, and gender should be documented.
with Hodgkin Lymphoma
These prognostic factors may be able to identify a
group of patients with an extremely low risk of relapse for A number of randomized clinical trials have investigated
whom therapy may be minimized. Because the treatment the benefit of adding chemotherapy to radiation therapy
of HL has improved and fewer patients relapse after initial in the treatment of HL. While the radiation therapy acts to
therapy, many previously reported clinical factors have lost control known sites of tumor, the chemotherapy is aimed
their prognostic significance. Also, these factors may be at occult disease outside the radiation field. The judicious
interrelated in the sense that disease stage, bulk, and biologic combination of the two modalities allows for a decrease
aggressiveness are frequently codependent. Consequently, in the dose and size of the radiation field used and a
no uniform system of prognostic strati fication exists in reduction in the intensity or duration of chemotherapy
Table 3 Prognostic factors and risk stratification in pediatric Hodgkin lymphoma

Study group/Trial Low risk Intermediate risk High risk
Children’s Oncology Group • IA/IIA no bulk or extranodal extension • IA bulk or “E” extension • IIIB, IVB
• IB
• IIA bulk or “E” extension
• IIB
• IIIA
• IVA
German studies/Euronet PHL • IA/B • IIB • IIEB
• IIA • IIEA • IIIEA/B
• IIB • IIIB
• IV
St Jude/Stanford/Dana Farber† • IA/IIA no bulk • IA bulk
• IB
• IIA bulk
• IIB
• III
• IV
Children’s Cancer Group 5942 • IA/B patients no adverse features* • IA/B patients with adverse features* • IV
• IIA patients no adverse features* • IIA patients with adverse features*
• IIB
• IIIA/B
*Adverse factors include hilar lymphadenopathy, >4 sites of nodal disease, or bulky disease.
†
Patients categorized as favorable or unfavorable risk.
below toxicity thresholds that would not be possible if contrast to adult HL, pediatric chemotherapy approaches
single modality chemotherapy or RT were used, thus have focused on 2 unique strategies.
decreasing overall acute and late toxicities. The use of RT
in pediatric HL permits reduction in dose-related toxicity Avoidance of Late Toxicity
of anthracyclines, alkylating agents, and bleomycin that
may preserve cardiopulmonary as well as gonadal function Non-crossresistant regimen like ABVD has demonstrated
and reduce the risk of secondary leukemia. The results of superior efficacy (i.e. freedom from progression) and less
prospective and controlled randomized trials indicate that toxicity when compared with MOPP in adults with Hodgkin
combined modality therapy, compared with chemotherapy lymphoma.28 However, due to cardiac and pulmonary
alone, produces a superior event-free survival (EFS). Thus, toxicity of ABVD in children, many investigators have
combined modality treatment approach has become the evaluated regimens either devoid of anthracyclines,
preferred initial therapy for children with HL.27 alkylators and/or bleomycin such as VAMP (St Jude), VBVP
(French MDH-90 study) or hybrid regimens that utilize
Chemotherapy Regimens in Children with lower total cumulative doses of alkylators, doxorubicin,
and bleomycin such as COPP/ABV (CCG) or ABVE-PC
Hodgkin Lymphoma (POG). VAMP like less-toxic regimens can be safely used in
Contemporary chemotherapy regimens in HL combine favorable risk HL but not in intermediate or high-risk HL.29
non-crossresistant agents wherein each agent is indi
vidually active against tumor but targets different
Gender-Adapted Chemotherapy
cellular events to prevent drug-resistance and have non-
overlapping toxicities which allow delivery of each agent In an effort to decrease risk for male infertility, etoposide
at full dose which are detailed in Table 4. All of the agents has been substituted for procarbazine in the initial
in original MOPP and ABVD regimens continue to be used courses of therapy in studies of the German pediatric HL
in contemporary pediatric treatment regimens. However, group (OPEA) and Pediatric Oncology Group (DBVE and
COPP with less leukemogenic and gonadotoxic potential DBVE-PC)30 and dacarbazine (COPDAC) has been used to
(substituting cyclophosphamide for mechlorethamine) replace procarbazine (COPP) with preservation of efficacy
has replaced MOPP as the preferred alkylator regimen. In and minimization of infertility.31
Table 4 Contemporary chemotherapy regimens for children with Hodgkin lymphoma

Name Drugs
COPP Cyclophosphamide, vincristine (oncovin), procarbazine, prednisone
COPDAC Dacarbazine substituted for procarbazine in COPP
OPPA Vincristine (Oncovin), procarbazine, prednisone, doxorubicin (Adriamycin)
OEPA Vincristine (Oncovin), etoposide, prednisone, doxorubicin (Adriamycin)
ABVD Doxorubicin (Adriamycin), bleomycin, vinblastine, dacarbazine
COPP/ABV Cyclophosphamide, vincristine (Oncovin), procarbazine, prednisone, doxorubicin (Adriamycin), bleomycin, vinblastine
VAMP Vinblastine, doxorubicin (Adriamycin), methotrexate, prednisone
DBVE Doxorubicin, bleomycin, vincristine (Oncovin), etoposide
ABVE-PC Doxorubicin (Adriamycin), bleomycin, vincristine (Oncovin), etoposide, prednisone
cyclophosphamide
BEACOPP Bleomycin, etoposide, doxorubicin (Adriamycin), cyclophosphamide, vincristine (Oncovin), prednisone, procarbazine
Radiotherapy regression with chemotherapy and avoids extensive

inclusion of uninvolved regions. With contemporary
Consolidative radiation therapy (RT) after risk-adapted low-dose RT, treatment of contralateral uninvolved sites
chemotherapy is an integral part of treatment of children is not necessary in most children. Currently, targeted
with HL.27 Radiation has been used as an adjunct to RT, which entails restricting RT to areas of initial bulky
multiagent chemotherapy in intermediate/high-risk pedi disease (generally defined as ≥5 cm at the time of disease
atric HL with the goal of reducing risk of relapse in initially presentation) or post-chemotherapy residual disease
involved sites and preventing toxicity associated with (generally defined as ≥2.5 cm or residual PET-avidity),
second-line therapy. Compared with chemotherapy alone, and involved-nodal RT, which treats only the initially
adjuvant radiation produces superior EFS for children involved nodes with a margin (typically 2 cm), are under
with intermediate/high-risk HL who achieve a complete investigation.
remission (CR) to multiagent chemotherapy, but it does
not affect overall survival (OS) because of the success of
second-line therapy.29,30 Since, adjuvant radiation therapy Radiation Therapy Technique
may be associated with excess late effects or mortality; While CT-based two-dimensional RT remains the standard
there has been a movement to decrease the field of technique for radiation delivery, three-dimensional
radiation as well as dose of radiation therapy in order to conformal RT (3D CRT) or intensity-modulated radiation
limit toxicities while maintaining survival rates. therapy (IMRT) are often used in situations where
the more conformal techniques would reduce dose to
Radiation Dose surrounding normal critical structures. Proton therapy is
The dose of radiation is variously defined and often currently being investigated and may further decrease the
protocol specific. However, doses of 15 to 25 Gy are mean dose to the surrounding normal tissue compared
typically used with modifications based on patient age, the with IMRT or 3D CRT.
presence of bulky or residual (postchemotherapy) disease,
and normal tissue concerns.37 Some protocols prescribe Response-Adapted Therapy
a boost of 5 Gy in regions with suboptimal response to
chemotherapy. Response to therapy is one of the most robust prognostic
factors in HL as in many other pediatric tumors. The concept
of tailoring the extent as well as dose of radiotherapy and
Radiation Therapy Volume
duration of chemotherapy based on response to therapy
Involved field radiation therapy (IFRT) is the current in HL is the focus of many recent and future trials.
standard of care in children in place of total nodal RT. With regards to avoiding radiotherapy in patients with
The IFRT treats the clinically involved region(s), with early stage favorable risk HL, finding from three recent
coverage of the whole nodal region but accounts for tumor studies32,33 (COG-9542, GPOH HD-95 and Euronet PHL
C-1) have shown that RT can be safely avoided in patients given in an effort to reduce breast cancer risk. Rapid early
who achieve CR after initial chemotherapy. However, responding boys received two cycles of ABVD followed
omission of RT was found to be detrimental in intermediate by IFRT. Slow early responders received four additional
and high-risk subgroups except in a recent North American courses of BEACOPP and IFRT. RER was achieved by 74
study (COG AHOD 0031) which showed that RT can be percent of patients after four BEACOPP cycles and 5-year
avoided in rapid early responders in intermediate risk HL.34 EFS among the cohort was 94 percent.36
In advanced HL, a recent POG study (P9425) has
shown that in rapid early responders to a dose dense RECOMMENDATIONS FOR TREATMENT OF
chemotherapy, further chemotherapy can be safely PEDIATRIC HODGKIN LYMPHOMA
curtailed. In this study, 216 children with intermediate or
high-risk HL received ABVE-PC every 21 days. Rapid early Treatment of Low-risk Classical Hodgkin
responders (RER, 63% of patients) to 3 cycles received 21 Lymphoma
Gy RT to involved regions. Slow early responders received The preferred treatment option for early stage, favorable
two additional cycles before 21 Gy radiation.35 Five-year prognosis HL (stages I–IIA; no bulky disease; no B
EFS was 86 percent for the RER and 83 percent for the symptoms, less than four sites of disease) is combined
slow early responders (P = 0.85). Five-year OS was 95 modality treatment including hybrid chemotherapy with
percent. Cumulative doses of alkylators, anthracyclines, less or no alkylators and anthracyclines (VAMP/VBVP/
and epipodophyllotoxins were below thresholds usually OPPA/OPEA or equivalent) for two to four cycles with
associated with significant long-term toxicity. or without low-dose IFRT of 15 to 25 Gy. IFRT can be
Similarly, Children’s Cancer Group (CCG) (CCG- safely avoided in children who achieve CR after initial
59704) evaluated response-adapted therapy featuring four chemotherapy in some regimens (Table 5). Ongoing
cycles of the dose-intensive BEACOPP regimen followed trials for patients with low-risk HL are evaluating the
by a gender-tailored consolidation for pediatric patients effectiveness of treatment with fewer cycles of combination
with high-risk HL. For rapid early responding girls, an chemotherapy alone that limit doses of anthracyclines
additional four courses of COPP/ABV (without IFRT) was and alkylating agents.29-31
Table 5 Risk-adapted treatment of newly diagnosed Hodgkin lymphoma in children

Risk group 5 yr EFS 5 yr OS
Low risk disease
• Four cycles of VAMP with LD-IFRT (if not in CR after 2 cycles) or without IFRT ( if in CR post 2 cycles)
• Four cycles of COP/ABV plus LD-IFRT
92% 98%
• ABVE, administered for two to four courses depending on response, followed by LD-IFRT
• Two cycles of OEPA or OPPA with LD-IFRT (if not in CR after two cycles) or without IFRT (if in CR post two cycles)
Intermediate risk disease
• Six cycles of COPP/ABV plus LD-IFRT
• ABVE-PC, administered for three to five courses depending upon response, with or without LD-IFRT 85% 95%
• Two cycles of OPPA (for males) or OEPA (for females), followed by two cycles of COPP (for females) or COPDAC
(for males) plus LD-IFRT
High-risk disease
ABVE-PC, administered for three to five courses depending upon response, followed by LD-IFRT
Two cycles of OPPA (for males) or OEPA (for females), followed by four cycles of COPP (for females)
or COPDAC (for males) plus LD-IFRT
83% 94%
Two cycles of cytarabine/etoposide, COPP/ABV, and CHOP plus LD-IFRT
Four cycles of BEACOPP with subsequent dependent upon response; rapid responders: four cycles of COPP/ABV
without IFRT (for females) or two cycles ABVD with IFRT (for males); slow responders: four additional cycles of
BEACOPP plus IFRT
Treatment of Intermediate Risk without treatment following excision in children with

Classical Hodgkin Lymphoma stage I NLPHL. The overall survival (OS) in most series is
100 percent, with lower progression-free survival (PFS)
Patients with intermediate risk HL (all stage I and stage II and EFS in series with surgery alone (67 to 82 percent)38,39
patients not classified as early stage; stage IIIA) generally or with CVP (cyclophosphamide, vincristine, prednisone)
qualify for combined modality treatment. However, chemotherapy (74 percent).40
the ideal chemotherapy and radiation combinations
are not yet clearly defined, and there is an ongoing Relapsed or Refractory Disease
desire to optimize treatment in this risk group. These
children require 3 to 6 cycles of dose-intense alkylator Most relapses in children with HL occur in first 3 years.
based chemotherapy followed by low-dose IFRT. In Treatment and prognosis after relapse depends upon
some studies, chemotherapy is given to maximal tumor the timing of relapse, the initial stage of disease, and the
response, as judged by CT scan and PET, after which initial treatment given. Also, presence of B symptoms,
two additional cycles of consolidation chemotherapy are extranodal disease and inadequate response to second-
given followed by limited RT (Table 5). The patients with line therapy portend poor prognosis. Relapsed patients
rapid early response may be treated with less number of may be classified in 2 groups for prognostication and
chemotherapy cycles as shown in the recent studies (POG treatment planning.
9425)35 or may not require RT (COG AHOD 0031).34
Low-risk (Favorable) Group
Treatment of High-risk Classical Children with localized late ( ≥12 months after completing
Hodgkin Lymphoma therapy) recurrences after limited risk-adapted therapy or
with chemotherapy alone and/or IFRT have a high likeli-
Children with high-risk (Stages IIIB, IV) HL require 6 to
hood of achieving long-term survival following treatment
8 cycles of dose-intense alkylator based chemotherapy
with more intensive conventional chemotherapy alone.
regimens followed by low-dose IFRT (Table 5).37 Current
Intensive non-crossresistant regimens using agents not
trials for patients with intermediate/high-risk HL are
part of initial treatment such as cytarabine at moder-
testing, if chemotherapy and radiation therapy can be
ate or high doses, carboplatin and cisplatin, ifosfamide,
limited in patients who achieve a rapid early response to
etoposide, vinorelbine, gemcitabine, and vinblastine are
dose-intensive chemotherapy regimens.
used. Approximately, two-third of these patients may be
salvaged with second-line chemotherapy.
Treatment of Nodular Lymphocyte-
Predominant Hodgkin Lymphoma High-risk Group
NLPHL, an uncommon subtype, represents a more indo Children who develop refractory disease during therapy
lent disease than classical HL, and is therefore managed or relapsed disease within 1 year after completing
uniquely. Most information concerning its therapy therapy require aggressive salvage chemotherapy and
has come from reports of single institutions or pooled, consolidation with high dose chemotherapy and sub
multi-institutional retrospective analyses in children and sequent autologous hematopoietic cell transplantation
adults. Generally, patients with stage I/II NLPHL without (HCT).43,44 Autologous source of stem cells is preferable to
B symptoms are treated with less intensive therapy than allogeneic because of the high transplant-related mortality
patients with classical HL. In contrast, patients with stage (TRM) associated with allogeneic transplantation.
III/IV are treated in a similar fashion to patients with Following autologous HCT, the projected overall survival
classical HL. The current strategies for treating NLPHL in rate is 45 to 70 percent and progression-free survival (PFS)
children are modest intensity chemotherapy regimens, is 30 to 89 percent. Patients who fail autologous HCT or
some without anthracyclines, with or without IFRT. for patients with chemoresistant disease, allogeneic HCT
Given the indolent nature of NLPHL and because has been used with encouraging results. Salvage rates
deaths observed among individuals with this histological for patients with primary refractory HL are poor even
subtype are more frequently related to complications with autologous HCT and range from 20 to 40 percent.
from cytotoxic therapy, several pediatric study groups Brentuximab vedotin (Anti-CD30 monoclonal antibody)
have evaluated treatment de-escalation in an attempt to has been evaluated in adults with relapsed/refractory
avoid toxicities associated with treatment.38,41,42 Some have HL and has shown promising response rates of 50 to 70
evaluated the use of chemotherapy alone or observation percent in phase-I/II studies.
Table 6 Late effects in Hodgkin lymphoma survivors

Adverse effects Predisposing therapy Clinical features
Thyroid Radiation to thyroid Hypothyroidism, hyperthyroidism, thyroid nodules
Cardiovascular Radiation to heart Left ventricular dysfunction, cardiomyopathy pericarditis, heart valve
dysfunction, conduction disorders, vascular disease, myocardial
infarction, stroke
Anthracyclines Left ventricular dysfunction, cardiomyopathy, congestive heart
failure
Pulmonary Radiation to lungs Subclinical pulmonary dysfunction
Bleomycin Pulmonary fibrosis
Musculoskeletal Radiation to musculoskeletal tissues Growth impairment
Glucocorticosteroids Bone mineral density deficit
Reproductive Alkylating agents Hypogonadism
Gonadal irradiation Infertility
Subsequent neoplasm Alkylating agents Myelodysplasia/acute myeloid leukemia
or disease Epipodophyllotoxins Myelodysplasia/acute myeloid leukemia
Radiation Solid benign and malignant neoplasms
FOLLOW-UP AND LATE EFFECTS SUMMARY

Current 5-year relative survival for HL is approximately 90 Pediatric Hodgkin lymphoma is currently one of the
percent with higher rates reported in younger populations.45 most curable childhood malignancies with more than 90
However, emergence of late toxicity among survivors can percent cure rates in recent studies. Risk and response-
limit long-term survival and affect quality of life. Mortality adapted combined modality therapy is the current stan
in the first 15 years after diagnosis relates to the primary dard of care and is stratified based on disease stage and
disease and following that to second cancers (SMNs) the presence of adverse prognostic factors. Long-term
and cardiovascular disease (CVD).46 Hence, children follow-up of pediatric HL patients should take place in
treated with HL should be closely followed for relapse a comprehensive pediatric-oncology center, where late
as well as late effects. Imaging is not recommended for complications can be anticipated, monitored, and treated.
routine follow-up, as a recent Children’s Oncology Group Recent trials are exploring to reduce toxicities of treatment
Study, which evaluated surveillance CT for detection of with the selective elimination of radiotherapy, devising
relapse, found that most relapses were detected based on less toxic chemotherapy regimens and assessing the role
symptoms, laboratory, or physical findings without any of functional imaging with PET in assessing response and
incremental value of imaging. The method of detection predicting outcome.
of late relapse, whether by imaging or clinical change, did
not affect overall survival.47
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C H A P T E R 44
Non-Hodgkin Lymphoma in Children
and Adolescents
Non-Hodgkin lymphoma (NHL) is diverse collection of neoplasms of lymphoid system derived from numerous cell types comprising
the immune system including B-cell progenitors, T-cell progenitors, mature B-cells, or mature T-cells. Lymphomas are systemic diseases
and have patterns of spread that mimic the migration patterns of their normal cellular counterparts. Progress in therapy of childhood
NHL is one of the greatest success stories of the pediatric oncology in past two decades. More than 75 percent of children with NHL
can now be cured with modern therapy. These extraordinary advances in treatment have resulted from enhanced understanding of
the biology, immunology, and molecular biology of the NHL; improvements in imaging and staging systems; advances in supportive
care; and more rational application of risk adapted chemotherapy by cooperative group trials. Consequent to such high cure rates, the
current focus is on optimization of therapy to reduce the acute and long-term consequences of treatment.
Childhood NHL is a heterogeneous collection of diseases tumors. Although nearly all pathologic subtypes of NHL
derived from both mature and immature cells of B- can be seen in children, majority of NHL that occur in
and T-lineage. Early morphology based classification children appear in four major categories in the WHO
systems have given way to a practical approach classification systems which are detailed in Table 1.
utilizing available immunologic and molecular genetic Unlike adults where more than 60 percent lymphomas
techniques in addition to the standard morphologic are indolent, childhood NHL are diffuse, intermediate-
criteria in the current World Health Organization high grade, clinically aggressive and are predominantly
(WHO) classification of hematopoietic and lymphoid multifocal and disseminated at diagnosis.1
Table 1 Major biologic subgroups in childhood NHL

Histology Immunology Clinical features Cytogenetics
Burkitt and Burkitt-like B-cell Abdominal masses, GIT tumors, t(8;14)(q24;q32)
Waldeyer’s ring t(2;8)(p11;q24)
t(8;22)(q24;q11)
Diffuse large B-cell lymphoma B-cells (germinal center or post- Abdominal masses, GIT tumors, t(8;14)(q24;q32)
(DLBCL) germinal center) Waldeyer’s ring t(2;17)(p23;q23)
Primary mediastinal DLBCL B-cells (medullary thymus) Mediastinum
Anaplastic large cell T-cell, null cell or NK cell (CD30+) Skin, nodes, bone, lung t (2;5) (p23;q35)
t (1;2) (q21;p23)
t (2;3) (p23;q21)
Precursor T lymphoblastic T-cell Anterior mediastinal mass t (1;14) (p32;q11)
t (11;14) (p13;q11)
t (10;14) (q24;q11)
t (7;19) (q35;p13)
Precursor B lymphoblastic B-cell precursors Skin, lymph node
EPIDEMIOLOGY AND ETIOLOGY OF translocations results in the inappropriate expression of

CHILDHOOD NHL cMYC, the gene involved in cellular proliferation due to
juxtaposition of the cMYC oncogene on chromosome
Lymphoma (Hodgkin and non-Hodgkin) is the third 8 and immunoglobulin locus regulatory elements
most common childhood malignancy, and NHL accounts on chromosome 14, 2 or 22. Endemic BL possesses
for approximately 7 to 10 percent of cancers in children breakpoints upstream of cMYC while sporadic BL have
younger than 20 years. NHL occurs most commonly in breakpoints within cMYC. The two most common primary
the second decade of life, and occurs less frequently in sites of disease are the abdomen and head-neck region.
children younger than 3 years. Other sites of involvement include testes, bone, peripheral
Immunodeficiency, both congenital and acquired (HIV lymph nodes, skin, bone marrow (BM), and central
infection or post-transplant), increases the risk of NHL nervous system (CNS).1,4
more than 100-fold compared with general population.
Epstein-Barr virus (EBV) has been shown to transform
Diffuse Large B-cell Lymphoma
human B-cells and has been associated with lymphomas
in immunocompromised hosts. However, its role in Diffuse large B-cell lymphoma (DLBCL) represents 10 to 20
pathogenesis of NHL in immunocompetent individuals percent of pediatric NHL. DLBCL occurs more frequently
is unproven. EBV DNA has been found in more than 95 during the second decade of life. Pediatric DLBCL is
percent of tumor cells in endemic Burkitt lymphoma (BL) clinically similar to BL, though it is more often localized
in Africa in contrast to only 15 to 20 percent in sporadic BL and less often involves the BM or CNS. These cells have
in Europe and North America.1,2 a mature B-cell immunophenotype with expression of
The incidence and relative frequency of various subtypes CD19, CD20, CD22, CD79a, and PAX5. Most cells express
of lymphoma in children varies considerably in different monoclonal surface immunoglobulin light chain. They
world regions. In India, the estimated incidence is between have a high mitotic rate but MIB-1 index is less than 90
6 to 10/million/year with an almost equal distribution of percent. In contrast to adults, pediatric DLBCL have
B- and T-cell tumors.1,2 In India, there is no population- high expression of cMYC and low expression of BCL-2.
based study with sufficient immunohistochemical backup Majority of pediatric DLBCL have a germinal center B-cell
to allow assignment according to the WHO classification. phenotype with expression of normal germinal center
However, data from lymphoma registry at Tata Memorial markers such as the BCL6 gene product and CD10. Unlike
Hospital (TMH) suggests that B-cell lymphomas form 48.1 adult DLBCL, pediatric diffuse DLBCL rarely demonstrates
percent of NHLs whereas T-cell lymphomas form 44.3 the t(14;18) translocation but 30 percent of will have a gene
percent of all the lymphomas. Of B-cell, diffuse large B-cell signature similar to BL.
lymphoma (DLBCL) is the most common (22.9%) followed About 20 percent of pediatric DLBCL present as
by BL (15.3%) and in T-cell, lymphoblastic lymphoma primary mediastinal disease (primary mediastinal B-cell
(LL) is the most common (31.5%) followed by anaplastic lymphoma [PMBCL]). This presentation is more common
large cell lymphoma (ALCL) seen in 11.1 percent cases. in older children and adolescents. These tumors arise
Overall, there seems to be a higher prevalence of DLBCL from thymic B-cells and show diffuse large cell prolife
and LL and lower frequency of BL compared to western ration with classic compartmentalizing sclerosis. Cells
countries.3 have surface markers similar to DLBCL but lack surface
immunoglobulins and commonly express CD30. It is
associated with distinctive chromosomal aberrations
CLINICAL FEATURES (gains in chromosome 9p and 2p in regions that involve
Burkitt Lymphoma JAK2 and c-REL, respectively) and has an inferior outcome
compared with other pediatric DLBCL.1,4
Burkitt lymphoma (BL) is the most common subtype and
accounts for about 30 to 50 percent of childhood NHL.
Lymphoblastic Lymphoma
It exhibits consistent, aggressive clinical behavior. The
malignant cells display a mature B-cell phenotype with Lymphoblastic lymphoma (LL) makes up approximately
expression of surface immunoglobulin M with either 20 percent of childhood NHL. More than 75 percent of
kappa or lambda light chains, CD20, CD22, CD10 and LL usually have a T-cell immunophenotype (T-LL) and
are negative for the enzyme terminal deoxynucleotidyl the remainders have a precursor B-cell phenotype (B-
transferase (TdT). BL expresses a characteristic chromo LL). These are part of a spectrum of precursor blast cell
somal translocation, t(8;14) in 80 percent cases and t(8;22) neoplasms seen in children. By definition, patients with
or t(2;8) in rest 20 percent of children; which is considered more than 25 percent marrow blasts are considered to have
the gold standard for diagnosis of BL. Each of these leukemia, and those with fewer than 25 percent marrow
Chapter-44 Non-Hodgkin Lymphoma in Children and Adolescents 453
blasts are considered to have lymphoma. However, this is WHO as unique entities. Pediatric follicular lymphoma
arbitrary and current WHO classification labels them in the predominantly occurs as cervical adenopathy and tonsillar
category of lymphoblastic lymphoma/leukemia. Notably, enlargement generally in males, and is more likely to be
despite the clinicopathologic overlap between ALL and LL, localized disease. It can rarely involve extranodal sites such
there is suggestion of different gene expression profile and as the testis, kidney, gastrointestinal tract, and parotid.
loss of heterozygosity at 6q indicating biologic differences The outcome of pediatric follicular lymphoma is excellent.
between them. Cytologically, cells have a high mitotic rate MALT lymphomas present as low-stage (stage I or II)
and express TdT. T-LL display cortical thymocyte origin disease both in nodal and extranodal sites such as stomach
and express CD1a, CD2, CD5 and CD7 along with co- (associated with H. pylori infection) and conjunctiva
expression of CD4 and/or CD8. B-LL displays early pre-B (associated with chlamydial psittaci infections).4-6
or pre-B immunophenotype with expression of CD19,
CD10 and TdT. Majority of patients with T-LL present with
Diagnostic and Staging Evaluation
an anterior mediastinal mass and may have symptoms
of dyspnea, wheezing, stridor, dysphagia, or swelling of The diagnosis of NHL is based upon the pathologic
the head and neck due to compression of mediastinal evaluation of involved tissue, usually an abdominal mass,
structures which is called superior vena cava syndrome extranodal site, or lymph node, interpreted within the
(SVCS). Pleural effusions and supradiaphragmatic lymph clinical context. Subtypes of NHL are identified using
nodes may also be present. There may also be involvement histology, immuno phenotype, and genetic studies.
of bone, skin, bone marrow, CNS, abdominal organs Most children present with advanced-stage disease
(but rarely bowel), and occasionally other sites such as including BM invasion or/and malignant effusions. In
lymphoid tissue of Waldeyer ring and testes.1,4 such cases, correct diagnosis can be made by cytology
and immunophenotyping by flow cytometry. If this is not
Anaplastic Large Cell Lymphoma possible, diagnosis is based on biopsy, and most cases are
correctly classified by cytology of tumor touch imprints,
Anaplastic large cell lymphoma (ALCL) is a peripheral
histomorphology, and immunohistochemistry. In most
T-cell lymphoma (PTCL) as per WHO classification and
cases, these enable correct classification and allocation
accounts for approximately 10 percent of childhood NHL.
of patients to appropriate treatment subgroups. In certain
Majority of ALCL are mature T-cell, but 20 percent may
cases, cytogenetics is also required for diagnosis, such
have null-cell disease (i.e. no T-cell, B-cell, or NK-cell
as variant BL/BL-like lymphomas. Fluorescence in situ
surface antigen expression). However, ALK positive null
hybridization (FISH), which can be performed on tumor
ALCL shows TCR gene rearrangements. Morphologically,
touch preparations, or paraffin sections, is a standard
classic variant shows large anaplastic cells and horseshoe
method for confirming most of the chromosomal
like multinucleate “hallmark” cells. Ten percent would
have lymphohistiocytic variant with lots of benign translocations.1,4
• Routine staging of pediatric NHL should include
histiocytes and a small percentage would show the small
contrast enhanced computerized tomographic (CT)
cell variant. The latter two variants have relatively poor
imaging of the neck, chest, abdomen, and pelvis.
prognosis. Immunophenotypically, tumors cells variably
Baseline CT serves to help determine disease stage at
express CD30, CD45, epithelial membrane antigen and
diagnosis and to provide a baseline for comparison
ALK protein. More than 90 percent of ALCL cases have the
to determine response to treatment. Examination
translocation t(2;5)(p23;q35) leading to the expression of
of cerebrospinal fluid and BM is crucial for staging
the fusion protein NPM/ALK. Clinically, ALCL has protein
evaluation. Laboratory tests also may be abnormal
presentations, including involvement of lymph nodes and
in patients with newly diagnosed pediatric NHL
a variety of extranodal sites, particularly skin and bone and,
such as unexplained anemia, thrombocytopenia, or
less often, gastrointestinal tract, lung, pleura, and muscle.
leukopenia due to extensive bone marrow infiltration,
Involvement of the CNS and bone marrow is uncommon.
hyperuricemia as well as other features of tumor
ALCL is often associated with systemic symptoms (e.g.
lysis syndrome and elevated level of serum lactate
fever, weight loss) and a prolonged waxing and waning
dehydrogenase (LDH) due to high tumor burden.1,4
course, making diagnosis difficult and often delayed.1,4
• Emerging role of PET-scan: PET-CT scan of whole body
for staging and response evaluation in children is
Rare Lymphomas in Children
currently investigational and being evaluated in many
Indolent mature B-cell lymphomas, are rare in children current studies. Although PET-CT is recognized to be
but follicular lymphoma (FL) and nodal marginal zone advantageous in the primary staging of adult NHL,
lymphoma (MALT) have been described and accepted by this has not been demonstrated in childhood NHL.
This may be, because majority of children present Currently used St. Jude children research hospital
with advanced disease (stages III or IV) which is easily (Murphy’s) staging classification takes into consi
detectable by CT-scan. However, PET-CT appears to deration increased extranodal involvement, metastatic
have a higher level of sensitivity than bone marrow spread to the BM or CNS and noncontiguous spread
biopsy in the detection of bone marrow infiltration of disease in this group (Table 2).1,4 However, with the
and hence may be useful as a noninvasive modality evolution of more intensive therapy, it is becoming
for detecting bone marrow involvement in pediatric redundant. For example, in BL, the cure rates for
NHL.1,4,7 stages 2, 3 and 4 (CNS-negative) have become
Similarly, early response assessment to chemo almost equal. Hence, the French Society of Pediatric
therapy with an interim PET is now routine done in the Oncology (SFOP) and BFM group have modified the
management of adults with NHL; this is not regarded St. Jude’s system with incorporation of other clinical
as standard practice in children due to limited data. and biological parameters for better risk-assignment
However, PET may be potentially useful for assessing (Table 3). This classification is being applied in the
the speed of response and confirmation of post- ongoing B-NHL international study (FAB-LMB).8-13
therapy remission (CR).7 St. Jude’s system is also not ideal for LL and ALCL. In
• Staging and risk stratification (Tables 2 and 3): The LL, patients presenting with stage 1 or 2 disease are rare
Ann Arbor staging classification used for HL does and majority present in stage 3. Moreover, there is no
not adequately reflect prognosis in childhood NHL significant difference in outcome in those with stage 3 and
because of the unique biology, clinical behavior and stage 4 disease.1,4 Similarly, ALCL frequently involves sites
outcome of the four major subtypes of NHL seen in atypical of childhood lymphoma (such as skin, bone and
children. lung). Le Deley evaluated prognostic factors for ALCL in
Table 2 St. Jude’s staging system for childhood NHL

Stage Definition
I Single tumor (extranodal)
Single anatomic area (nodal) excluding mediastinum or abdomen
II Single tumor (extranodal) with regional node involvement
Primary gastrointestinal tumor with or without involvement of mesenteric node only
On same side of diaphragm:
(a) Two or more nodal areas
(b) Two single extranodal tumors with or without regional node involvement
III All primary intrathoracic tumors
All extensive primary intra-abdominal disease
Two or more nodal or extranodal areas on both sides of diaphragm
IV Any of the above with CNS or bone marrow involvement
Table 3 Pediatric B-NHL—current risk grouping

Protocol Group Definition 5 years EFS
A Completely resected stage-1 and abdominal stage 2 98%
B-NHL
(LMB89) B Unresected stage-1, nonabdominal stage 2
All stages 3 and 4 92%
B-ALL <25% blasts, CNS –ve
C B-ALL >25% blast or CNS +ve 84%

B-NHL R1 Stage I, II initial complete resection 94%
(BFM) R2 Stage I, II unresected, stage III with LDH <500 U/L 94%
R3 Stage III with LDH <500–999 U/L 85%
BM+ve and LDH <1000 U/L
R4 LDH >1000 U/L and/or CNS +ve 81%
culled data from BFM, SFOP and UKCCSG studies and least invasive procedure should be used to establish the
found that, mediastinal involvement (p=0.004), lung, diagnosis of lymphoma such as pleural tap, bone marrow
spleen and/or hepatic disease (p=0.006) and skin lesions examination, a lymph node biopsy under local anesthesia
(p=0.02) were associated with a significantly poorer or a computed tomography–guided core needle biopsy
outcome. Based on this, two risk groups were delineated: should be contemplated. These children should be closely
standard (EFS 87%), and high risk (skin, mediastinal and/ monitored in intensive care units in propped-up lateral
or visceral disease; EFS 61%).14 position and may be started on steroids if it is unsafe
Furthermore, in B-NHL, adolescent age, primary to perform a diagnostic biopsy because of the risk of
medi astinal DLBCL subtype, involvement of CNS with anesthesia or sedation. Biopsy should be obtained as soon
bone marrow, high LDH (more than 2.5 times upper limit as patient is able to undergo the procedure safely.1,4
of normal), and poor response to COP prophase (<20% Tumor lysis syndrome (TLS) results from rapid
reduction in tumor burden) are associated with poor prog breakdown of malignant cells resulting in a number of
nosis.15 Also, secondary cytogenetic abnormalities, other metabolic abnormalities, most notably hyperuricemia,
than cMYC rearrangement including gain of 7q or deletion hyperkalemia, and hyperphosphatemia. Hyperhydration
of 13q have been shown to be strong adverse factors in and allopurinol or rasburicase (urate oxidase) are essential
two recent studies in BL. Similarly, deletion of 6q has been components of therapy. Rasburicase, a recombinant
demonstrated to be a poor prognostic factor in LL.16 urate oxidase rapidly lowers serum uric acid levels and
• Upcoming role of minimal residual disease (MRD) prevents the metabolic problems associated with TLS.
evaluation: Monitoring residual clonal lymphoma Use of rasburicase (0.05 to 0.1 mg/kg IV [max 1.5 mg])
cells in the blood and/or BM by means of aberrant preserves renal function and allows early administration of
immunophenotype or PCR-based identification of planned therapy. The use of rasburicase has dramatically
specific fusion gene products is an emerging tool reduced the requirement for dialysis in this population.
for evaluating the kinetics of treatment response in Gastrointestinal bleeding, obstruction, and (rarely)
childhood NHL. The cumulative incidence of relapse perforation may also occur during the initial phase of
was 71 percent in children with ALCL having > 10 therapy in B-NHL with gut involvement.19
copies of NPM-ALK/10,000 copies ABL in BM or blood.
Quantitative PCR for NPM-ALK in BM or blood allowed Principles of Chemotherapy in NHL
identification of 20 percent of patients experiencing 60
percent of all relapses with an event-free survival of Many studies including the seminal Children’s Oncology
only 20 percent in one study. Similarly, in BL, a assay Group (COG) trial that randomized all children with NHL
that can detect the t(8;14) has been used at diagnosis to be treated with short duration pulse intensive COMP
or during therapy and has been found superior to BM regimen (cyclophosphamide, vincristine, methotrexate,
aspirate and BM biopsy in the assessment of MRD.17,18 and prednisone) or to a long duration modified LSA2L2
regimen (used for acute lymphoblastic leukemia) have
shown that LL fare better when treated with long duration
TREATMENT
LSA2L2 leukemia regimen and short duration COMP was
Principles of Management better for patients with B-cell NHL.20,21
DLBCL has a similar pattern of initial disease
Childhood NHL are extremely chemosensitive tumors. distribution and the same rapid response to chemotherapy
Surgery plays a very limited role, mainly for arriving at a as BL. Results from the large studies such as LMB and BFM
diagnosis. Radiation of primary sites is used very rarely in suggest that with similar therapy there is no difference in
emergency situations. Hence, multiagent chemotherapy outcome between BL and DLBCL and these should be
directed to the histologic subtype and stage of the disease treated with the same approach. In B-NHL, in view of high
remains the cornerstone of therapy. growth fraction and short doubling time; short, pulse-
There are two potentially life-threatening clinical intensive, multi-agent chemotherapy is given in courses of
situations that are often seen in children with NHL at 3 to 5 days with a schedule characterized by fractionation or
presentation: continuous infusion of drugs. To prevent rapid re-growth,
• Superior vena cava syndrome (or mediastinal tumor courses are administrated at shortest intervals. Treatment
with airway obstruction), most often seen in LL intensity is adapted to tumor burden (stage, LDH level,
• Tumor lysis syndrome, most often seen in lympho BM involvement, CNS involvement) and response to COP
blastic and BL. These emergent situations should be pre-phase. In addition, intensive CNS directed therapy
anticipated and addressed immediately. using high dose methotrexate or cytosine-arabinoside
Patients with large mediastinal masses are at risk of and intrathecal therapy (single agent methotrexate +/-
cardiac or respiratory arrest during general anesthesia or ara-c or triple intrathecal) is usually necessary. Cranial
heavy sedation. If peripheral blood counts are normal, the radiotherapy is not necessary.8-13
T-cell Lymphomas (Table 4)
Table 4 Treatment and outcome of T-NHL

ALCL ( T-cell type approach)
Protocol Number Stage EFS (%) Duration /number of cycles
LSA2L2 19 III/IV 56 14–36 months
CCG-5941 86 III/IV 78 12 months
POG 9315 86 III/IV 72 12 months
AIEOP 34 II/III/IV 65 24 months
(LNH-92)
ALCL (B-Cell type approach)
BFM 90 8 I 100 Stage I/II (completely resected): 3 cycles
20 II 79 Stage II (unresected)/stage III: 6 cycles
55 III 74 Stage IV/bone disease: 6 intensified cycles
6 IV 50
SFOP-HM 82 I/II 94 7–8 months
89/91 III/IV 55
UKCCSG 72 III/IV 59 Stage III/IV (CNS–neg): 5 cycles
CNS positive: Intensified 5 cycles
MCP-842 27 I/II 67 6–8 (alternating A and B) cycles
III/IV 40
Lymphoblastic lymphoma
LSA2L2/ADCOMP 281 I/II 84 18 months
with LSA2L2 III/IV 64
POG8704 218 III / IV 67 24 months
LMT 81 76 I / II 76 12 months
8 III / IV 73
UKCCSG 59 III / IV 65 24 months
BFM- 90 82 III 90 24 months
19 IV 95
BFM- 95 22 I / II 95 24 months
169 III / IV 78
Precursor T Lymphoblastic Lymphoma • Advanced stage LL: Advanced LL have event free
survival (EFS) rates higher than 80 percent with
• Localized LL: For localized LL patients (stage I/II protocols designed for high-risk ALL consisting of
disease), induction therapy with short, pulsed a four-drug induction, consolidation incorporating
chemotherapy (CHOP) results in a 95 percent CR rate. high-dose methotrexate, cyclophosphamide, cytara
However, only 60 percent can achieve long-term DFS bine, CNS directed therapy including intrathecal
due to late relapses in bone marrow. Majority of these therapy and long maintenance with total 24 months
patients can be salvaged, giving an overall survival of therapy.21-26 Some recent studies have shown that
(OS) of >90 percent at 5 years. There is no survival patients receiving high-dose asparaginase regimen
benefit of involved field irradiation. Using a leukemia had a superior outcome.23 Several studies also suggest
like approach with induction, consolidation, and that high dose methotrexate (MTX) results in survival
maintenance therapy for a total of 24 months, most advantage. No benefit on outcome has been observed
studies have shown more than 90 percent survival with use of high dose cytarabine. The results of the
for localized LL. No reinduction therapy and local or various chemotherapeutic regimens are shown in
cranial radiation is given for stage I and II patients.21,22 Table 4.21-26
Until recently, CNS directed therapy included the • Localized ALCL: For localized ALCL (grossly resected,
combined use of cranial irradiation and intrathecal i.e. >90% stage I/II disease), the best results have come
chemo therapy. However, recent studies have demon from using pulsed chemotherapy similar to B-NHL
strated that cranial irradiation can be safely omitted if therapy. BFM group has shown results similar to those
systemic high-dose methotrexate combined with intra obtained with the BFM-90 regimen for B-NHL with use
thecal chemotherapy is administered. However, cranial of 3 cycles after cytoreduction. In POG studies, Stage I
irradiation may be necessary for children who present and II disease was very effectively treated with CHOP
with CNS disease (fewer than 5% of children).26 Irradiation for three cycles without RT. The recent ALCL-99 trial
of primary sites such as mediastinum has not been shown used three cycles of chemotherapy following prophase
to improve outcome when added to chemotherapy. Even for patients with stage I completely resected disease
in patients with testicular disease at diagnosis, testicular with good results. Primary cutaneous ALCL may be
radiation is only indicated for residual disease after treated successfully with surgical resection and/or
systemic therapy incorporating high-dose MTX.21-26 local radiotherapy without systemic chemotherapy.

In NHL-BFM-95, prophylactic cranial radiation was Thus, children with standard risk disease (Stage I/II
omitted, and the intensity of induction therapy was completely resected with no high risk features such
modified (reduction of asparaginase and/or doxorubicin). as involvement of skin, mediastinum, viscera, CNS or
There was no significant increase in CNS relapses, BM) can be managed with 3 cycles (10–12 weeks) of
suggesting cranial radiation may be reserved for patients B-cell type regimen and intrathecal therapy.28-32
with CNS disease at diagnosis. However, survival was • Disseminated ALCL: Children with disseminated ALCL
worse in BFM-95 than in BFM-90 (90% vs. 82%), possibly have EFS of approximately 60 to 75 percent. Majority
due to reduced intensity induction and increased number of European studies (BFM, SFOP, UKCCSG, ALCL-99)
of secondary malignancies in BFM-95.26 Currently, have used short duration (5–8 months) pulse intensive
ongoing BFM-based trials are trying to determine whether B-cell type approach with good results while American
dexamethasone instead of prednisone during induction (CCG, POG) and Italian groups (AIEOP) have used
can further improve outcome of patients with LL, as was leukemia type long duration (12–24 months) less
observed in children with ALL. intensive approach with almost equivalent survival
but increased hematological toxicity. The recent
B-Precursor LL POG studies demonstrated no benefit of adding
methotrexate and high-dose cytarabine to 52 weeks
The correct treatment for B-lineage LL constituting around of cyclic chemotherapy. Cranial prophylaxis using
20 percent of LL has not been clearly defined because high or intermediate dose methotrexate with (BFM) or
of rarity of this disease. The results of the largest review without intrathecal (SFOP) or only intrathecal therapy
of 98 patients (64% <18 years old) showed that majority (COG, MCP-842) have shown equivalent results with
had skin (with or without adjacent nodal disease), lymph less than 1 percent incidence of CNS relapses. Cranial
node, bone, head and neck and retroperitoneal disease. radiotherapy (RT) is not recommended.28-32
Mediastinal disease was uncommon. The disease free Recently conducted international ALCL 99 study
survival was 74 percent at a median follow-up of 28 months. has shown that HD MTX (3 g/m² intravenously over 3
In BFM-NHL trials, 27 children with precursor B-cell LL hours) is sufficient to protect the CNS in CNS-negative
were treated; 21 on ALL-type therapy (<10% relapses) and patients in the absence of additional intrathecal
6 on Burkitt type therapy (50% relapses). All relapses on chemotherapy and addition of vinblastine during
the latter regimen were salvaged with ALL-type therapy induction and as maintenance for a total treatment
leading to 73 percent EFS and 92 percent OS for the group duration of 1 year did not reduce the risk of failure.31,32
at 10 years. This suggests that patients with B-lineage LL Thus, disseminated ALCL may be managed with 6
should be treated with ALL like therapy duration of 18 to cycles (6 months) of B-cell type regimen using short
24 months.27 infusion HD MTX.
• Anaplastic large cell lymphoma (Table 4): There
is no consensus on management of ALCL due to B-Cell Lymphoma (Table 5)
small number of patients treated in various studies,
heterogeneity in inclusion criteria, different staging • Treatment of limited stage B-NHL: Children with
systems and diverse treatment approaches used in limited stage B-cell NHL (stage I or stage II, BFM R1 or
past trials. However, following broad principles can be FAB group A) have a good prognosis with an estimated
derived. five-year EFS of 90 to 95 percent with minimal
Table 5 Outcome of B-NHL in International Studies

Protocol Number Stage EFS (%) Duration/number of cycles
COMP 57 I/II 84 6 months
135 III/IV 53
LMB 89 52 Gp A 98 Group A: 2 cycles, No IT
386 Gp B 92 Group B: 5 cycles
123 Gp C 84 Group C: 8 cycles
FAB-LMB96 136 Gp A 98 Group A and C: As LMB 89
760 Gp B 90 Group B: 4 cycles with reduced dose
238 Gp C 79 cyclophosphamide
Group C: 8 cycles
BFM-95 98 R1 94 R1: 2 cycles
233 R2 94 R2: 4 cycles
82 R3 85 R3: 5 intensified cycles
142 R4 81 R4: 6 intensified cycles
CHOP 266 I/II 90 6 cycles
Orange (CCG) 34 III/IV 77 5–7 months
chemotherapy (range 6 weeks to 6 months). There are response rate of 41.4 percent in a phase II window study
several multiagent chemotherapy regimens that have in newly diagnosed B-cell NHL and Burkitt leukemia.
resulted in this excellent outcome, including 6 weeks of Based on results of a COG pilot study (ANHL01P1)
COPAD (FAB), 3 to 6 months of COMP (CCG and POG), that it is feasible and safe to include rituximab in the
or two cycles of multiagent chemotherapy (BFM). In current chemotherapy backbone, current studies are
the most recent BFM study (BFM-95), it was shown that evaluating rituximab in high-risk B-NHL patients.33
reducing the dose or duration of methotrexate infusion • Primary mediastinal DLBCL (PMBL): The response to
did not affect the results for localized disease.8-13,21 chemotherapy is slow and outcome is poor in PMBL.
• Treatment of advanced B-NHL: The prognosis of In one CCG series of 20 children with PMBCL, where
advanced B-NHL has improved significantly over almost half received local irradiation, the 5-year EFS
the past decade with the use of short intensive was only 75 percent. In a BFM report of 30 children,
chemotherapy regimens such as FAB/LMB 96 (FAB), the 5-year EFS was 70 percent using chemotherapy
Orange (CCG) or BFM NHL 95 with more than 90 alone. In FAB/LMB-96 study of stage III primary
percent 5-year disease-free survival except in patients mediastinal large B-cell lymphoma, the 5-year event-
with CNS disease.4-9 Recent studies have demonstrated free survival (EFS) was 66 percent, versus 85 percent
that cranial irradiation can be eliminated in patients for adolescents with nonmediastinal DLBCL. Recently,
with CNS-positive disease with the substitution of more a single-arm study in adults showed excellent event-
aggressive high-dose methotrexate and additional free survival utilizing the DA-EPOCH-R regimen (dose-
intrathecal chemotherapy. 8-13,21 adjusted etoposide, doxorubicin, cyclophosphamide,
Recently conducted FAB/LMB 96 study showed vincristine, prednisone, and rituximab; usually six
that intermediate-risk patients (group B) with and cycles) with filgrastim and no radiation therapy.
good response to prophase COP can be treated with The 5-year EFS was 93 percent and overall survival
reduced intensity therapy with 4 courses but high- (OS) was 97 percent. This is currently being tested in
risk patients should receive standard FAB/LMB pediatric clinical trials. Early mediastinal irradiation in
therapy (8 courses).12,13 Children with BM and/or CNS incomplete initial responders may be considered.34
involvement have an inferior outcome if they have a • Outcome of pediatric NHL in India (Table 6): Before
poor response to reduction chemotherapy with COP 1986, survival of pediatric NHL patients in India was
prophase or have combined BM and CNS disease.15 less than 30 percent. To improve survival rates and
Rituximab, a mouse/human chimeric monoclonal to overcome the barriers of limited resources, sub-
antibody targeting the CD20 antigen, has shown optimal supportive care, high prevalence of infectious
good responses in relapsed B-NHL and a promising diseases, impaired nutritional status and delayed
Table 6 Outcome with MCP-842 (1986–2006) at Tata Memorial Hospital

Histology Number Stage Modified MCP-842 OS (%) (10 years)
EFS (%) (10 years)
Burkitt-lymphoma 107 I/II 100% 83%

III/IV 78%
DLBL 53 I/II 96% 82%
III/IV 82%
ALCL 27 All stages 75% 71%
diagnosis; a moderately intensive, short duration be active in the relapse setting. Current investigational
protocol (MCP842) was designed in 1984. Protocol protocols combine chemotherapy with rituximab followed
consisted of 6 to 8 alternating cycles of two different by allogeneic or autologous SCT. The outcome is more
drug combinations designated as regimens A and B favorable for patients who achieve a second remission
with intrathecal therapy during the first four cycles. before proceeding to SCT.38
The drugs used in regimen A were cyclophosphamide, Children with relapsed DLBCL are often treated with
vincristine, doxorubicin and cytarabine, while regimen salvage chemoimmunotherapy regimens such as ICE,
B included ifosfamide, etoposide and MTX. Intrathecal GDP (Gemcitabine, dexamethasone and cisplatinum) and
MTX and cytarabine were used for central nervous DECAL (dexamethasone, etoposide, cisplatin, HD cytara-
system (CNS) prophylaxis. Neither cranial nor local bine, and L-Asp) with rituximab followed by autologous
radiotherapy were included in the treatment protocol. SCT based on the adult experience. The outcome is quite
Over last 2 decades, MCP 842 has been found to be an favorable for those children who have chemosensitive dis-
effective protocol for the management of patients with ease at the time of relapse.38
B-cell NHL and may be an ideal protocol for patients in The outcome for survival after relapse of ALCL is
centers with limited resources since it involves no high relatively favorable in contrast to the less optimistic
dose chemotherapy, there is no need for methotrexate outcome for children with relapsed LL and BL. A French
level monitoring, no central line/hyperalimentation study demonstrated excellent responses to single-agent
requirement and there is minimum blood component vinblastine followed by some very durable second remiss
usage. This protocol has been recently modified with ions. A survival rate of 69 percent at 3 years with courses of
addition of vinblastine as well as maintenance (6–12 CCNU, vinblastine, bleomycin or cytarabine followed by
months) for ALCL, addition of COP prephase (for autologous HSCT in some of the patients has been reported.40
patients with bulky disease or poor GC), use of urate Even in high-risk patients with on-therapy relapse or
oxidase in tumor lysis. The survival with modified relapse after autologous HSCT, long-term remissions
MCP-842 is significantly better compared to standard have been observed after allogeneic HSCT.41 The potential
MCP-842.The results of this protocol are summarized benefit of vinblastine and anti-CD30 antibody combined
in Table 5.35-37 with either an APO or BFM-like regimen is currently under
investigation. Phase 1 study of ALK oncogenic tyrosine
Management of Relapse kinase inhibitor (Crizotinib) in pediatric patients with
anaplastic large-cell lymphoma has shown response rate of
Relapse is a significant obstacle to long-term survival for
88 percent in ALK positive patients.42 Brentuximab vedotin
children with advanced-stage NHL. In LL, most relapses
is a novel antibody-drug conjugate that targets CD30, a cell
occur within 2 years of diagnosis, but occasional late
surface antigen expressed by HL and ALCL. A phase II trial
relapse is observed. In contrast to relapse for early-
in adults with relapsed anaplastic large cell lymphoma has
stage disease, the outcome after salvage chemotherapy
shown CR rates of approximately 55 to 60 percent and PR
is poor for children with advanced-stage disease at
rates of 29 percent.43
initial presentation. However, survival rates of 30 to 50
percent have been reported after allogenic stem cell
CONCLUSION
transplantation (SCT).38,39
The outcome of relapsed patients with BL is dismal Refinements in systemic chemotherapy fuelled by better
because most relapses tend to occur early during active understanding of NHL biology in children have led to
chemotherapy, and drug resistance is a major obstacle cure in approximately 80 to 85 percent of all patients. This
to successful salvage. Rituximab have been reported to improved outlook for childhood NHL, however, has come
with a certain price. The use of intense chemotherapy has 10. Woessmann W, Seidemann K, Mann G, et al. The impact
resulted in long hospitalizations, severe hematopoietic as of the methotrexate administration schedule and dose
well as non-hematopoietic toxicity and late effects, such in the treatment of children and adolescents with B-cell
neoplasms: a report of the BFM Group Study NHL-BFM95.
as sterility, cardiomyopathy, and secondary malignancies.
Blood. 2005;105:948-58.
Consequently, the emphasis for the near future is to 11. Gerrard M, Cairo MS, Weston C, et al. Results of the FAB
decrease the therapy in good risk patients as well as LMB 96 international study in children and adolescents
better identification and development of new therapeutic (C+A) with localised, resected B cell lymphoma (large cell
approaches for high-risk cases. In future, as the molecular [LCL], Burkitt’s [BL] and Burkitt-like [BLL]). J Clin Oncol.
pathogenesis of the malignant lymphomas is better 2003;22:795-801.
elucidated using molecular diagnostic tools, new targets 12. Patte C, Auperin A, Gerrard M, et al. Results of the
randomized international FAB/LMB96 trial for inter
for therapy will emerge. Also, it is likely that targeted
mediate risk B-cell non-Hodgkin lymphoma in children
therapy will substitute for some of the toxic chemotherapy and adolescents: it is possible to reduce treatment for the
and thereby minimize the chemotherapy related morbi early responding patients. Blood. 2007;109:2773-80.
dity. This novel molecular biologic information will also 13. Cairo MS, Gerrard M, Sposto R, et al. Results of a
be valuable for developing more sensitive diagnostic randomized international study of high-risk central
tools, measurement of early response to therapy as well as nervous system B non-Hodgkin lymphoma and B acute
submicroscopic disease and for identifying new prognostic lymphoblastic leukemia in children and adolescents.
subgroups. Superior risk-adapted therapy based on these Blood. 2007;109:2736-43.
14. Le Deley MC, Reiter A, Williams D, et al. Prognostic factors
advances would maximize the chance for cure while in childhood anaplastic large cell lymphoma: results of a
avoiding both acute and chronic toxicities of treatment. large European intergroup study. Blood. 2008;111:1560-6.
15. Cairo MS, Sposto R, Gerrard M, et al. Advanced stage,
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treatment results in childhood B-cell neoplasms with Treatment of children and young adults with early-stage
tailored intensification of therapy: a report of the Berlin- non-Hodgkin’s lymphoma. N Engl J Med. 1997;337:1259-66.
Frankfurt-Munster Group Trial NHL-BFM 90. Blood. 22. Reiter A, Schrappe M, Ludwig WD, et al. Intensive ALL-
1999;94:3294-306. type therapy without local radiotherapy provides a 90%
event-free survival for children with T-cell lymphoblastic 34. Seidemann K, Tiemann M, Lauterbach I, et al. Primary
lymphoma: a BFM group report. Blood. 2000;95:416-21. mediastinal large B-cell lymphoma with sclerosis in
23. Amylon MD, Shuster J, Pullen J, et al. Intensive high-dose pediatric and adolescent patients: treatment and results
asparaginase consolidation improves survival for pediatric from three therapeutic studies of the Berlin-Frankfurt-
patients with T cell acute lymphoblastic leukemia and Munster Group. J Clin Oncol. 2003;21:1782-9.
advanced stage lymphoblastic lymphoma: a Pediatric 35. Arora B, Khadwal A, Bhagwat R, Pai SK, Nair CN, Parikh
Oncology Group study. Leukemia. 1999;13:335-42. PM, Naresh KN, Kolhatkar B, Adde M, Magrath I, Banavali
24. Eden OB, Hann I, Imeson J, et al. Treatment of advanced SD. Short duration intensive chemotherapy protocol for
stage T cell lymphoblastic lymphoma: results of the United treatment of small noncleaved cell lymphoma (SNCL) in
Kingdom Children’s Cancer Study Group (UKCCSG) children and young adults. SIOP XXXVI meeting Abstracts
protocol 8503. Br J Haematol. 1992;82:310-6. PJ036. Pediatr Blood Cancer. 2005;45:533.
25. Patte C, Kalifa C, Flamant F, et al. Results of the LMT81 36. Banavali SD, Arora B, Vora T, Bansal S, Hingmire S, Pai SK,
protocol, a modified LSA2L2 protocol with high dose metho Parikh PM, Kolhatkar B, Adde M, Magrath I. Vinblastine
trexate, on 84 children with non-B-cell (lymphoblastic)
improves outcome in children with anaplastic large cell
lymphoma. Med Pediatr Oncol. 1992;20:105-13.
lymphoma (ALCL) treated with a uniform short duration
26. Burkhardt B, Woessmann W, Zimmermann M, et al.
intensive protocol. SIOP XXXIX meeting Abstracts PJ038.
Impact of cranial radiotherapy on central nervous system
Pediatr Blood Cancer. 2007;49:532.
prophylaxis in children and adolescents with central
nervous system-negative stage III or IV lymphoblastic 37. Arora B, Vora T, Bansal S, Hingmire S, Pai SK, Parikh PM,
lymphoma. J Clin Oncol. 2006;24:491-9. Kolhatkar B, Adde M, Magrath I, Banavali SD. Clinical
27. Neth O, Seidemann K, Jansen P, et al. Precursor B-cell presentation and outcome of children with diffuse large
lymphoblastic lymphoma in childhood and adolescence: B-cell lymphoma treated with a uniform short duration
clinical features, treatment, and results in trials NHL-BFM intensive protocol in India. SIOP XXXIX meeting Abstracts
86 and 90. Med Pediatr Oncol. 2000;35:20-7. PJ04. Pediatr Blood Cancer. 2005;45:524.
28. Seidemann K, Tiemann M, Schrappe M, et al. Short- 38. Attarbaschi A, Dworzak M, Steiner M, et al. Outcome of
pulse B-non-Hodgkin lymphoma-type chemotherapy is children with primary resistant or relapsed non-Hodgkin
efficacious treatment for pediatric anaplastic large cell lymphoma and mature B-cell leukemia after intensive
lymphoma: a report of the Berlin-Frankfurt-Munster first-line treatment: a population-based analysis of the
Group Trial NHL-BFM 90. Blood. 2001;97:3699-706. Austrian Cooperative Study Group. Pediatr Blood Cancer.
29. Bugieres L, Deley MC, Pacquement H, et al. CD30 (+) 2005;44:70-6.
anaplastic large-cell lymphoma in children: analysis of T- 39. Burkhardt B, Reiter A, Landmann E, et al. Poor outcome
and NK-cell lymphomas in children and adolescents of 82 for children and adolescents with progressive disease
patients enrolled in two consecutive studies of the French or relapse of lymphoblastic lymphoma: a report from
Society of Pediatric Oncology. Blood. 1998;92:3591-8. the berlin-frankfurt-muenster group. J Clin Oncol.
30. Williams DM, Hobson R, Imeson J, et al. Anaplastic large 2009;27:3363-9.
cell lymphoma in childhood: analysis of 72 patients treated 40. Brugières L, Pacquement H, Le Deley MC, et al. Single-
on The United Kingdom Children’s Cancer Study Group drug vinblastine as salvage treatment for refractory or
chemotherapy regimens. Br J Haematol. 2002;117:812-20. relapsed anaplastic large-cell lymphoma: a report from
31. Brugieres L, Le Deley MC, Rosolen A, et al. Anaplastic large the French Society of Pediatric Oncology. J Clin Oncol.
cell lympoma (ALCL) in children: equal efficacy but greater 2009;27:5056-61.
toxicity of chemotherapy including methotrexate (MTX) 41. Woessmann W, Zimmermann M, Lenhard M, et al.
1 g/m2 in 24-hour infusion with intrathecal injection (IT) Relapsed or refractory anaplastic large-cell lymphoma in
than chemotherapy with MTX 3 g/m2 in 3-hour infusion children and adolescents after Berlin-Frankfurt-Muenster
without IT: results of the ALCL99–R1 randomized trial (BFM)-type first-line therapy: a BFM-group study. J Clin
[abstract]. Blood. 2006;108:122a.
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32. Marie-Cećile Le Deley, Angelo Rosolen, Denise M
42. Yael P Mossé, Megan S Lim, Stephan D, Voss MD, et al.
Williams, et al. Vinblastine in Children and Adolescents
Safety and activity of crizotinib for paediatric patients
with High-Risk Anaplastic Large-Cell Lymphoma: Results
of the Randomized ALCL99-Vinblastine Trial. J Clin Oncol. with refractory solid tumours or anaplastic large-cell
2010;28:3987-93. lymphoma: a Children’s Oncology Group phase 1
33. Andrea Meinhardt, Birgit Burkhardt, Martin Zimmermann consortium study. Lancet Oncology. 2013.pp.472-80.
et al. Phase II Window Study on Rituximab in Newly 43. Pro B, Advani R, Brice P, et al. Brentuximab vedotin
Diagnosed Pediatric Mature B-Cell Non-Hodgkin’s (SGN-35) in patients with relapsed or refractory systemic
Lymphoma and Burkitt Leukemia. J Clin Oncol. 2010;28: anaplastic large-cell lymphoma: results of a phase II study.
3115-21. J Clin Oncol. 2012;30:2190-6.
C H A P T E R 45
Langerhans Cell Histiocytosis
Langerhans cell histiocytosis (LCH) is an enigmatic disorder occurring due to a reactive clonal proliferation of dendritic cells, which are
immunophenotypically and functionally immature and comprises a group of idiopathic disorders characterized by the presence of
these cells in a background of hematopoietic cells, including T-cells, macrophages, eosinophils and occasional multinucleated giant
cells.1 Accumulation of these cells at various sites in the body are responsible for its clinical manifestations. More recent work has
shown that the pathologic LCH cells have a gene expression profile of a myeloid dendritic cells rather than the skin Langerhans cells
(LC) that are closer to the sites where the disease usually occurs. Controversy too exists whether the clonal proliferation of LCH cells
results from a malignant transformation or due to immune dysregulation of LCs.2
Earlier, LCH was subcategorized based upon clinical EPIDEMIOLOGY

presentation (i.e. Letterer-Siwe disease, Hand-Schüller-
Christian, eosinophilic granuloma, etc.). Lichtenstein The reported incidence of LCH from various studies
was the first to suggest integration of these disorders is two to ten cases per million children aged 15 years
under the common term histiocytosis X, the first or younger.8,9 The male/female (M/F) ratio is close to
word recognizing their common origin, while the “X” one and the median age of presentation is 30 months.10
underlined his uncertainty as to what the origin was!3 Solvent exposures in parents, family history of cancer,
However, over the past few decades since then, there and perinatal infections have been weakly associated with
has been unprecedented progress in the understanding LCH.11,12
of this enigmatic disease by means of collaborative trials
notably the LCH trials by the Histiocyte Study Groups, and
CLINICAL FEATURES
large single institutional studies.4-6 The common etiology
between Lichtenstein’s groups is now well understood. LCH is a rare childhood malignancy. In a large Indian
Minimum diagnostic criteria have been evolved to ensure cancer center, only 52 cases were registered over a 17-years-
clarity and uniformity allowing comparison between period.7 In addition to its rarity is the fact that it has myriad
various studies—a prerequisite all the more essential for presentations and its clinical course can range from low
rare disorders like LCH. For treatment purposes too, the grade chronic and persistent to the rapidly progressive
disorders are now clubbed and categorized by the number and fatal, making its behavior unpredictable to most
and nature of organs involved at diagnosis. Involvement of individual clinicians. Collaborative trials and large single
the liver, spleen, lung, and bone marrow, and their degree institutional studies therefore, have been the major means
of dysfunction has been shown to be the key determinant of shedding light on the understanding of this disease and
to outcome irrespective of the therapies offered. This was deciding its management. The most important concept
even corroborated when the new risk classifications were that has evolved has been that multiorgan involvement
applied retrospectively to past cases, to see how they fared, has worse outcome than single-organ disease and needs
in a large single institution study from India, showing the some form of cytotoxic therapy. Also, among those with
robustness of these risk stratifications.7 multiorgan disease, some patients are more “at risk”
Chapter-45 Langerhans Cell Histiocytosis 463
for disease progression, sequelae, and death. High-risk • Oral cavity: The oral cavity lesions may precede
organs include liver, spleen, and bone marrow. Low-risk evidence of LCH in other organs. Presenting symptoms
organs include skin, bone, lymph nodes, gastrointestinal may be gingival hypertrophy, and ulcers of the soft or
tract, pituitary gland, and central nervous system hard palate, buccal mucosa, or on the tongue and lips.
(CNS).4-7,9,10,13,14 Hypermobile teeth (floating teeth) and tooth loss may
LCH in children usually presents with a skin rash or occur.18
painful bone lesions. Systemic symptoms of fever, weight • Bone: Bones are the most common site of involvement
loss, diarrhea, edema, dyspnea, polydipsia, and polyuria in LCH. Skeletal involvement with or without other
may relate to specific organ involvement by disease.7, 9, 10 sites occurs in 80 to 100 percent of patients in most
Patients may present with a single organ involvement large series.7,10,13 Hands and feet are often spared, but it
(single-system LCH), which may be a single site (unifocal) can involve almost any other bone, the most frequent
or involve multiple sites (multifocal). Involvement of more site being the skull. It presents as a lytic bony lesion
than one organ is categorized as multisystem LCH. In this with associated soft tissue swelling, which may be
group, involvement may be in a limited number of organs asymptomatic or painful. In the skull, the soft tissue
or it may be disseminated. Patients may present with mass may impinge inwards on the dura.19 Other
LCH of the skin, bone, lymph nodes, and pituitary in any frequent sites involved are the femur, ribs, humerus,
combination and are still considered at low-risk of death. and vertebra.19,20 Amongst the vertebra, the cervical
Multisystem LCH patients’ have relatively high-risk for are the most common to be involved. Vertebral lesions
long-term consequences of the disease.7, 9, 10, 13, 14 may result in collapse of the vertebral body (vertebra
plana). Vertebral lesions with soft tissue extension may
Single-System Disease present with pain and neurologic deficits.20 Disease of
the facial bones or anterior or middle cranial fossae
In single-system LCH, the patient presents with involve (e.g. temporal, sphenoid, ethmoid, zygomatic) with
ment of a single site or organ, including skin and nails, oral intracranial tumor extension comprise part of a CNS-
cavity, bone, lymph nodes and thymus, pituitary gland, risk group. These children have a threefold increased
and thyroid. risk of developing diabetes insipidus and an increased
• Skin and nails: Dermal involvement occurs in 35 to risk of other CNS disease.21, 22
50 perent cases in most series.10,15 However, in Indian • Lymph nodes and thymus: Lymphadenopathy has
literature, the reported incidence was lower at 25 been reported more commonly in a large Indian
percent. This may have been due to under-reporting series than in western literature, where it is reported
of minor lesions such as seborrheic dermatitis, the to be less than 10 percent.7,22 Cervical nodes are
data being from a large referral center.7 In infants, most frequently involved. Nodes may be soft- or
seborrheic involvement of the scalp may be mistaken hard-matted with accompanying lymphedema. An
for prolonged cradle cap. Infants may also present enlarged thymus or mediastinal node due to LCH
with brown to purplish papules over any part of can mimic lymphoma or an infectious process, or
their body (Hashimoto-Pritzker disease). These asthma.
lesions in infants may be self-limited as the lesions • Pituitary gland and thyroid: Posterior pituitary gland
often disappear without treatment during the first involvement presents with central diabetes insipidus
year of life; however, they need to be followed up (DI). Involvement of anterior pituitary results in
closely for systemic disease manifestations which growth failure and delayed or precocious puberty.
may present later on after the initial skin lesions.15-17 DI more commonly manifests in multisystem LCH
Children may present with a red papular rash in where it can afflict nearly 1/3rd of all such patients
the groin, abdomen, back, or chest that resembles a and can develop at any time during the course of the
diffuse candidal rash. Seborrheic involvement of the illness.4,7,21,22
scalp may be mistaken for a severe case of dandruff
in older children. Ulcerative lesions behind the ears,
Multisystem Disease
involving the scalp, under the breasts, or genitalia or
perianal region may be misdiagnosed as bacterial or In this presentation, multiple organs are involved at the
fungal infections on presentation. Involvement of nails outset. The involvement of certain organs like the liver,
is an unusual presentation. They may present as a spleen and hematological system put the patients into a
single site or in conjunction with other sites, and often higher risk category. Other organs that can be involved
show longitudinal, discolored grooves with loss of nail are the same as in single system disease but in various
tissue.15-17 combinations.
• Bone and other organ systems: LCH patients may Endoscopic examination with multiple biopsies is
present with multiple bone lesions (single-system usually needed.
multifocal bone) or bone lesions with other organ • Lung: Lung is less frequently involved in children than
involvement (multisystem including bone). As already in adults, in whom smoking is an etiologic factor.29
discussed above, the later group has a higher incidence It was seen in 15 percent cases in an Indian series.
of diabetes insipidus, probably due to the higher Criteria used to diagnose pulmonary lesions play a
frequency of lesions in the facial bones (temporal major role in frequencies reported in various series
bone, mastoid/petrous bone, orbit, and zygomatic and these have been the most diverse criteria used
bone). among all organ involvements ranging from plain
• Abdominal/gastrointestinal system: Liver and spleen radiographs to more sophisticated anatomical and
are considered high-risk organs. Enlargement of these functional imaging and even pulmonary function
organs may be due to direct infiltration of LCH cells tests. The incidence was as high as 50 percent
or as a secondary phenomenon of excess cytokines in a large series that also included patients with
leading to macrophage activation or infiltration of isolated abnormal pulmonary function tests.30 The
lymphocytes around bile ducts. lung is usually involved in a symmetrical manner
Hepatomegaly is often present in systemic disease and predominantly involves the upper and middle
and pathological changes might be present in the liver lung fields, while sparing the costophrenic angle.31
on histology, even in the absence of liver dysfunction.23 Confluence of cysts may cause bullous formation,
LCH in liver has a portal (bile duct) tropism and which can sometimes rupture spontaneously leading
can cause biliary damage and ductal sclerosis. He to a pneumothorax. Occasionally this may be the first
patomegaly may be accompanied by hypoalbumine sign of LCH involvement of the lung. As the disease
mia with ascites, hyperbilirubinemia, clotting fac progresses, widespread fibrosis and destruction of
tor deficiencies, elevated alkaline phosphatase, liver lung tissue leads to severe pulmonary insufficiency,
transaminases, and gamma glutamyl transpeptidase and patients can present with progressive tachypnea
levels. Cholestasis and sclerosing cholangitis is one of or dyspnea. Eventually declining diffusion capacity
the most serious complications of liver involvement may lead to pulmonary hypertension.32, 33
in LCH.23, 24 Sonography, computed tomography (CT), • Bone marrow: Patients with bone marrow involvement
or MRI of the liver will show hypoechoic or low-signal are usually younger, with multisystem disease and often
intensity along the portal veins or biliary tracts when have diffuse disease in the liver, spleen, lymph nodes,
the liver is involved with LCH.25 This usually occurs and skin. They present with variable thrombocytopenia
months after initial presentation, but occasionally and anemia with or without neutropenia.34 Patients with
may present at diagnosis. Children with sclerosing LCH may sometimes present with hemophagocytosis
cholangitis will not respond to chemotherapy as the involving the bone marrow.35
disease is not active and the fibrosis and sclerosis • Endocrine system: The most frequent endocrine
remain. Liver transplantation is the only treatment manifestation in LCH is diabetes insipidus. This
when hepatic function worsens.24 Rare cases of LCH is caused by damage to the antidiuretic hormone
infiltration of the pancreas and kidneys has been (ADH)—secreting cells of the posterior pituitary.
reported.26 MRI scans show nodularity and/or thickening of the
• Splenic involvement has been noted to be much higher pituitary stalk with loss of the pituitary bright spot on
(25%), at presentation in a large Indian center,7 while T2-weighted images. Pituitary biopsies are rarely done
the French in a series of nearly 350 patients found it for diagnosis, and are only indicated when the stalk is
in only 5 percent at presentation.10 This may represent greater than 6.5 mm or there is a hypothalamic mass.
natural evolution in a country like ours where many Most often the diagnosis is established by biopsying
patients tend to present late. Massive splenomegaly the other sites of involvement in patients who also have
may lead to cytopenias due to hypersplenism and pituitary abnormalities. LCH patients with diabetes
may cause respiratory compromise. Splenectomy may insipidus have a 50 to 80 percent chance of developing
provide transient relief of cytopenias, but should be other organ involvement diagnostic of the disease
done only as a life-saving measure. within one year of onset of diabetes insipidus.7, 21, 23, 36-38
• Other gastrointestinal manifestations in LCH are • Ocular: Ocular involvement in LCH is very rare.
diarrhea, hematochezia, perianal fistulas, or malab Sometimes it may lead to blindness. Patients may have
sorption.27,28 Diagnosis of gastrointestinal involvement other organ systems involved, and this form of LCH
in LCH is difficult because of patchy involvement. rarely responds well to conventional chemotherapy.39
• Central nervous system: Apart from mass lesions Tests and Procedures
in the hypothalamic-pituitary region, LCH may also
involve the choroid plexus, the gray matter, or white • Blood tests: These include complete blood count
matter. CD1a-positive LCH cells and CD8-positive and biochemical evaluations that include liver
lymphocytes are present in these lesions. and renal function tests and serum electrolytes. A
Chronic neurodegenerative syndrome manifested coagulation work-up with prothrombin time/partial
by dysarthria, ataxia, dysmetria, and sometimes beha thromboplastin time in patients with hepatomegaly
vioral changes may develop in one to four percent of and jaundice should also be done.
LCH patients, and sometimes, these neuropsychologic • Urine tests: Apart from routine urinalysis, a water-
dysfunctions may be severe.37 MRI scan may show deprivation test must be done if diabetes insipidus is
hyperintensity of the dentate nucleus and white suspected.
matter of the cerebellum on T2-weighted images • Bone marrow aspirate and biopsy: This is indicated
or hyperintense lesions of the basal ganglia on T1- in all patients with multisystem disease who have
weighted images and/or atrophy of the cerebellum.38 unexplained anemia or thrombocytopenia. The bone
These radiologic findings may precede the onset marrow biopsy sample should be stained with anti-
of symptoms by many years or found coincidently. CD1a and/or anti-CD207 (langerin) and anti-CD163
The neurodegenerative form of the disease has immunostains for the detection of LCH cells.
been compared to a paraneoplastic inflammatory • Radiologic and imaging evaluation: This is the
response.37, 38 most important aspect of work-up for a case of
LCH. Mandatory in all cases as a first screening, is
DIAGNOSTIC EVALUATION OF LANGERHANS a complete skeletal survey with skull series, bone
scans, and a chest X-ray. Radionuclide scanning
CELL HISTIOCYTOSIS has been shown to provide no additional benefit as
Diagnostic evaluation of LCH must proceed along logical against its suggested complimentary role in earlier
and established lines. This helps to correctly identify risk studies.40- 42
groups as well established by the LCH trials. Incorrectly
assigning risk groups due to faulty or incorrect assessment Fludeoxyglucose F18 (18F-FDG)
test or overdiagnosis due to a very sensitive test makes
comparisons difficult in what still remains a rare disorder. The PET scans have proved to be the most sensitive
The strength of the diagnostic criteria established by the technique in detection of involvement and for assessing
LCH trials has been validated even when applied in a response to treatment and in follow-up of patients with
retrospective analysis, proving there robustness.7 The LCH,43 but is as yet not recommended to replace the
current recommendations have been recently summarized standard tests14 as its exact role is still being evaluated.
in an exhaustive review.14 Depending on the clinical scenario in a given case,
Table 1 Recommended additional diagnostic testing in a case of LCH40

Clinical scenario and recommended additional testing
History of polyuria or polydipsia
• Early morning urine specific gravity and osmolality
• Blood electrolytes
• Water deprivation test if possible
• MRI of the head
Bicytopenia, pancytopenia, or persistent unexplained single cytopenia
• Other causes of anemia or thrombocytopenia has to be ruled out according to standard medical practice. If no other causes are
found, the cytopenia is considered LCH-related
• Bone marrow aspirate and trephine biopsy to exclude causes other than LCH
• Evaluation for features of macrophage activation and hemophagocytic syndrome (triglycerides and ferritin in addition to coagulation
studies)
Liver dysfunction
• If frank liver dysfunction (liver enzymes >5-fold upper limit of normal/bilirubin >5-fold upper limit of normal): consult a hepatologist
and consider liver MRI which is preferable to retrograde cholangiography
• Liver biopsy is only recommended if there is clinically significant liver involvement and the result will alter treatment (i.e. to
differentiate between active LCH and sclerosing cholangitis)
Contd...
Contd...
Lung involvement
• Further testing is only needed in case of abnormal chest X-ray or symptoms/signs suggestive of lung involvement, or pulmonary
findings not characteristic of LCH or suspicion of an atypical infection
• High resolution-computed tomography (HR-CT) is preferred mode
• Only cysts and nodules are typical of LCH; all other lesions are not diagnostic
• In children already diagnosed with MS-LCH, low dose CT is sufficient to assess extent of pulmonary involvement, and reduce radiation
exposure
• Lung function tests (if age appropriate)
• Bronchoalveolar lavage (BAL): >5% CD1a + cells in BAL fluid may be diagnostic in a nonsmoker
• Lung biopsy (if BAL is not diagnostic)
Suspected craniofacial bone lesions including maxilla and mandible
• MRI of head including the brain, hypothalamus-pituitary axis, and all craniofacial bones. If MRI not available, CT of the involved bone
and the skull base is recommended
Aural discharge or suspected hearing impairment/mastoid involvement
• Formal hearing assessment
• MRI of head or HR-CT of temporal bone
Vertebral lesions (even if only suspected)
• MRI of spine to assess for soft tissue masses and to exclude spinal cord compression
Visual or neurological abnormalities
• MRI of head
• Neurological assessment
• Neuropsychometric assessment
Suspected other endocrine abnormality (i.e. short stature, growth failure, hypothalamic syndromes, or delayed puberty)
• Endocrine assessment (including dynamic tests of the anterior pituitary and thyroid)
• MRI of head
Unexplained chronic diarrhea, failure to thrive, or evidence of malabsorption
• Endoscopy
• Biopsy
Adapted from Haupt, et al40
additional specific testing is required. These are included surgery, radiation therapy, or oral and topical
summarized in Table 1. medications. Later intravenous chemotherapy was also
• Biopsy: This remains the gold standard for establishing used. The earliest chemotherapy trials were from the
LCH, and is indeed mandatory for a confirmed German-Austrian-Dutch (Deutsche Arbeitsgemeinschaft
diagnosis, except in the cases of isolated vertebra plana für Leukämieforschung und-therapie im Kindesalter
without a soft tissue mass or isolated pituitary stalk [DAL]) Group trials.44 In updated results from these trials
disease when the risk outweighs the benefits.14 guidelines for formulations for single-system and multi-
Lytic bone lesions, skin, and lymph nodes are the most system disease were made.4,22 However, since the mid
frequent sites biopsied.7,14 Biopsies from other sites are nineties, most of the European study groups merged under
indicated only in specific situations already summarized the umbrella of the LCH trials, and were increasingly joined
in Table 1. by the North American groups. By the time of the launch
The LCH cells are large cells with abundant pink of the LCH IV trial in 2013–14, will become a truly global
cytoplasm on hematoxylin and eosin staining with a bean- study group, with representation from all continents. Most
shaped folded nucleus. LCH cells stain with anti-CD1a or of the current recommendations on treatment are based
anti-langerin (CD207) and any one of these is essential to on the LCH I, II and III trials.
confirm the diagnosis of LCH. Other types of histiocytes
and macrophages may stain with S-100, which is not Low-Risk Disease (Single-System or
considered sufficient to establish the diagnosis of LCH.14 Multisystem)
TREATMENT OF LANGERHANS CELL • Isolated skin involvement has been historically
treated with topical steroids, oral methotrexate, oral
HISTIOCYTOSIS thalidomide, topical application of nitrogen mustard,
Treatment of langerhans cell histiocytosis (LCH) depends and later with psoralen and UV light. However, if no other
on the site(s) and extent of disease. Treatment historically site is involved, most lesions resolve spontaneously and
only observation would be required. Single-site, single So drugs that cross the blood-brain barrier, such as
lesion disease similarly requires observation only.14, 22 cladribine (2-CdA), or other nucleoside analogs, such
• Skull lesions in the mastoid, temporal, or orbital bones as cytarabine, seem to be the best option for active CNS
form a risk group for later CNS involvement and DI LCH lesions.48-50 For treatment of symptoms of LCH CNS
(CNS-risk lesions) and need to be treated with 6 to 12 neurodegenerative syndrome, dexamethasone, retinoic
months of vinblastine and prednisone to decrease the acid, intravenous immunoglobulin (IVIg), infliximab,
risk of developing DI.5 with or without vincristine have been used. 51, 52
For instability of the cervical vertebrae and in patients
with neurologic symptoms bracing or spinal fusion Treatment of Recurrent, Refractory, or
may be needed. Chemotherapy is often successful in Progressive Childhood Langerhans Cell
patients with soft tissue extension from the vertebral
lesions.20, 22
Histiocytosis
• Multiple bone lesions; or combinations of skin, lymph Various strategies have been evolved to manage LCH
node, or pituitary gland with or without bone lesions— patients with recurrent, refractory, or progressive
should be treated with 12 months of vinblastine and LCH. Optimal therapy for these patients has not been
prednisone. A short (≤ 6 months) treatment course determined. Low-risk recurrence occurring after comple
with only a single agent (e.g. prednisone) results in a tion of planned treatment can be treated with a reinduction
higher number of relapses compared to combination of vinblastine and prednisone for 6 weeks. Cladribine (2-
chemotherapy.22,45 Pamidronate is also effective in CdA) has also been used effectively for recurrent low-risk
LCH with bone lesions.46 LCH (multifocal bone and low-risk multisystem LCH).53
For patients having refractory high-risk organ
High-Risk Multisystem Disease involvement therapy needs to be changed early. Evaluation
points at 6 and 12 weeks post initiation of induction are
• The standard therapy length recommended for LCH predictive of outcome. For example, those with progressive
involving the spleen, liver, or bone marrow (high-risk disease after 6 weeks of standard treatment, or partial
organs) is based upon LCH-I, LCH-II, and the DAL- response by 12 weeks require new treatment plan, as
HX-83 studies and varies from 6 months (LCH-I and they have only a 10 to 50 percent chance of surviving.5,6,47
LCH-II) to 1 year (DAL-HX-83).4-6,44 The LCH-II and Patients with refractory high-risk organ (liver, spleen, or
LCH-III studies used a standard arm consisting of bone marrow) involvement and resistant multisystem
vinblastine and prednisone but 6-mercaptopurine low-risk organ involvement have been treated with an
was added to the continuation phase of the proto intensive acute myeloid leukemia–like protocol. Prompt
col. These two studies also conclusively proved change of therapy to cladribine (2-CdA) and/or cytosine
that treatment intensification,47 and prolongation,6 arabinoside may provide an improvement in overall
works better for multisystem LCH. The LCH-II survival (OS).54, 55
study was a randomized trial which compared treat Hematopoietic stem cell transplantation (HSCT)
ment of patients with vinblastine, prednisone, and has been used for multisystem high-risk organ disease
mercaptopurine or vinblastine, prednisone, mercap refractory to chemotherapy.56, 57
topurine, and etoposide.47 There was no statistical
significance in outcomes (response at 6 weeks, 5-year
probability of survival, relapses, and permanent
REFERENCES
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Hence, etoposide has not been used in subsequent histiocytosis ‘insight into DC biology’. Trends Immunol.
Histiocyte Society trials. The LCH-III study rando 2003;24(4):190-6.
mized risk organ-affected patients to either velban/ 2. Allen CE, Li L, Peters TL, et al. Cell-specific gene expression
in Langerhans cell histiocytosis lesions reveals a distinct
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C H A P T E R 46
Hemophagocytic
Lymphohistiocytosis: Revisited
INTRODUCTION To understand HLH we must understand the function

of NK cell and cytotoxic T cells. Both are designed to kill
Hemophagocytosis lymphohistiocytosis (HLH) is a virally infected cells.
disorder of immune dysregulation and is not an uncommon Natural killer cells are our innate immune system
disorder encountered at a tertiary care. It is a potentially cell that comprise 10 to 15 percent of peripheral blood
fatal hyperinflammatory syndrome with high-grade fever, lymphocytes and are like Rambo’s of the immune system
organomegaly and characteristic laboratory abnormalities with ready to kill receptors but have to be inhibited by
like pancytopenia, coagulopathy, hyperferritinemia, receptors for MHC class I molecule, which override the
hypertriglyceridemia and hemophagocytosis.1-4 kill signal. Thus, MHC class I molecule protect us from NK
It was first described in 1939 by Scott and Robb Smith cell toxicity. Cytotoxic T cells are produced as an adaptive
and was initially considered a malignant histiocytic disorder response to any infection. They recognize cytosolic protein
called Malignant Histiocytic Reticulosis.5 Subsequently antigen (e.g. viral proteins) which are presented on the
Farquhar and Claireaux provided its correct description major histocompatibility complex (MHC) class I molecule.
in 1952.6 Risdall et al. in 1979 was the 1st to recognize Cytotoxic T cells are specific for that infection and are
this as a reactive hemophagocytosis secondary to viral produced later after 4 to 5 days of infection. They are the
infection in a cohort of 19 highly immunocompromised atypical lymphocytes seen in infectious mononucleosis
postrenal transplant patients and he coined the term and express Cd3+ and Cd8+ on their surface.11
virus-associated hemophagocytic syndrome (VAHS). He
observed that though it was a benign disorder the mortality
was extremely high and most had infections with herpes
Familial Hemophagocytic
group viruses like EBV and CMV.7,8 Lymphohistiocytosis
Acquired HLH is commonly seen with infections (IAHS– Inheritance of familial HLH is autosomal recessive. Up
Infection associated Hemophagocytic Syndrome). The till now there are 5 genes discovered in familial HLH. Of
most common infections, which trigger HLH are EBV, CMV, which most common is perforin gene (10q21) i.e. PRF 1
HSV, HHV, Koch’s, Salmonella, Malaria, Kala azar in the mutations seen in 20–40 percent of cases.12 NK cell and
Indian set up but virtually any infection can trigger HLH.9,10 cytotoxic T cells eliminate a virally infected target cell via
the Perforin Granzyme pathway. Perforin is a protein like
PATHOGENESIS Complement C5-9, it perforates the target cell membrane
forming a channel thus allowing Granzyme B to enter the
The hallmark of HLH is defective NK cell and cytotoxic T
target cell and induce apoptosis by activating the apoptotic
cell activity. NK cells and cytotoxic T cells can be recognized
mechanism.13-15 Recent studies suggest that granzyme B
morphologically as large lymphocytes with azurophilic
can enter into target cells, independent of perforin,16-18
granules in wright preparation. These granules contain
but granzyme alone is not sufficient to induce toxicity.
apoptosis inducing machinery the Perforin protein and
Once NK cell or Cytotoxic T cell binds the virally infected
Granzyme B.
Chapter-46 Hemophagocytic Lymphohistiocytosis: Revisited 471
cell they form an immunologic synapse at the contact and results in further activation of immune system by
site. All the subsequent events occur at the immunologic antigen presenting cell. The cytokine IFNG activate the
synapse. The azurophilic granule undergoes steps of macrophages that result in hemophagocytosis, thus giving
vesicle maturation (LYST protein), polarization to the the name to this syndrome30 (Fig. 2).
immunologic synapse (AP3B1 and SH2D1A), docking Not only NK cells and cytotoxic CD8+ve T cells but
(RAB 27α), and priming (Unc13)19, vesicle fusion (Syntaxin also other cells with cytotoxic activity like CD4+ cytotoxic
11), vesicle docking, priming and fusion (MUNC 18-2) T cells and iNKT cells participate in pathogenesis of HLH.
before it fuses with the surface membrane and releases There is some correlation between hyper cytokinemia and
its content with in the immunological synapse (Fig. 1). the diverse clinical manifestations in HLH.
XLP (X-linked lymphoproliferative disorder) is of two
types XLP1 due to defect in SAP protein and XLP2 due Secondary Hemophagocytic
to defect in XIAP (X-linked Inhibitor of Apoptosis) gene. Lymphohistiocytosis
60% of XLP1 patients develop EBV induced HLH while
In most cases of secondary HLH cytotoxicity and cytotoxic
90% of XLP2 develop HLH. Currently it XLP2 is being
lymphocyte degranulation are not impaired31 (Figs 3 and 4).
reclassified as HLH causing disease rather than causing
There is increases APC activation that disrupt the balance
lymphoproliferation.20-25
between APC activation and CTL-mediated control.
APC can directly activated by intracellular pathogens for
Genes Associated with Familial example via toll-like receptor (TLR) activation.
Hemophagocytic Lymphohistiocytosis Based on in vitro analyses, four distinct pathways of
macrophage activation have been described. Classical
(Table 1, Fig. 2)
activation via interferon-γ or lipopolysaccharide induces
Inability to kill target cells by NK cell and cytotoxic T cells microbicidal activities and upregulation of expression of
following events can occur. There is excessive stimulation class II MHC. Alternative activation by interleukin 4 or 13
of the immune system, increase in antigen presentation induces the expression of genes that are involved in tissue
and increase in T cell proliferation with infiltration of repair or suppression of inflammation. Innate activation
various organs like CNS, liver, spleen, and lymph nodes. via toll-like receptor ligands also, not surprisingly, induces
Ultimately this results in hyper cytokine storm producing microbicidal activities. Deactivation by stimulation of
mainly TNF alpha, INFG (most important), IL1, Gm-CSF. interleukin-10 or transforming growth factor-β reduces
The Antigen Presenting Cell needs to be culled by NK cells class II expression and increases secretion of anti-
to achieve immune homeostasis. This does not happen inflammatory cytokines.32-34
Fig. 1 Pathogenesis of hemophagocytic lymphohistiocytosis. Depicts the events occurring at the immunologic synapse and highlights
molecules important in process of granule exocytosis. Abbreviations: CHS: Chédiak-Higashi syndrome; FHL: Familial hemophagocytic
lymphohistiocytosis; GS: Griscelli syndrome; HP: Hermansky–Pudlak; NK: Natural killer cell; CTL: Cytotoxic T lymphocyte1, 29
Table 1 Genes associated with familial HLH

Disease Locus Gene Gene symbol Function
FHLH 1 9q22.1-23 Unknown Unknown
FHLH 2 10q22 Perforin PRF 1 Pore forming protein
FHLH 3 17q25 C. elegans Unc13 MUNC 13-4 Vesicle priming
FHLH 4 6q24 Syntaxin 11 STX11 Vesicle fusion
FHLH 5 c. 1697G > A MUNC 18-2 (STXBP2 MUNC 18-2 Vesicle Bocking, priming and
p. G566D gene) fusion
Griscelli syndrome type 2 5q21 Ras ass protein RAB27A Vesicle docking
HSP (Hermansky-Pudlak type II) Chr. 10 AP3B1 AP3B1 Granule polarization
CHS (Chédiak-Higashi syndrome) 1q42.1–42.2 Lysosomal trafficking LYST Vesicle maturation
regulator
XLP 1 Xq25 SLAM ass protein (SAP) SHD2D1A Granule polarization
XLP 2 Xq24-25 XIAP BIRC4 Inhibition of apoptosis
Abbreviations: EBV: Epstein-Barr virus; FHLH: Familial hemophagocytic lymphohistiocytosis. Adopted from1,26-28
Fig. 2 Patients of HLH due to genetic defect; there is inability to kill virally infected cell as well as antigen presenting cells resulting in
massive clonal expansion of CTLs, secretion of their cytokines like interferon gamma (IFNg), tumor necrosis factor (TNF) alpha, IL6, IL18 and
granulocyte macrophage colony stimulating factor (GM-CSF). IFNg activate macrophages, which then phagocytose blood cells resulting
in hemophagocytosis. Since the cytokines are produced in a massive amount by macrophages, T cells and NK cells, hyperstimulation
continues and the patient has a cytokine storm with signs and symptoms of HLH. The normal contraction of the immune system also
does not take place resulting in persistent cytokine secretion, infiltration of various organs by T cells, massive tissue necrosis and organ
failure
Fig. 3 Natural killer cell and cytotoxic T lymphocyte (CTL) response to virally infected cells. In normal patients there is clonal expansion,
secretion of interferon gamma and killing of virally infected cell resulting in control of viral infection. Once infection is controlled the CTL
are culled and immune homeostasis achieved. Some of these cells become memory cells
A B
Figs 4A and B Neutrophil phagocytosis and erythrophagocytosis respectively
Types and Causes of HLH35 sensorium seen in 30% of cases.36,37 In early course of the
disease, hemophagocytosis may not be obvious on bone
• Genetic causes of HLH
marrow examination study and may need repeat bone
– Familial HLH
marrow if clinical suspicion is strong38 (Figs 4A and B).
– Pigmentary dilution disorders
Incidence of FHLH is 1 in 5000 live births.39 Ratio of
- Chédiak-Higashi syndrome
male and female affection is equal. Almost 70% of FHLH
- Griscelli syndrome type 2
are diagnosed in first year of life, with peak age between
- Hermansky-Pudlak syndrome type II
1 and 6 month. With availiblity of genetic testing, it is
– X-linked lymphoproliferative (XLP) disease type 1
possible to point out the first significant episode of FHLH
and 2
throughout life,40 including in utero.
• Infection associated HLH
In 1987, the Histiocyte Society adopted the unifying
– Viruses-EBV, CMV, HHV-6, HHV-8, HIV, adeno,
term hemophagocytic lymphohistiocytosis (HLH) and
hepatitis, parvo virus.
defined a set of diagnostic criteria to assist clinicians and
– Bacteria numerous including Koch’s, Salmonella.
researchers (Table 2). The criteria have subsequently been
– Parasites-Malaria, Kala azar in the Indian set-up
refined to account for advances in our understanding of
– Spirochetal, and fungal-associated infections.
the syndrome, and to simplify it for practical usage. In 2009,
• Malignancy associated HLH: Leukemia lymphoma,
Filipovich et al has revised the HLH diagnostic criteria,
GCT.
which are practical usage and very much applicable in our
• Macrophage activation syndrome (MAS) associated
set-up as shown in Table 3.
with autoimmune disease like rheumatoid arthritis,
The HLH must be suspected in setting of rapidly
SLE.
evolving cytopenias, LFT dysfunction, organomegaly,
coagulopathy. It is prudent to ask for serum ferritin and
Clinical Features triglycerides with ‘D’ dimer. If ferritin is >500 ng/mL and
Presentation of HLH in initial period is nonspecific and specially >3000 ng/mL a BMA done to rule out HLH.
easily confused with common infection, autoimmune
disorders and malignancy.1 HLH typically presents Macrophage Activation Syndrome Associated
with prolonged fever; unresponsive to antibiotics, with Autoimmune Disease
hepatosplenomegaly, rash (6–65%), lymphadenopathy,
cytopenias, liver dysfunction, hypofibrinogenemia, Macrophage activation syndrome (MAS) is observed in
hypertriglyceridemia, hypoalbuminemia, hyponatremia. number of autoimmune disorders, infections and neo-
In initial course,CNS manifestations in form of irritability, plasms. It is caused by an excessive proliferation and activa-
hypo- or hypertonia, cranial nerve palsies, meningismus, tion of macrophages. Incidence of MAS in systemic onset
signs of increased intracranial pressure and altered juvenile inflammatory arthritis is 7-30% and usual triggers
Table 2 Diagnostic guidelines for hemophagocytic lymphohistiocytosis (2004)35,38

The diagnosis of HLH can be established if one of either 1 or 2 below is fulfilled
1. A molecular diagnosis consistent with HLH
2. Diagnostic criteria for HLH fulfilled (five out of the eight criteria below)
Clinical criteria
i. Fever
ii. Splenomegaly
Laboratory criteria
i. Cytopenias (affecting >2 of 3 lineages in the peripheral blood): HB<9 g/dL (in infants <4 weeks: HB<10.0 g/dL), platelets
<100,000/mm3, neutrophils <1,000/mm3.
ii. Hypertriglyceridemia and/or hypofibrinogenemia: Fasting triglycerides > 3 SD, fibrinogen < 3 SD
iii. Hemophagocytosis in bone marrow or spleen or lymph nodes. No evidence of malignancy.
New diagnostic criteria
i. Low or absent NK-cell activity (according to local laboratory reference)
ii. Ferritin >500 μg/L
iii. Soluble CD25 (i.e. soluble IL-2 receptor) >2,400 U/mL.
Adapted from Treatment Protocol of the 2nd International HLH Study, 200435,38
Table 3 Filipovich HLH diagnostic criteria 200941 – Lymph node biopsy

Molecular diagnosis of HLH or XLP OR • Sophisticated lab investigations
At least 3 of 4 – sCD25 >2400 IU/L
• Fever – NK cell activity (may be normal in 30% of cases)
• Splenomegaly – Serum Beta 2 microglobulin
• Hepatitis • Etiological work-up
• Cytopenias – Anti-EBV VCA IgM, PCR
And at least 1 of 4
– Anti-CMV Abs, antigen, PCR
• Hemophagocytosis
– Appropriate microbiological cultures
• Hyperferritinemia
– Hair mount studies for pigmentary dilution
• Increased soluble IL2R alpha
disorders.
• Absent or very decreased NK cell function
• Work-up for familial HLH (Flow chart 1)
– Perforin by flow cytometry
Supportive of HLH
– Granule release assay (GRA)
• Hypertriglyceridemia
– Gene sequencing to identify mutations.
• Hypofibrinogenemia
• Hyponatremia
Adapted from hematology ash education book. 2009;1:127-31. MANAGEMENT OF HEMOPHAGOCYTIC
LYMPHOHISTIOCYTOSIS31
Principle of Treatment
are gold therapy, aspirin, viral infection and some reports
with anti TNF α antibodies.41 Other diseases associated The immediate aim of treatment is to suppress the severe
with MAS are SLE, Kawasaki disease and other rheumatic hyperinflammation. The secondary aim is to eliminate
diseases.42,43 Specific parameters taken into consideration pathogen activated antigen presenting cells (APCs) so as
are falling WBC and platelet counts, hyperferritinemia, to remove the stimulus for ineffective activation of T cells.
hypofibrinogenemia, hemophagocytosis in bone marrow, The treatment recommended is HLH 2004 protocol that is
elevated liver enzymes, elevated erythrocyte sedimenta- devised by Histiocytic Society. It essentially consists of 3
tion rate, and hypertriglyceridemia.44,45 It can be presenting drugs:
manifestation of autoimmune disorder and features of such • Dexamethasone is lympholytic, inhibit expression of
diseases (such as arthritis or rash) should therefore be care- cytokines, and suppresses maturation of APCs, better
fully looked in patient with HLH. CNS penetration hence preferred over prednisolone.
• Cyclosporine A prevents T cell activation and
Work-up for Patient of Hemophagocytic proliferation.
Lymphohistiocytosis38
Laboratory evaluation of HLH is directed with following Flow chart 1 Algorithm for investigation of HLH
considerations:
• Establish diagnosis of HLH
– CBC platelet
– ESR
– PS (look for peripheral blood HLH)
– LFT
– Creatinine
– LDH
– Serum electrolytes
– Serum ferritin
– Serum triglycerides
– Coagulation profile (PT, PTT, plasma fibrinogen
and ‘D’ dimer)
– CSF for pleocytosis and elevated proteins (50% of
cases).
• Supportive evidence for HLH
– PB or bone marrow aspiration
– Liver biopsy
• Etoposide (VP-16) has activity against monocytes • HLA mismatched unrelated donor: 54 percent ± 27
and macrophages, inhibits EBNA synthesis and EBV percent
infected cells. • Haploidentical related donor: 50 percent ± 24 percent.
• IV gammaglobulins provide cytokine and pathogen
specific antibodies and immunomodulation. Disease Directed Therapy
Antileishmania therapy, antilymphoma therapy. Please
HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS note in secondary HLH pathogen directed therapy is
2004 PROTOCOL not sufficient to control the hyperinflammation. Leish-
maniasis treated with liposomal amphotericin B is the
Initial Therapy (8 Weeks) only exception. In patients with EBV-induced HLH or XLP
• Dexamethasone, 10 mg/m2/day for 2 weeks followed with fulminant EBV induced HLH use injection rituximab
by a decrease every 2 weeks to 5 mg/m2, 2.5 mg/m2 and (monoclonal Ab to CD 20) at doses of 375 mg/m2 weekly for
1.25 mg/m2 for a total of 8 weeks. 4 weeks, to knock out the B cells harboring EBV in addition
• Etoposide IV, etoposide 150 mg/m2 IV, twice weekly to HLH directed therapy. This strategy works very well to
for the first two weeks, then weekly during the initial control EBV induced HLH and in fulminant infectious
therapy. Paradoxically even if ANC <0.5 × 109/L and mononucleosis. In desperate situations, anti-TNF alpha-
the bone marrow is hypocellular, at least the first two receptor blocking agents have been tried.47-50
doses should be given. Macrophage activation syndrome: In addition to
• Cyclosporine A, aiming at levels around 200 microg/L corticosteroids, CSA has been found effective in patients
(monoclonal, trough value). Start with 6 mg/kg daily with corticosteroid-resistant MAS.51
orally (divide in 2 daily doses), if normal kidney The French group does not use etoposide and they use
function. anti-thymocyte globulin (ATG) to control HLH.52
• Intrathecal methotrexate (IT MTX), age-adjusted doses Once inflammation is controlled a search for a potential
of intrathecal methotrexate weekly for 3 to 6 weeks as bone marrow donor is done and if a match is available the
follows if there are progressive neurological symptoms child should be transplanted to achieve a cure. In HLH,
or if abnormal cells persist in the CSF. 1994 protocol median survival was 64 percent with overall
• Supportive therapy: Cotrimoxazole eq 5 mg/kg survival (OS) of 55 percent.46
of trimethoprim 2 to 3 times weekly (week 1 and If there is poor or no response to 4 weeks of HLH
onwards), an oral antimycotic (from week 1 to 9), IV 2004 protocol treatment, HLH is probably refractory
immunoglobulin (0.5 g/kg) every 4 weeks. and continuing HLH 2004 protocol will be of no further
benefit. Salvage treatment options that can be tried in
Continuation Therapy (9–40 Weeks) such a situation are ATG, fludarabine, alemtuzumab,53,54
Daclizumab, anti TNF-alpha receptor blocking agents,
The continuation therapy is a continuation of the initial anti-interferon gamma antibodies, chemotherapeutic
therapy with the major aim to keep the disease nonactive agents. There is no established salvage regime. ATG is
week 9 to 40. Increasing disease activity may make it rarely effective, if etoposide-based regimen has been
necessary to intensify the treatment in some children. ineffective. Search for BM donor should be done and one
Patients with nonfamilial disease and no genetic evidence should attempt to transplant these patients.
of HLH, are suggested to start continuation therapy only In secondary HLH treatment is given for 8 weeks. HLH
if the disease is active after the initial therapy. Etoposide re-evaluation is done and if normal treatment is stopped
150 mg/m2 IV, every second week. Dexamethasone pulses and close follow-up including signs of reactivation
every second week, 10 mg/m2 for 3 days. Cyclosporin A are warranted (such as fever, hepatosplenomegaly,
aims for blood levels around 200 microgram/L, as above. neurological abnormalities; hemoglobin, platelets, WBC,
Monitor GFR. ANC, ferritin, transaminases). Once hyperinflammation
is controlled there can be reactivation of HLH or
Indications for BMT development of CNS events. Intrathecal MTX would
benefit for CNS activation. The HLH therapy is reinforced
• Familial hemophagocytic lymphohistiocytosis (FHLH) in case of systemic reactivation. If the patient develops
• Relapsing secondary HLH. a reactivation, it is recommended to intensify therapy,
Results of BMT are:46 such as to restart from week 2, but the initial therapy may
• Matched related donor: 71 percent ± 16 percent be less than 8 weeks, and then continue with modified
• Matched unrelated donor 71 percent continuation therapy. Add intrathecal therapy in case of
CNS reactivation. Consider dexamethasone daily, also N, Révész T, Egeler RM, Jahnukainen K, Storm-Mathiesen
between the dexa-pulses, in continuation therapy, but be I, Haraldsson A, Poole J, de Saint Basile G, Nordenskjöld
aware that it may lead to severe side-effects, so an early M, Henter J-I. Spectrum of perforin gene mutations in
SCT is then suggested. familial hemophagocytic lymphohistiocytosis. Am J Hum
Genet. 2001;68(3):590-7.
Poor prognostic factors: Degree CSF pleocytosis, 13. Lichtenheld MG, Podack ER. Structure of the human
severity of thrombocytopenia, hyperbilirubinemia and perforin gene. Simple gene organization with interesting
hyperferritinemia are important risk factors for outcome potential regulatory sequences. J Immunol. 1989;143:4267-
in HLH. HLH associated with EBV infection with high 74.
viral load is associated with poor outcome.56 Persistent 14. Tschopp J, Nabholz M. Perforin-mediated target cell
fever and thrombocytopenia also associated with poor lysis by cytolytic T lymphocytes. Annu Rev Immunol.
outcome.55 1990;8:279-302.
15. Lowin B, Peitsch MC, Tschopp J. Perforin and granzymes:
Increasing awareness among pediatricians regarding
crucial effector molecules in cytolytic T lymphocyte
HLH; with early diagnosis and initiation of early treatment
and natural killer cell-mediated cytotoxicity. Curr Top
decreases morbidity and mortality. Bone marrow Microbiol Immunol. 1995;198:1-24.
transplantation cures disease with good success rate. 16. Darmon AJ, Nicholson DW, Bleackley RC. Activation of
the apoptotic protease CPP32 by cytotoxic T-cell-derived
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C H A P T E R 47
Bone Marrow Transplantation
Hematopoietic Stem Cells

Hematopoietic stem cells reside primarily in the bone marrow but do circulate in the peripheral blood. These cells may replenish
damaged or missing components of the hematopoietic and immunologic system.1
Hematopoietic stem cell transplantation was originally conceived more than 50 years ago. Initial studies done in animals showed
that transplantation of genetically identical material or the animal’s own marrow averted death. Studies in animals later translated to
work in humans and a team led by Dr E Donnall Thomas pioneered this. In 1959, he reported that a patient with end stage leukemia
sustained a remission for more than 3 months following total body irradiation and infusion of bone marrow from her identical twin.
Later in 1970s the same group performed identical sibling transplant in leukemic patients. This work laid the foundation for further
advances in hematopoietic stem cell transplantation and this was recognized by the 1990 Nobel Prize awarded to Dr E Donnall
Thomas.2
GRAFT TYPES IN HEMOTOPOIETIC Allogeneic transplants are hemopoietic stem cells

STEM CELLS from the bone marrow, peripheral blood, or umbilical
cord blood of a healthy donor matched for HLA type,
There are three different graft types that can be used for who may be a family member or an unrelated volunteer.
bone marrow transplant.3 Initially, allogeneic was developed for treatment of
1. Autologous hematological malignancies. Now it is being utilized
2. Allogeneic for a variety of hematological disorders and for non-
3. Syngeneic hematological disorders like inborn errors of metabolism
and autoimmune diseases.1
Autologous Transplants Syngeneic transplant: Syngeneic transplant involves
transplantation from a person sharing identical genetic
Autologous transplants use stem cells derived from the material, i.e. an identical twin.
patient’s own marrow or peripheral blood. Initially, this
was developed in order to rescue the bone marrow of
SOURCES OF STEM CELLS
patients undergoing chemotherapy.1
Autologous transplants are being increasingly incorpo Bone marrow: Bone marrow obtained by repeated
rated in protocols for solid tumors like neuroblastoma. aspiration of posterior iliac crests while donor is under
This now is the most important form of stem cell general or local anesthesia is the traditional source of stem
transplantation performed worldwide. Stem cells can be cells (Fig. 1). This does not cause any side effects to the
stored without loss of viability and mortality is low with donor except for slight discomfort at the site of aspiration
this procedure. In addition, there is no risk graft versus and the requirement of packed cell transfusion in some
host disease. cases of pediatric donors.
Fig. 1 Bone marrow harvesting Fig. 2 Peripheral blood stem cell apheresis
Peripheral blood stem cells (PBSC): It was noted in early

1980s that marrow stem cells circulated in the peripheral
blood. Stem cell yield from peripheral blood can be
increased by giving bone marrow growth factors like
granulocyte colony stimulating factor. Stem cells are then
harvested by leukapheresis (Fig. 2). CD 34 cell surface
molecule is used as a surrogate marker of stem cells.4
G-CSF increases the proliferation of neutrophils and
causes release of proteases. PBSC causes the most rapid
hematopoietic reconstitution. But, it contains more T
cells than bone marrow. Peripheral blood stem cells are
Fig. 3 Umbilical cord blood stem cells
associated with increased risk of chronic GVHD.
Peripheral Blood Stem Cell Apheresis minorities and countries where marrow donor registries
do not exist.
Umbilical cord blood stem cells (Fig. 3): Cord blood of
neonates contains substantial number of hematopoietic Cord blood is also associated with:
stem cells that can be harvested at delivery, frozen and • Minimal GVHD due to naïve T cells in it.
then transplanted into a patient (Fig. 3). They can be • The disadvantage with cord blood is delayed
matched with potential donors without much delay. Cord engraftment due to the small number of stem cells
blood requires less stringent HLA matching than marrow/ • Hence infections are more common during the
peripheral blood stem cells. The first umbilical cord prolonged neutropenic period.
transplantation was done in 1988 for a child with Fanconi • The cell dose in one cord blood unit may not be
anemia from cryopreserved cord stem cells from an HLA sufficient for an older child or an adult.
matched sibling.5 With the emergence of cord blood The use of double cords has overcome this problem.6
banks with public cord blood banking facilities, they form A comparative analysis of the various sources of stem
an important source of stem cells especially for ethnic cells is given in Table 1.
Table 1 Comparison of various sources of stem cells

Bone marrow Peripheral blood Umbilical cord
Stem cell content Usually adequate Good can be increased, if needed Fixed source
T cell content Low High Low, naïve
HLA matching Close matching required Close matching required Close matching not very important
Engraftment Fast Fastest Slowest
Chapter-47 Bone Marrow Transplantation 481
HUMAN LEUKOCYTE ANTIGEN MATCHING Table 2 Common indications for hematopoietic

Allogeneic transplantations became feasible with the stem cell transplantation
identification and typing of human leukocyte antigen (HLA) Diseases commonly treated with hematopoietic
located on major histocompatibility locus on chromosome stem cell transplantation
6. There are two sets of genes on both alleles and hence they Malignant conditions Nonmalignant conditions
are inherited as haplotypes. Thus two siblings have one Autologous
chance in four of being HLA identical. The HLA loci that are
Acute myeloid leukemia Autoimmune diseases
important in the transplant setting include Class I antigens Hodgkin’s disease
(HLA A, B and C) and Class II antigens (HLA DR). For a Non-Hodgkin’s lymphoma
successful transplant, it is necessary to have matching at all Neuroblastoma, Ewing’s
these loci.1 The immune reaction against HLA molecules sarcoma
can cause problems. Mismatching at Class I antigenic loci, Allogenic
increase the chance of rejection of the donor, where as that
Acute myeloid leukemia Aplastic anemia
at Class II locus increases graft vs host disease.
Acute lymphoblastic leukemia Fanconi’s anemia
Chronic myeloid leukemia Paroxysmal nocturnal
DONOR REGISTRIES Myelodysplastic syndromes Hemoglobinuria
Myeloproliferative syndromes Diamond blackfan anemia
Only 20 to 25 percent of patients eligible for allogeneic
Non-Hodgkin’s lymphoma Dyskeratosis congenita
transplantation will have suitable sibling donors. To
Thalassemia
make transplants available to a greater number of eligible Sickle cell disease
patients, bone marrow donor registries have been esta Glanzmann thrombasthenia
blished in several countries. This will identify unrelated Severe combined
but matched donors for prospective patients. With the immunodeficiency
establishment of international marrow donor registries Wiskott-Aldrich syndrome
there are good chances of finding a matched unrelated Chronic granulomatous disease
donor depending on the ethnic group. For patients from Congenital neutropenia
Asia and Indian subcontinent, the probability of finding a Congenital megakaryocytosis
donor of Asian origin is low due to the poor representation Inborn errors of metabolism
in these registries and due to the absence of local
registries. The strongest transplant reactions occur when
the major histocompatibility antigens of the donor and of
the recipient are incompatible. gently shaken during the procedure to avoid the formation
The HSCT has resulted in sustained remission in of clots. The puncture site is changed after 10 to 15 mL is
patients with autoimmune diseases. SCT results in aspirated from the site. After collection of the desired
re-education of the immune system and hence is now volume, bony spicules and clumps are filtered out and the
used for refractory rheumatoid arthritis and other final product is made ready for infusion.7
autoimmune diseases. HSCT cures many genetic diseases Peripheral blood stem cells can be collected after
like thalassemia and sickle cell disease in developing being mobilized from the bone marrow by G-CSF given at
countries such ‘one shot’ treatments are highly desirable a dose of 10 µg/kg/day for 4 to 5 days. PB stem cells are
because chronic treatments often are difficult to sustain. then collected by leukapheresis. Neither anesthesia nor
Among the above mentioned indications, immuno hospitalization is required for the donor.
deficiencies, certain genetic disorders and severe aplastic Children may experience minor side effects related
anemia deserves urgent referral to a transplant center for to G-CSF use like body aches and influenza like illness.
consideration for an early transplant (Table 2). Adequate vascular access and extracorporeal volume in
the circuit of leukapheresis are the main limiting factors
COLLECTION OF HEMATOPOIETIC for peripheral blood stem cell collection in small children.
Central venous catheter is usually inserted in subclavian
STEM CELLS
or femoral veins and should be sufficiently stiff to avoid
Bone marrow is harvested from posterior iliac crest and is collapse under the negative pressure while drawing
generally well tolerated. The donor needs to be admitted blood into the apheresis machine. In children adequate
and the procedure is done under general anesthesia. The priming of the extracorporeal circuit may be required
harvested marrow is collected in a special harvest bag in order to avoid hypotension.8 As the anticoagulation
with adequate anticoagulation. The harvest bag should be most commonly used during is citrate dextrose (ACD A),
hypocalcemia should be anticipated and managed with ing to the plasma levels or by using intravenous instead of
calcium boluses given under proper monitoring. Heparin oral busulfan.
may also be used for anticoagulation, but has been Non-myeloablative regimens are those that usechemo
observed to have higher frequency of bleeding during therapy agents/radiation in lower doses than men
catheter removal. tioned above. These regimens are immunosuppressive,
Umbilical cord blood is collected at the time of delivery but do not destroy the entire recipient’s marrow. The
by clamping the cord and cutting the umbilical cord. The advantage of this regimen is that the toxicity associated
median volume collected is around 60 mL. Once collected, with the conditioning regimen is significantly less. It is
it is then processed and stored in liquid nitrogen till further immunosuppressive as well and can be used in patients
use. with comorbidities. So also in case of malignancies the
Umbilical cord blood is collected after clamping residual cells of the recipient can exert a graft versus tumor
the cord. The collected blood is tested, processed and effect. However, the risk of rejection of the graft increased
cryopreserved. At the time of use, the cord blood cassette with non-myeloablative regimens.1
is transported in liquid nitrogen.
After collection of the marrow/peripheral blood STEM CELL INFUSION
stem cells, the product can be infused immediately, or
may be cryopreserved and stored till need arises. Graft After the preparative regimen, the processed stem cells
manipulation like T cell depletion may be done when are infused intravenously. The patients are kept in HEPA
required. In case of major or minor ABO incompatibility, it filtered rooms and are on prophylactic antifungals,
is necessary to either deplete the red cells or plasma in the antibiotics and antiviral agents (Fig. 4).
product as required. Engraftment is defined as absolute neutrophil count
more than 500/cumm for more that 3 days consecutively.
PREPARATIVE REGIMENS/CONDITIONING
COMPLICATIONS (FIG. 6)
REGIMENS
The chemotherapy or irradiation given prior to stem cell Early Effects
infusion is called conditioning regimen. The regimen
is intended to be myeloablative as well as immuno Mucositis: It is the most common complication of myelo
suppressive. The objective of myeloablation of the reci ablative preparative regimes especially with the use of
pient prior to transplant is to eradicate the recipient’s total body irradiation and melphalan. Other factors that
own bone marrow stem cells. In case of malignancies, contribute to mucositis include GVHD, use of methotrexate
such high doses of chemotherapy help in eradicating the for GVHD prophylaxis and co-existent infections. Oropha
cancer cells.1 The preparative regimen also augments the ryngeal mucositis results in painful ulcers in the mouth
antitumor immune response by causing a breakdown of and throat. It can also lead to mucoid diarrhea and pain
tumor cells, which results in flood of tumor antigens into abdomen. In addition to the considerable pain and need
the antigen presenting cells. This results in proliferation of
T cells that attack the surviving malignant cells.
Total body irradiation: It is both myeloablative and
immunosuppressive. The effects are independent of blood
supply, and the effects reach sites that are not accessible
by chemotherapy. It is also not associated with cross-
resistance to chemotherapy. Local shielding of organs
and fractionation of the total dose can reduce toxicity.
The toxicity and scarcity of facilities for TBI have led to
development of radiation free regimens.
Busulfan-Cyclophosphamide (Bu-Cy): In 1983, a regimen
of Bu with high doses of Cy proved effective in treatment
of acute myeloid leukemia. Acute adverse effects are
associated with high plasma levels of busulfan and me
tabolites of cyclophosphamide. The dose of cyclophos
phamide was later lowered to reduce toxicity. Toxicity can
also be reduced by adjusting the dose of busulfan accord Fig. 4 Bone marrow transplantation room (HEPA filtered)
for narcotics, mucositis leads to compromised enteral dependent on many factors including the condition
nutrition and predisposition to infections due to breach in ing regimen used. The pathogenesis of acute GVHD is
mucous membranes. Management includes prophylaxis described as a three-step process that includes
against herpes infections with acyclovir as well as against • Conditioning induced tissue damage phase:
candidal infections. There are several ongoing studies • Donor lymphocyte activation phase
with agents like glutamine, palifermin, medicated pastes, • Cellular and inflammatory effector phase.
topical lidocaine which have not yet been of proven The first phase consists of tissue damage induced by
benefit.9 conditioning regimen and infection. As a result of the
inflammatory cytokines released, there is maturation/
Sinusoidal obstruction syndrome/veno-occlusive disease
activation of host dendritic cells with subsequent recog
(SOS/VOD): Hepatic veno-occlusive disease is an organ
nition of host major and minor histocompatibility antigens
injury syndrome that occurs after high dose chemotherapy
by mature donor T cells. Despite matching of major HLA
employed in HSCT. After myeloablative conditioning
antigens, there may be minor HLA mismatches that may
in allogeneic transplantation VOD is seen in up to 10
be recognized as foreign. This results in activation of
percent of patients. It is potentially fatal syndrome of
the donor T cells against the recipient antigens. The IL
painful hepatomegaly, jaundice and fluid retention. Total
2 and IFN gamma produced by T helper 1 cells result in
body irradiation, busulfan, cyclophosphamide and many
activation of NK cells and cytotoxic T lymphocytes. Thus
other preparative regimens cause SOS. The metabolites
various cytokines and T cell subtypes are involved in the
of these drugs and irradiation result in sloughing of the
pathogenesis of aGVHD and they are potential targets for
sinusoidal endothelium, which results in obstruction
intervention.11
of hepatic circulation and injury to the centrilobular
Acute GVHD is characterized by manifestations in the
hepatocytes.9 Recognized risk factors for VOD includes
skin, liver, and GI tract. Grading of the severity of aGVHD
older transplant age, HLA disparity between donor and
is based on evaluation of the degree of involvement in
recipient, preexisting liver disease, etc. The type and
each of these organs (Table 3).
intensity of transplant conditioning regimen are probably
• Skin involvement in GVHD starts as redness and
the greatest determining factor for development of severe
maculopapular rash, initially of face, ears and palms
VOD. High plasma levels of Busulfan or metabolites of
and soles. Later the rash may progress to other parts
cyclophosphamide are associated with increased risk of
of the trunk and may progress to bullae formation and
VOD.
desquamation (Figs 5A to C).
Because there is no effective treatment of this
• Hyperbilirubinemia is the primary hepatic manifesta
complication, prevention is critical. Use of reduced
tion of liver involvement in GVHD. The other causes of
intensity conditioning regimens and the substitution of
jaundice in transplant patients include veno-occlusive
fludarabine for cyclophosphamide appears to reduce
disease, drug toxicity and infections.
the risk. Defibrotide is a mixture of single-stranded
• The GIT involvement is characterized by diarrhea,
oligonucleotides that have local antithrombotic, anti-
nausea and food intolerance.
ischemic and anti-inflammatory properties. It protects
• Other organs may also be involved in GVHD, e.g.
the sinusoidal endothelium without compromising the
ocular GVHD is manifested by hemorrhagic conjunc
cytotoxic therapy. Defibrotide modulates endothelial
tivitis and pseudomembrane formation.12
cell injury and protects the sinusoidal endothelium. It
also modulates platelet activity and enhances fibrinolytic MANAGEMENT OF GVHD
activity. Hence, it is used now for prophylaxis as well as
treatment of SOS. Other agents used with variable results Over the years, there have been several strategies deve
include tissue plasminogen activator, antithrombin III loped for management of GVHD. Prophylactic measures
and prostaglandin E1, low molecular weight heparin, include methotrexate, steroids and cyclosporine. Cyclo
Ursodeoxycholic acid, etc. Despite emerging therapies, sporine is a calcineurin inhibitor that interferes with T
VOD remains a much feared transplant complication and lymphocyte functions. Agents used for treatment include
severe cases are associated with dismal prognosis.10 steroids, agents like tacrolimus, mycophenolate mofetil,
monoclonal antibodies like Infliximab (TNF alpha
inhibitor) and photopheresis. The principal risk factor
ACUTE GRAFT VERSUS HOST DISEASE for aGVHD is HLA mismatch, but it can occur despite
The graft versus host disease (GVHD) is the most im a full HLA match. The incidence of GVHD can also be
portant complication of allogeneic transplantation. The reduced by in vitro T cell depletion of the graft before
development of GVHD is a complex process that is transplantation.12
Table 3 Grading of graft versus host disease

Clinical staging Stage I Stage II Stage III Stage IV
Skin Rash <25% BSA Rash 25–50% BSA Rash 50–100% BSA Desquamation and bulla
formation
GIT Persistent nausea or Diarrhea 10–15 mL/kg/ Diarrhea > 15 mL/kg/day Pain +/– ileus
Diarrhea 5–10 mL/kg/day day
Liver Bilirubin 2–3 mg/dL Bilirubin 3–6 mg/dL Bilirubin 6–15 mg/dL Bilirubin >15 mg/dL
Clinical grading Skin GIT Liver Functional impairment

0 0 0 0 0
I I–II 0 0 0
II I–III I I I
III II–IIII II–III II–III II
IV II–IV II–IV II–IV III
A B C
Figs 5A to C Skin rash in GVHD
Interstitial Pneumonitis DELAYED EFFECTS

Transplantation associated lung injury usually occurs Chronic GVHD: The risk of chronic GVHD increases
within four months of the procedure, and the mortality with recipient and donor age. Chronic GVHD is asso
exceeds 60 percent. Risk factors include TBI, allogeneic ciated with loss of self-tolerance and often resembles
transplantation and acute GVHD suggesting that donor Sjögren’s syndrome or scleroderma. Chronic GVHD
lymphocytes target the lung. Treatment with etanercept can cause bronchiolitis, keratoconjunctivitis sicca,
that blocks tumor necrosis factor, combined with corti esophageal stricture, malabsorption, cholestasis,
costeroids may reduce the injury promptly.9 hematocytopenia, and generalized immunosuppres
sion. Treatment with corticosteroids may be needed for
two years or longer.1
Infections
Growth and development are impaired in children
Transplant related infections result from damage to the who undergo transplantation as a result of myeloablative
mouth, gut and skin from preparative regimens as well preparative regimens. Growth hormone therapy increases
as from catheters, neutropenia and immunodeficiency. height in these children.
Prolonged neutropenia, GVHD and the administration of Fertility in adulthood may be impaired in children
corticosteroids predispose patients to fungal infections.13 undergoing transplantation. Young men may recover
Cytomegalovirus is an important cause of morbidity their fertility later in life. If sperms are present before
during this period. The various infections which occur in transplantation, semen can be cryopreserved and used
the course of transplantation are given in Figure 1. later. Women can also go in for cryopreserved oocytes.
Fig. 6 Timeline of infections in a patient undergoing transplantation
Secondary cancers increase after transplantation: Myelo experience, has divided patients with thalassemia into 3
dysplasia and acute leukemia are complications of auto major risk classes based on the presence of hepatomegaly,
logous transplantation for Hodgkin’s and non-Hodgkin’s inadequate chelation and portal fibrosis.14
lymphoma. Survivors of transplantation should be followed
indefinitely to detect early cancer or precursor lesions. Pesaro Thalassemia Risk Classification
Risk Factors
Beta Thalassemia
• Hepatomegaly: Determined by physical examination
The median survival of patients with beta thalassemia • Hepatic fibrosis: Liver biopsy
major who are on regular transfusion and chelation • Inadequate chelation: History, ferritin value, liver iron
program is around 35 years. By the fourth decade of life quantitation.
most of the patients succumb to complications of the
disease. At present, the only curative approach in this
illness is allogeneic stem cell transplant. The first transplant Risk Classification
was performed in 1982 and ever since, the transplant • Class 1 risk: No risk factors present
group from Pesaro, Italy headed by Dr Guido Lucarelli • Class 2 risk: 1 or 2 risk factors present
has led the subsequent way. The Pesaro group with their • Class 3 risk: All 3 risk factors present.
The conditioning regimen used was Bu 14 to 16 mg/kg evolution of clonal hematopoietic disorders like PNH and
and Cy 200 mg/kg. myelodysplasia.17
On analysis of their data of 1003 patients, the overall
thalassemia free survival observed was 68 percent (Class LEUKEMIA
1: 87%, Class 2: 84%, Class 3: 58%). The major problem
observed was with Class 3 patients, as many could Allogeneic stem cell transplantation is used for pediatric
not tolerate the toxicity of the regimen. In 1997, a new patients with acute lymphoblastic leukemia (ALL), acute
preparative regimen was developed that used myelo myeloid leukemia (AML) as well as for chronic myeloid
suppression and immunosuppression with azathioprine, and juvenile myelomonocytic leukemia. In addition to the
hydroxyurea and fludarabine followed by conditioning stem cells, the donor graft also has T cells and NK cells.
with Bu 14 mg/kg and Cy 160 mg/kg. This strategy They populate in the recipient’s hematopoietic system
named Protocol 26 has shown good results. In 33 class 3 and give rise to a new immune system. This can help in
thalassemics <17 years of age, the thalassemia free survival eliminating the remaining leukemia cells that escape
has increased to 85 percent and the rate of rejection has the conditioning regimen. This is called the Graft versus
dropped from 30 to 8 percent. Leukemia effect. The GVL effect is exerted through T cell
Thus allogeneic transplantation is increasingly beco mediated allo reactivity.18
ming a feasible option across the globe. The availability Acute lymphoblastic leukemia is the most common
of HLA matched sibling donor is the limiting factor as it is indication for stem cell transplantation in pediatric age
seen only in 25 to 30 percent of cases. Recently, there have group. In ALL transplantation is done in first remission
been reports of use of alternative donors like umbilical for patients at very high-risk of a relapse. This includes
cord blood, HLA mismatched related donors, etc. such high risk cytogenetic features like t(9; 22) and t(4; 11). In
techniques need to be perfected before they can be other patients who relapse while on or after completion
accepted as a standard of care.15 of primary treatment, transplantation is indicated in
second remission. Conditioning regimens that utilize total
APLASTIC ANEMIA body irradiation are associated with better survival than
with busulfan/cyclophosphamide alone. The estimated
Severe aplastic anemia is defined as ANC <500/mL, probability for event free survival for patients transplanted
Absolute reticulocyte count <20,000/mL and platelet count in first and second remission is around 65 percent and 50
<20,000/mL. Currently, the frontline therapy consists of percent respectively.1
either immunosuppressive therapy or matched sibling Less intensive GVHD prophylaxis is employed to reap
transplantation. Stem cell transplantation provides the benefits of GVL effect.
curative therapy in SAA. Initial reports of success have Acute myeloid leukemia is another indication for
been with the use of syngeneic donors. The first successful allogeneic stem cell transplantation from an HLA identical
allogeneic transplantation was done in 1972. The donor. Subtypes of AML including acute promyelocytic
combination of cyclophosphamide with ATG was shown leukemia and good cytogenetic features like t(8; 21) and
to be a successful conditioning in these patients. Survival inv(16) are no longer considered for transplantation
for HLA matched sibling transplants has increased from in first remission due to good results with conventional
48 percent in the 1970s to 66 percent in the late 1980s and therapy.
to 70 to 90 percent in recent data. The rate of graft rejection In chronic myeloid leukemia, the earlier treatment of
has fallen since cyclophosphamide with ATG has become choice was allogeneic SCT. With the advent of targeted
the standard conditioning regimen.16 therapy directed against t(9; 22), i.e. imatinib mesylate,
The comparison of hematological response rates, as the treatment has been revolutionized. Transplant is
well as long-term responses strongly supports SCT as the indicated only for those patients who fail this treatment or
treatment of choice in SAA, in cases where an HLA matched progress to blast crisis.
related donor is available. However, transplantation
is also associated with risk of early mortality. Data indicates
IMMUNODEFICIENCIES
that in patients younger than 40 years of age, SCT is always
superior to immunosuppressive therapy. In older subjects Bone marrow transplantation for lethal congenital
immunosuppression may be a more feasible option consi immunodeficiencies has been established as a treatment
dering the comorbidities. In patients >40 years of age, modality ever since transplants done for severe combined
the decision should be made on an individual basis. The Immunodeficiency and Wiskott Aldrich syndrome were
response to immunosuppressive therapy may take as successful in 1968. In contrast to other indications, the goal
long as 6 to 12 months and also carries a 10 percent risk of of transplantation in these patients, is complete recovery of
immune function. Hence 100 percent donor engraftment died, out of which 2 AML patients died of relapse at 56 and
may not be needed inorder to cure the immunodeficiency. 90 days post-transplant. The main causes of death in 5 other
In certain diseases like chronic granulomatous disease, patients were sepsis (3 bacterial and 2 fungal), sinusoidal
stable donor chimerism of around 10 to 15 percent cells obstruction syndrome (1 case), acute GVHD (1 case) and
is enough to establish normal host defense mechanisms. chronic GVHD (1 case). Two thalassemic children rejected
Before taking up for transplantation, a detailed evaluation graft but are alive and transfusion dependent.
for infections must be undertaken and cure of underlying The main indications for 19 autologous HSCT were
infections must be attempted whenever possible. Myelo multiple myeloma-9, Non-Hodgkin’s lymphoma-4, meta
ablative chemotherapy conditioning regimens are utilized static neuroblastoma-2, relapsed Hodgkin’s lymphoma-1,
for patients with non-SCID primary immune deficiency relapsed rhabdomyosarcoma-1, relapsed primitive neuro
diseases undergoing SCT. Non-myeloablative or reduced- ectodermal tumor (PNET) of the kidney-1 and rejection
intensity conditioning regimens also have the potential for post cord blood transplant in thalassemia-1. The source
engraftment without the risk of morbidity and late sequelae of HSCT was peripheral blood in 16 patients and bone
associated with standard myeloablative regimens.20 marrow in 3. Ten patients (55.6%) are alive and disease-
free at a median follow up of 114 days (range 21-617 days).
INDIAN SCENARIO Seventeen patients engrafted neutrophils at a median
duration of 12 days (range 9 to 30).
The first Bone Marrow transplantation done in India was At a median follow-up of 200 days (range 21 to 1200 days),
in 1983 at Tata Memorial Hospital, Mumbai in 1983 for a the estimated overall survival and event free survival for all
patient with Acute Myeloid Leukemia. At present more the transplant population are 67.3 percent ± 8.6 percent and
than 15 centers in India have facility for BMT. India caters 63.5 ± 8.9 percent respectively. Overall transplant related
not only to patients within our country but also to patients mortality is 23.5%, with a decrease from 28.6 percent to
from neighboring countries. Since, its humble beginnings, 22.2 percent after a dedicated HSCT unit with HEPA filtered
HSCT has progressed to a successful modality of treatment. rooms became functional by mid 2007.20
The BMT Unit at Christian Medical College, Vellore has It is remarkable that, through bone marrow and HS-
been designated as center of excellence by ICMR.19 cell transplants, stem-cell therapies have brought about
As far as our experience of HSCT program at Sir Ganga permanent cures for many patients suffering from blood
Ram Hospital is concerned, over a period from January disorders. There are efforts underway to develop therapies
2006 to August 2009, 39 transplants (16 allogeneic and using alternative sources of stem cells, such as embryonic
23 autologous) were done. The median age of transplant stem cells. However, because HS cells are relatively
patients was 34 years (11 months-68 years). Children abundant and accessible, alternative sources might be
comprised 41.2 percent of the patients. The indications less crucial for treating common blood disorders than
for 16 allogeneic transplants were thalassemia major-6, for diseases of other organs and tissues. Advances in HS
acute myeloid leukemia (AML)/myelodysplastic cell based therapies would probably be the answer for the
syndrome-6, severe aplastic anemia-3 and high-risk acute many incurable diseases of today.21
lymphoblastic leukemia-1. Donors were HLA-matched
sibling in 13 cases, HLA-matched relative in 1 and
unrelated umbilical cord blood in 2. The source of HSCT REFERENCES
was peripheral blood in 8 patients, bone marrow in 6 and 1. Velardi A, Locatelli F. Hematopoietic stem cell trans
umbilical cord blood in 2. Fourteen patients underwent plantation. In: Kleigman RM, Behrman RE, Jenson HB,
myeloablative transplants and two were given reduced Stanton BF (eds): Nelson Textbook of Pediatrics, 18th edn.
intensity conditioning. Three Donor Lymphocyte infusions Elsevier. 2007.pp.924-33.
were given to two patients. Seven patients (44.8%) are alive 2. Allogeneic bone marrow/stem cell transplantation. A
and disease free at a median follow-up of 453 days (65–591 medical and educational handbook. (Online) 2001. Avail
days). Thirteen patients showed neutrophil engraftment able at URL http://www.bonemarrow.org/downloads/
at a median duration of 14 days (range 11 to 44). AllogeneicBook.pdf
3. Fairview-University Blood and Marrow Transplant
Acute graft versus host disease (GVHD) was seen
Services. Blood and Marrow Transplantation: a Patient’s
in 6 patients, grade I-II was seen in 5 patients, which Guide to Hematopoietic Stem Cell Transplantation.
responded to steroids. Steroid-refractory grade IV GVHD Minneapolis, MN: Fairview Press. 2004.
was seen in one child; it did not respond to mycophenolate 4. Guidelines for the clinical use of blood cell separators.
and infliximab. Three patients developed sinusoidal Prepared by a joint working party of the transfusion and
obstruction syndrome. All were given defibrotide and two clinical hematology task forces of the British Committee for
responded. A total of 7 allogeneic HSCT recipients have Standards in Hematology. Clin Lab Haem. 1998;20:265-78.
5. Grewal SS, Wagner JE. Umbilical Cord Blood Trans 14. Boulad F. Hematopoietic Stem-Cell Transplantation
plantation. In: Kline RM (ed): Pediatric Hematopoietic for the Treatment of Beta Thalassemia. In: Kline RM
stem cell transplantation. Informa Healthcare USA. 2006. (ed). Pediatric hematopoietic stem cell transplantation.
pp.161-87. Informa Healthcare USA. 2006.pp.383-96.
6. Gluckman EG, Rocha V, Chastang C. The use of cord blood 15. Lucarelli G, Gaziev J. Advances in the allogeneic trans
cells for banking and transplant. Oncologist. 1997;2:340-3. plantation for thalassemia. Blood Rev. 2008;22:53-63.
7. Blood and Marrow Stem Cell Transplantation (Online). 16. Paley C, Vlachos A, Lipton JM. Hematopoietic Stem-Cell
The Leukemia and Lymphoma Society. 2001. Transplantation for acquired aplastic anemia. In: Kline RM
8. Takaue Y, Kawano Y, Abe T, Okamoto Y, Suzue T, Shimizu (ed). Pediatric hematopoietic stem cell transplantation.
T, et al. Collection and transplantation of peripheral blood Informa Healthcare USA. 2006.pp.369-81.
stem cells in very small children weighing 20 kg or less. 17. Marsh JCW, Ball ES, Cavenagh J, Darbyshire P, Dokal I,
Blood. 1995;86(1):372-80. Gordon-Smith EC, et al. Guidelines for diagnosis and
9. Aquino VM, Sandler ES. Supportive Care of the Pediatric management of aplastic anemia. Br J Haematol. 2009;147:
Hematopoietic Stem-Cell Transplant Patient. In: Kline RM 43-70.
(ed): Pediatric hematopoietic stem cell transplantation. 18. Haining WN, Duncan C, Lehmann LE. Principles of bone
Informa Healthcare USA. 2006.pp.1-26. marrow and stem cell transplantation. In: Orkin SH,
10. Ho VT, Revta C, Richardson PG. Hepatic veno-occlusive Nathan DG, Ginsburg D, Look AT, Fisher DE, Lux SE (eds).
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update on defibrotide and other current investigational 7th edn. Elsevier. 2009.pp.395-452.
therapies. Bone Marrow Transplant. 2008;41:229-37. 19. Loechelt BJ, Kamani NR. Primary immunodeficienies. In:
11. Moore TB, Feig SA. Acute graft versus host disease. Kline RM (ed): Pediatric hematopoietic stem cell trans
In: Kline RM (ed). Pediatric hematopoietic stem cell plantation. Informa Healthcare USA. 2006.pp.321-36.
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12. Socie G, Blazar BR. Acute graft versus host disease: from Choudhary D, et al. The experience of hematopoietic stem
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Pediatric hematopoietic stem cell transplantation. Informa transplantation. Indian Journal of Practical Pediatrics.
Healthcare USA. 2006.pp.27-63. 2009;11(2):127-35.
S E C T I O N 7
General
CHAPTERS OUTLINE
48. Gene Therapy
49. Monoclonal Antibodies in Pediatric Hematology and Oncology
50. Biological Response Modifiers
C H A P T E R 48
Gene Therapy
INTRODUCTION is the introduction of the target gene into the zygote, a

change that is transmissible to the offspring. Before the
Normal as well as some defective genes are present in initiation of gene therapy the candidate gene needs to be
all individuals. The genes usually remain dormant until identified. Diseases that are amenable to probable gene
a disease associated with the gene manifests in a case. therapy are enumerated in Table 1. The complexity of gene
Genetic defects can lead to more than four thousand therapy lies in the mechanisms to deliver the therapeutic
diseases. Apart from the genotype of an individual, the gene into the target organ in an accurate, controlled and
environment in which the individual lives also affect the effective way.
manifestation of the disease. In 1990 in NIH, Maryand a four-year-old-boy with
Gene therapy is the introduction of a target gene into a severe combined immunodeficiency (SCID) received an
cell. Gene therapy can be somatic or germ-line. In somatic infusion of genetically modified stem cells. This was the
gene therapy the genetic make-up of the individual is not first instance of gene therapy and the recipient was later
altered and it is not transmissible to the off-spring. Somatic known as the bubble boy. Since then the field of gene
gene therapy aims at the introduction of the target gene to therapy has targeted many diseases and has expanded its
correct a defective organ or tissue. Germ-line gene therapy coverage.
Table 1 Diseases for which gene therapy is being explored

Disease Underlying defect Target cell for genetic manipulation
SCID ADA deficiency T-lymphocytes
Hemophilia Factor VIII or IX deficiency Hepatocytes, muscle fibroblasts or
hematopoietic cells
Cystic fibrosis CFTR gene mutation Airway epithelial cell in lungs
Hemoglobinopathies Globin chain defects Hematopoietic cells
Gaucher’s disease Defect in enzyme glucocerebrosidase Macrophages or hematopoietic cells
α-1 antitrypsin deficiency Lack of α-1 antitrypsin Lung and liver cells
Familial hypercholosterolemia Lack of LDL receptors Liver cells
Cancer Multiple causes Different cancer cell types
Neurological diseases Parkinson’s/Alzheimer’s, etc. Neuronal cells
Cardiovascular diseases Arteriosclerosis Endothelial cells of vessels
Infections HIV, Hepatitis B T-cells, liver, macrophages
492 Section-7 General
PROCEDURE OF GENE THERAPEUTICS Retroviruses are RNA viruses that can integrate its
nucleic acid into the host cells, using reverse transcriptase
Target Tissue enzyme that transcribes RNA into DNA . Other vectors used
Nature of the disease determines the somatic candidate in gene therapy can be adenoviruses, retrotransposons
organ for gene therapy. The target cell needs to be clearly and liposomes (Fig. 1). Engineered vectors are used for
defined. For example in cystic fibrosis the target tissue is gene therapy where the detrimental gene is removed and
the lung where delivery of the therapeutic gene is being the corrective gene is added. The properties of an ideal
tried by the aerosolized route. In a clotting disorders vector are enumerated in Table 2.
like hemophilia the deficient factor can be provided by
introduction of the candidate gene into the myocytes or Type of Vectors
the hepatocytes. The choice of the target tissue depends • Retrovirus
upon factors such as protein modification, gene delivery • Lentivirus
efficacy of the vector and immunological factors. • Adenovirus
• Herpes viruses
Vectors • Plasmids, retrotransposons and liposomes.
The method of delivery of the target gene into the tissue is Low host immunogenicity and allowance for large
of vital importance. Naked genes can be delivered into the scale production are advantages of non viral methods over
cells but the method has low efficiency. Vectors which are the viral methods. Non viral methods however have the
usually plasmids or viruses, can move recombinant DNA disadvantage of low levels of transfection and subsequent
from one cell to the other. Special synthetic vectors have gene expression, but these have been now overcome with
also been designed for gene transfer. modern vector technologies that yield molecules and
Fig. 1 Gene therapy (a simplified representation)

Chapter-48 Gene Therapy 493
Table 2 Properties of a good vector for gene therapy

All vectors used in gene therapy—viral or nonviral have certain limitations. The disease type often dictates the type of vector to be
employed. Adenoviral vectors are useful in situations where a short term expression of the gene product is needed (e.g. products that
may be toxic to malignant cells). In case of sustained gene expression the integrating vector that leads to minimal immunological
response is desirable. An ideal vector should have
• A good concentration in minimal amount of injection so that a maximum number of target cells are infected
• Easily reproducibility
• Ability of a stable and site specific integration into the host genome
• Specificity for the target cell
• Minimal immunogenic potential
• Ability of its transcriptional unit to respond to external manipulation.
Presently such a desirable vector that possesses the advantages of both the synthetic and viral vectors is not available. Availability of
an ideal vector will make gene therapeutics grow by leaps and bounds.
techniques with transfection efficiencies similar to the antibodies. In vivo certain promoters undergo inactivation
viral vectors. Injection of naked DNA, electroporation, hampering long term factor VIII or factor IX expression.
the gene gun, sonoporation and magnetofection and Several phase I clinical trials are underway presently
the use of dendrimers, lipoplexes, oligo-nucleotides and for hemophilia and some subjects have reported lower
inorganic nanoparticles are some non viral methods of bleeding episodes and detectable clotting factor activity.
gene delivery.
Gene Therapy for Hemoglobinopathies
GENE THERAPY AND ITS USE IN PEDIATRIC A lentiviral vector has been used for inserting the gene for
HEMATOLOGY AND ONCOLOGY a normal hemoglobin expression in hemoglobinopathies
As hematopoietic cells can be easily collected and are into stem cells from the bone marrow, in mice cells cultured
amenable to in vitro manipulation they are ideal targets for in vitro. These cells have then been re-introduced into the
gene therapy. Genetic manipulation of the hematopoietic mice. The mice that have received this genetic treatment
stem cells can be utilized for the cure of many acquired are no different from the normal counterparts. As the stem
and inherited hematological and oncological diseases. cells used here were autologous, their rejection has been
The target cells can be the red cells, leukocytes or any other avoided. Although this therapy is still untested in humans
mature blood element. The desired type of blood cell can it is possible that the mutations in the β-globin gene be
be continuously produced for the lifetime of an individual corrected by gene targeting in induced pluripotent stem
by integrating the desired transgene into the chromatid cells (IPS) derived from somatic cells. Skin fibroblasts have
of the concerned pleuripotent stem cell. For example the been differentiated into IPS cells. The IPS cells in future
sources of deficient clotting factors could be cells such as could be utilized to create hematopoietic stem cells that
the hepatocytes and the myocytes. synthesize hemoglobin.
Gene Therapy for Hemophilia Gene Therapy for Immunodeficiencies

In mice, expression of factor VIII in blood cells and In mice models the restoration of lymphocyte function
platelets has been achieved by the use of ex vivo transduced in SCID mice secondary to JAK deficiency has been
hematopoietic stem cells. In vivo, transposons expressing reported as early as 1998. The same has however proved
factor VIII can be transferred into the endothelial challenging in larger animals. The transduction efficiency
cells or hepatocytes. In canine models the neonatal of human hematopoietic stem cells is being optimized
administration of reteroviral vectors expressing the canine by use of alternative envelope proteins to pseudotype
factor VIII completely corrected hemophilia in dogs. vector particles including hat derived from Gibbon ape
Similarly in dogs with hemophilia B factor IX levels that leukemia virus (GAVL). Cytokine combinations and use of
were 28 times more were achieved using double stranded fragments of fibronectin, retronectin to co-localize vector
adenoviruses in comparison to the single stranded ones. particles and target cells also help the cause.
The factor IX expression however was short lived due to In chronic granulomatous disease success has been
probable immune destruction of the modified cells. Stable achieved in two patients where genetically modified
phenotypic correction is often hampered by neutralizing hematopoietic cells have been successfully engrafted after
partial myeloablation. In these two patients the vector Suicide Gene Therapy for Graft Versus Host
used was the spleen focus forming virus (SFFV) as its Disease (GVHD)
enhancer-promoter region is active in myeloid cells.
A suicide gene can be inserted into a target cell making
Gene Therapy for Hematological Malignancies it susceptible to drug induced cell death. The target cell
can be the donor lymphocytes and this can be used to
Inhibitory signals to the host immune response by tumor control alloreactivity as seen in allogenic hematopoietic
cells helps in their survival. This can be blocked by the transplants. As shown in Figure 2 , a suicide gene is
use of gene products or cytokines released from the site introduced into the allogenic donor lymphocytes and
of vaccination (i.e. the therapeutic tumor vaccine). The this in turn has the potential to convert a prodrug into
methods used could rely either on the use of genetically an active drug. Following HSCT the donor receives
modified autologous tumor cells or allogenic tumor cells these genetically modified lymphocytes for immune
which may provide a paracrine stimulus. Skin fibroblasts reconstitution. In event of a GVHD occurring the prodrug
expressing IL-2 and CD-40 ligand have been mixed can be administered resulting in the ablation of these
with irradiated tumor cells and injected into patients of alloreactive lymphocytes.
refractory leukemias thereby inducing T cells reactive
against the blast cells. CHALLENGES WITH GENE THERAPY
Chimeric antigen receptors (CAR) are single chain
antibodies with specificity for the antigen expressed on the • Short-lived nature—As many cells are rapidly
human tumor cell is linked to an internal kinase domain multiplying ones, long term benefits after gene therapy
which mediates cell activation when the antibody is is difficult. Many rounds of gene therapy are needed to
engaged by the target antigen. In mice CARs targeting CD- achieve a substantial amount of benefit.
19 have been used to cure B cell leukemias. LMP-2 protein • Immune response to the inserted DNA material or the
of the EBV which is expressed in some human lymphomas virus per se could lead to fatal complications.
can be generated by gene transfer on population of • Viruses are the vectors of choice in most gene therapy
lymphoid cells. These can then be used to yield a studies. They however have a variety of potential
population of T cells with potent antitumor property. problems to the patient including toxicity, immune
Fig. 2 Gene therapy and its use in HSCT

Chapter-48 Gene Therapy 495
and inflammatory response. With these viral vectors 2. Cross D, Burmester JK. Gene therapy for cancer treatment:
gene control and targeting issues are also a problem. past, present and future. Clinical Medicine and Research.
The possibility of the viral vector regaining its virulence 2006;4(3):218-27.
once inside the host cell is always a danger. 3. Dubnar CE. Gene therapy for hematologic disease: don’t
throw the baby out with the bathwater!. Seminars in
• Multigene disorders are poor candidates for gene
Hematology. 2004;41(4):255-6.
therapy.
4. Dunbar CE, Wu T. Gene therapy for hematological
• The novel DNA may inadvertently impact the germline disorders. An introduction to molecular medicine and
by breach of the somatic-germline barrier against the gene therapy. 2001;6:133-52. Wiley-Liss, Inc.
intentions of the therapy. 5. Edelstein ML, Abedi MR, Wixon J. Gene therapy clinical
• Insertional mutagenesis can occur whereby a tumor trials worldwide to 2007—an update. J Gene Med. 2007;9:
can be induced if the DNA is integrated in a wrong 833-42.
place (e.g. in a tumor suppressor gene). Gene therapy 6. Mulherkar R. Gene and cell therapy in India. Current
in SCID has resulted in T cell leukemias in 3 out of 20 Science. 2010;99(11):1542-7.
patients due to insertional mutagenesis. 7. Nienhuis AW. Development of gene therapy for blood
disorders. Blood. 2008;111:4431-44.
8. Scollay R. Gene therapy a brief overview of the past,
BIBLIOGRAPHY present, and future. Annals New York Academy of
1. Borem A, Santos FR, Bowen DE. Gene therapy. Sciences. 2006;953a:26-30.
Understanding biotechnology. 2003.pp.87-98. Prentice 9. Verma IM, Somia N. Gene therapy-promises, problems
Hall. and prospects. Nature. 1997;18(389):239-42.
C H A P T E R 49
Monoclonal Antibodies in
Pediatric Hematology and Oncology
Despite the tremendous progress in the treatment of pediatric cancers in the past decade, current therapies are associated with wide
range of toxicities, which leads to treatment associated mortality and substantial morbidity in long-term survivors. Novel approaches
are needed to overcome resistance and to decrease adverse effects of standard treatment. Targeted therapies, which include
monoclonal antibodies (MoAbs) have significantly changed the treatment of adult cancers.1 During the last few years, progress has
been made in the therapeutic use of MoAbs in specific groups of pediatric cancers and hematologcal disorders.
In 1975, Kohler and Milstein demonstrated for the first • The target of the MoAb should not be shed from the
time that monoclonal antibodies (MoAb) could be tumor following MoAb binding; rather, the antigen-
generated from hybridomas.2 Because MoAbs can bind MoAb complex should be internalized by the tumor
to antigens expressed on the surface of malignant cells, cell.
it was proclaimed that these agents could be used as The ideal antigen should not undergo modulation (in
chemoimmunotherapy to specifically target and destroy which the antigen is no longer expressed on the cell surface
these cells. Moreover, by offering cytotoxic mechanisms after antibody binding). Its disappearance from the cell
different from conventional chemotherapy, MoAb therapy membrane can limit the effectiveness of treatment.
could potentially reduce the risk of tumor cell resistance to Despite the fact that many of the patients are inherently
the common chemotherapeutic agents. By 1979, the first immunosuppressed secondary to their malignancies and
patient was treated using MoAb therapy and in the ensuing extensive prior chemotherapy, development of neutra
decade over 100 patients with hematologic malignancies lizing antibodies have been seen in few patients. Its
have been similarly treated.3 Although progress in this field development can preclude the efficacious administration
has proceeded much slower than was initially anticipated, of MoAbs because of enhanced clearance of the antibody
recent clinical trials have also demonstrated antitumor from the circulation, the formation of antigen-antibody
activity in a variety of pediatric malignancies. complexes with subsequent end organ damage. Hence,
they must be rendered sufficiently nonimmunogenic to
MONOCLONAL ANTIBODY THERAPY
prevent development of neutralizing antibodies.
• Effective MoAb therapy for cancer requires the Recent advances in genetic engineering have allowed
identification of appropriate tumor—specific targets the development of chimeric antibodies and fully humani
expressed on the surface of the cancer cells. zed MoAbs which limit the likelihood of neutra lizing
• Ideally, the antibody should have minimal cross- antibody development.
reactivity with normal tissues and specifically target the
tumor cell. Nonspecific binding will reduce effective Mechanism
drug delivery. Moreover, any cross-reactive tissues
that are damaged may compromise patient’s function The overall success of MoAb therapy in cancer is
and survival. determined by the ability of antibody binding to result in
Chapter-49 Monoclonal Antibodies in Pediatric Hematology and Oncology 497
tumor cell death. A variety of mechanisms are thought to a year.8,9 International consensus report on the
play important roles in mediating the observed anti-tumor investigation and management of primary immune
effects.4 thrombocytopenia recommends a dose of 100 mg
• Antibody-dependent cellular cytotoxicity (ADCC) and or 375 mg/m2/week administered for four times as
complement-mediated cytolysis. Binding the antibody standard treatment strategies for children with chronic
to the tumor cell recruits cells with Fc receptors like NK ITP.10
cells and macrophages to the site of the tumor, which Rituximb has also been proved to be effective in
then kill the tumor cell, or complement is fixed and the treatment of various benign hematologic conditions like
tumor cell is killed. autoimmune hemolytic anemia (refractory to steroids
• Direct killing: Key process include interruption of a immune-suppressants and splenectomy),11 AIHA in
critical cell signaling cascade by inhibition of ligand setting of Evan’s syndrome, SLE and autoimmune lympho-
binding; downregulation of a receptor tyrosine kinase, proliferative syndrome12 and in patients with hemophilia
which transmits a necessary life signal; and induction who develop inhibitory antibodies to factor VIII and IX.13
of an apoptotic signal following ligation of the target by • Gemtuzumab ozogamicin (GO): It is a recombinant
the MoAb. humanized MoAb (IgG4) directed against the CD33
• Targeting via a conjugated antibody of antibody antigen that is conjugated to the derivative of the
receptor (e.g. radionuclide, immunotoxin, cell-based cytotoxic antibiotic calicheamicin.14 In pediatric acute
genetic fusion). This targets a lethal “hit” to the tumor myeloid leukemia (AML), the differentiation antigen
cell. CD33 is expressed in almost all patients. Following
The following gives an overview of the various MoAb binding of GO to the CD33 antigen, the antibody-
which are now in clinical use or in various phases of antigen complex gets internalized into the AML cells.
clinical trials. The calicheamicin conjugate is released inside the
• Rituximab: It is a chimeric unconjugated MoAb cell through hydrolysis and subsequently binds to the
directed to CD20, which is expressed on the surface of minor groove of DNA, inducing double strand breaks
malignant and normal B-cells, but not hematopoietic and leukemia cell apoptosis.
stem cells. It has been shown to significantly increase Currently, the agent is being tested both as a single
the response rate and survival of adult patients with agent and in combination with chemotherapy in children
CD20-positive B-cell lymphomas. It appears that with AML. Results of phase I and II clinical trials indicate
rituximab has efficacy in children with high-grade promise, with an overall remission response rate of 45
B-lymphoma/B-cell acute lymphocytic leukemia. percent and a 1-year event-free survival and overall survival
The mechanisms of action include inhibition of estimates of 38 percent and 53 percent, respectively.15,16
B-cell proliferation, antibody-dependent cellular Several earlier case reports found responses to anti-CD33
cytotoxicity, complement-dependent cytotoxicity in pediatric ALL and in a few cases of relapsed adult ALL.17
and possible induction of apoptosis.5 The Children’s Patients responding to gemtuzumab had very high (>90%)
Oncology Group is currently researching the efficacy of CD33 expression.17.
rituximab in recurrent and refractory CD20 lymphomas The US Food and Drug Administration (FDA) approved
in children (NCT01230788). With rituximab, a 96 anti-CD33 conjugated with calicheamicin (gemtuzumab
percent overall response rate was reported in one [Mylotarg]) for treatment of adult AML in 2000, but the
phase II trial for lymphocyte-predominant Hodgkin agent was withdrawn from the US market on June 21,
lymphoma, with 75 percent remaining in remission 2010. Apart from some infusional allergic reactions, the
after one year.6 Another phase II trial by Ekstrand primary toxicity has been bone marrow suppression
et al, 2003 showed 100 percent overall response rate caused by binding the MoAb-toxin conjugate to normal
(n = 22) with complete response (CR) in 41 percent, hematopoietic precursors that express CD33. Another
unconfirmed complete response in 5 percent, and still unexplained toxicity of anti-CD33–calicheamicin
partial response in 54 percent.7 A phase II pilot study conjugates is hepatic damage, which is characterized by
is underway through the Children’s Oncology Group transient increases in liver enzymes in approximately
to assess the toxicity of adding rituximab to upfront 25 percent of patients and, occasionally, a more severe
chemotherapy for B-cell leukemia and lymphoma complication consistent with veno-occlusive disease.
(NCT00324779). Children with refractory chronic • Epratuzumab: It is a humanized anti-CD22 MoAb that
immune thrombocytopenic purpura (ITP) have been binds to the extracellular domain of CD22. Epratu
treated with rituximab in various series with response zumab appears to modulate B-cell activation and
rates of 30 to 70 percent. Most of the responses were signaling. Proposed mechanisms of action include
obtained within 4 weeks and were maintained for antibody-dependent cell-mediated cytotoxicity, comp
lement dependent cytotoxicity and direct induc Neuroblastoma cells have been characterized for the
tion of apoptosis. CD22 is widely expressed in B-cell expression of tumor associated antigens recognized by
lymphomas and B-precursor ALL. It is rapidly antibodies. The identification of GD2 as a major target
internalized after antibody binding and re-expression for MoAb therapy has led to the production of both
on the cell surface is slow, occurring over the period of murine and chimeric anti-GD2 MoAbs in combination
several days. Internalization of CD22 has been shown with granulocyte-macrophage colony-stimulating factor
to directly induce apoptosis in malignant cells. (GM-CSF) and interleukin-2 (IL-2). The mechanisms
Epratuzumab has recently been studied by Children’s whereby anti-GD2 MoAbs kill tumor cells are likely related
Oncology Group in pediatric patients with first relapse to complement activation and antibody-dependent
of pre-B ALL (n = 15). The addition of epratuzumab to cell-mediated cytotoxicity (NCT00026312). The COG
re-induction chemotherapy was well tolerated, with no is currently investigating the efficacy of a humanized
apparent significant increase in toxicity.18 Although, it did MoAb in combination with a human recombinant
not improve the second remission rates, among patients interleukin-2 (Hu14.18-IL2) for the treatment of refractory
who attained CR, postinduction MRD-negative rates were neuroblastoma.
higher in comparison with those of historical controls Alice et al. 2010 compared between two treatment
treated with chemotherapy alone (42% versus 25%).11 groups in patients with high-risk neuroblastoma, the
• Bevacizumab: It is a humanized murine MoAb first group received the standard therapy of six cycles of
that binds to vascular endothelial growth factor-A isotretinoin. The second group received the new immuno
(VEGF-A) with high affinity and neutralizes its activity. therapy treatment: six cycles of isotretinoin plus five cycles
VEGF is one protein that plays a big role in the process of the monoclonal antibody ch14.18, in combination with
of angiogenesis. By cutting off the blood supply to the alternating GM-CSF and interleukin-2. Study results after
tumor, it is predicted that the tumor cells should die. two years showed, the rate of survival without relapse or
Bevacizumab is an antiangiogenesis agent approved disease progression was 20 percent greater in the children
for the treatment of colon cancer in adults and has who received immunotherapy (66% versus 46%).21
shown activity in carcinoma of the kidney, adeno In a recent phase II trial, Memorial Sloan-Kettering
carcinoma of the rectum and nonsmall-cell lung Cancer Center has shown promising result by the use of
cancer. VEGF is overexpressed in a number of solid anti-GD2 monoclonal antibody 3F8 and granulocyte-
tumors seen in children (NCT01218867), including macrophage colony-stimulating factor in neuroblastoma
Ewing sarcoma and glioblastomas, and is currently resistant to intensive induction therapy.22
being investigated in these and other pediatric solid • Trastuzumab: In a retrospective review of 53 osteo
tumors.19 sarcoma patients treated on the Memorial Sloan-
• Alemtuzumab: It is a humanized MoAb active against Kettering Cancer Center T12 protocol, higher freque
CD52; a cell surface co protein expressed by most T and ncies of HER2/erbB-2 expression were correlated with
B lymphoblasts. CD52 is neither shed nor internalized, metastatic disease at presentation, poor histologic
making it ideal for antibody directed immunotherapy. response to chemotherapy and significantly decreased
Most malignancies of B-cell origin and almost all T-cell event-free survival (47% versus 79% at 5 years, P = .05).23
malignancies strongly express the antigen. Binding of Trastuzumab, a MoAb directed against the human
alemtuzumab induces the lysis of lymhocytes, while epidermal growth factor receptor 2 (HER2), is being
monocytes and their precursors are less sensitive. attempted as a therapy for osteosarcoma.24,25 Although,
Alemtuzumab has shown antitumor activity in chronic it suggested that trastuzumab can be safely delivered in
lymphocytic leukemia, T-prolymphocytic leukemia, T-cell combination with anthracycline-based chemotherapy
non-Hodgkins lymphoma.17 In a few cases, clinical effects and dexrazoxane, the actual therapeutic benefit still
were observed in patients with single-drug treatment in remains uncertain.
relapsed adult ALL.20 It is being studied by COG in children
with ALL in second or greater relapse or primary induction
Adverse Effects of MoAbs
failure after two different regimens (PMC3120889).
Other antibodies that have demonstrated activity in Most reports of adverse events are from adult phase I and
T-cell leukemias, either in vitro or in vivo, include anti- II trials, since data for children are limited.
CD7-ricin, CD25 antigen (IL-2 receptor), anti-CD7-PAP, Acute infusion reactions are frequent, which can often
anti-CD2, OKT3, and a humanized anti-CD3 MoAb. be managed with antipyretics, antihistamines, and/or
Overall experience with MoAbs in T-cell ALL—with the corticosteroid. Immunosuppression and increased risk
exception of anti-CD52 alemtuzumab is scarce. of infection is not uncommon due to depletion of healthy
Chapter-49 Monoclonal Antibodies in Pediatric Hematology and Oncology 499
hematopoietic cell (rituximab, alemtuzumab). Rapid 2 trial of the German Hodgkin Lymphoma Study Group.
malignant cell kill can cause tumor lysis syndrome. Hepa Blood. 2003;101(2):420-4.
titis B virus reactivation with fulminant hepatic failure has 7. Ekstrand BC, Lucas JB, Horwitz SM, et al. Rituximab in
also been reported with rituximab. Other adverse effects of LP Hodgkin disease: results of a phase 2 trial. Blood.
2003;101(11):4285-9.
monoclonal antibodies documented are hemolytic uremic
8. Franchini M, Zaffenello M, Veneri D, Lippi G. Rituximab
syndrome, vascular leak syndrome, hypo albuminemia
for the treatment of childhood chronic idiopathic
and transaminitis, veno-occlusive disease, pulmonary thrombocytopenic purpura and hemophilia with
infiltrates and acute respiratory distress syndrome inhibitors. Pediatr Blood Cancer. 2007;49:6-10.
(ARDS).26 9. Mueller BU, Bennett CM, Feldman HA, Bussel JB, Abshire
Late effects include congestive heart failure, cardio TC, Moore TB, et al. One year follow-up of children and
myopathy, pericardial effusion, pericarditis, pulmonary adolescents with chronic immune thrombocytopenia
fibrosis and nephrotic syndrome. treated with rituximab. Pediatr Blood Cancer. 2009;52:
Monoclonal antibodies that contain high amounts of 59-62.
mouse protein result in various immunogenic responses, 10. Provan D, Stasi R, Newland AC, et al. International
including infusion-related reactions. More advanced consensus report on the investigation and management
of primary immune thrombocytopenia. Blood. 2010;115:
humanized antibodies contain only 5 to 10 percent of
168-86.
mouse protein sequences in an attempt to overcome
11. Zecca M, Nobili B, Ramenghi U, et al. Rituximab for the
the potential for dose-limiting or fatal hypersensitivity treatment of refractory autoimmune hemolytic anemia in
reactions. Recently, B-cell epitope mapping has been children. Blood. 2003;101:3857-61.
conducted to identify immunogenic amino acids, with 12. Bader-Meunier B, Aladjidi N, et al. Rituximab therapy
the goal to modify immunotoxin sequence to generate a for childhood Evan’s syndrome. Haematologica.
less immunogenic protein. Other techniques like coating 2007;92:1691-4.
immunotoxin with high molecular weight polyethylene 13. Collins PW, Mathias M, Hanley J, et al. Rituximab and
glycol (so called PEGylation) is also under investigation.27 immune tolerance in severe hemophilia A: a consecutive
national cohort. J Thromb Haemost. 2009;7:787-94.
14. Hamann PR, Hinman LM, Hollander I, et al. Gemtuzumab
Future ozogamicin, a potent and selective anti-CD33 antibody-
Modern recombinant techniques have made it possible to calicheamicin conjugate for treatment of acute myeloid
rapidly produce both chimeric and humanized antibodies. leukemia. Bioconjugate Chemistry. 2002;13:47-58.
Identification of surface receptors that are integral to 15. Aplenc R, Alonzo TA, Gerbing RB, et al. Safety and
efficacy of gemtuzumab ozogamicin in combination with
proliferation and apoptosis has also provided more targets
chemotherapy for pediatric acute myeloid leukemia: a
for monoclonal antibodies. At present, there are more
report from the Children‘s Oncology Group. J Clin Oncol.
than 100 monoclonal antibody based biologic drugs under 2008;26(14):2390-3295.
clinical trials and the optimal agents, dose, schedule, and 16. Arceci RJ, Sande J, Lange B, et al. Safety and efficacy
combination regimens have yet to be defined. of gemtuzumab ozogamicin in pediatric patients with
advanced CD33+ acute myeloid leukemia. Blood.
2005;106(4):1183-8.
REFERENCES 17. Pui CH, Evans WE. Treatment of acute lymphoblastic
1. Bassan R, Hoelzer D. Modern therapy of acute leukemia. N Engl J Med. 2006;354(2):166-78.
lymphoblastic leukemia. J Clin Oncol. 2011;29(5):532-43. 18. Raetz EA, Cairo MS, Borowitz M. Reinduction chemo
2. Kohler G, Milstein C. Continuous cultures of fused cells immunotherapy with epratuzumab in relapsed ALL in
secreting antibody of predefined specificity. Nature. 1975; children, adolescents and young adults: results from
256:495. children’s oncology group (COG) study ADVL04P2. Blood.
3. Nadler LM, Stashenko P, Hardy R, et al. Serotherapy of a 2011;573.
patient with a monoclonal antibody directed against a 19. Bender JL, Adamson PC, Reid JM, et al. Phase I trial and
human lymphoma-associated antigen. Cancer Res. 1980; pharmacokinetic study of bevacizumab in pediatric
40:3147-54. patients with refractory solid tumors: a Children‘s
4. Weiner GJ. Monoclonal antibody mechanisms of action in Oncology Group Study. J Clin Oncol. 2008;26(3):399-405.
cancer, Immunol Res. 2007;39(1-3):271-8. 20. Cheung KC, Wong LG, Yeung YM. Treatment of CD33
5. Maloney DG, Smith B, Rose A. Rituximab: mechanism of positive refractory acute lymphoblastic leukemia with
action and resistance. Semin Oncol. 2002;29:2-9. Mylotarg. Leuk Lymphoma. 2008;49(3):596-7.
6. Rehwald U, Schulz H, Reiser M, et al. Treatment of relapsed 21. Yu AL, Gilman AL, Ozkaynak MF, et al. Anti-GD2
CD20+ Hodgkin lymphoma with the monoclonal antibody antibody with GM-CSF, interleukin-2, and isotretinoin for
rituximab is effective and well tolerated: results of a phase neuroblastoma. N Engl J Med. 2010;363(14):1324-34.
22. Kushner BH, Kramer K, Cheung NK. Phase II trial of the 25. Ebb D, Meyers P, Grier H, et al. Phase II trial of trastuzumab
anti-G(D2) monoclonal antibody 3F8 and granulocyte- in combination with cytotoxic chemotherapy for treatment
macrophage colony-stimulating factor for neuroblastoma. of metastatic osteosarcoma with human epidermal growth
J Clin Oncol. 2001;19(22):4189-94. factor receptor 2 overexpression: a report from the children‘s
oncology group. J Clin Oncol. 2012;30(20):2545-51.
23. Gorlick R, Huvos AG, Heller G, et al. Expression of HER2/
26. Pastan I, Hassan R, FitzGerald DJ, et al. Immunotoxin
erbB-2 correlates with survival in osteosarcoma. J Clin
therapy of cancer. Nature Rev. 2006;6:559-65.
Oncol. 1999;17(9):2781-8. 27. Tsutsumi Y, Onda M, Nagata S, et al. Site-specific chemical
24. Scotlandi K, Manara MC, Hattinger CM, et al. Prognostic modification with polyethylene glycol of recombinant
and therapeutic relevance of HER 2 expression in immunotoxin anti-Tac(Fv)-PE38 (LMB-2) improves
osteosarcoma and Ewing’s sarcoma. European Journal of antitumor activity and reduces animal toxicity and
Cancer. 2005;41(9):1349-61. immunogenicity. Proc Natl Acad Sci USA. 2000;97:8548-53.
C H A P T E R 50
Biological Response Modifiers
Rapid advances in standard management approaches (surgery, chemotherapy and radiotherapy) for cancer, have led to remarkable
cure rates in children with cancer. Certain limitations include unresectability of the tumor, resistance to chemotherapeutic agents,
intolerability of vital structures to radiotherapy, and critical effect of radiotherapy on growth of pediatric patients. Consequently, new
therapeutic approaches are being explored, including the use of biologic response modifiers.
Cancer cells express a wide range of different proteins that DEFINITION

act as antigens. Some of these may be a result of oncogenic
transformation and are relatively specific to cancer cells. Biological response modifiers (BRMs) are natural or
These tumor-associated antigens are delivered to the synthetic substances used to boost or restore the ability of
immune system by antigen-presenting cells (APCs) the immune system to fight cancer, infections, and other
through major histocompatibility complex (MHC) diseases or used to lessen certain side effects that may
class I or class II pathways. In the class I pathway, the be caused by some cancer therapies. These substances
phagocytosed tumor cells are processed by proteosomes are also called as biological therapy, biotherapy or
and converted to short peptide fragments, which are then immunotherapy.
presented on class I MHC molecules. These are recognized
by CD8+ cytotoxic lymphocytes, which have direct
CLASSIFICATION OF BIOLOGICAL
cytotoxic effects leading to tumor cell lysis. In the class II RESPONSE MODIFIERS
pathway, the secreted products from tumor cells enter the Newer molecularly targeted drugs and biotherapeutic
APCs, which are then processed and presented to MHC agents have made the classification of anticancer drugs
class II molecules. These processed antigens are recognized more complicated. Xiong-Zhi Wu3 tried a simpler
by CD4+ helper lymphocytes, which enhance the CD8+ classification based on mechanism of action of the drugs.
cytotoxic responses as well as the humoral response For descriptive purpose, BRMs can be classified as below:
to surface antigens present on tumor cells. Lowered • Monoclonal antibodies
expression or lack of MHC antigens on the tumor cells may • Nucleic acid based agents
allow tumor cells to escape the host immune surveillance.1 • Small molecule agents
The goal of biologic response modifiers is to stimulate • Cytokines
the body’s own immune system to help eradicate tumor • Tumor vaccines.
cells. In 1884, Cooley2 observed absence of postsurgical The two sections that follow address monoclonal
recurrence of round cell sarcoma in one patient who had antibody and nucleic acid-based therapeutic approaches
erysipelas infection at surgical site. He directly inoculated with relevance to pediatric oncology. Examples of small
an infectious agent to a tumor site in hopes of stimulating molecule inhibitors directed at specific targets are
an immune response against tumor. Immunotherapy has discussed in later sections. Cytokines and tumor vaccines
evolved considerably since these early days. are discussed briefly thereafter.
Monoclonal Antibodies • Anti-GD2 antibodies tried in neuroblastoma patients

had shown limed success. These include murine
Monoclonal antibodies directed against unique tumor monoclonal 3F8, ch14.18 with GM-CSF and IL-2
antigens have efficacy against neoplastic cells. After and ch14.18-IL-2 fusion protein. Radiolabeled 3F8-
binding to their target antigen, monoclonal antibodies murine antibodies have been used to deliver up to 40
have multiple mechanisms of anticancer effect, including: Gy radiation to tumor. The molecule ch14.18 is being
• Antibody-dependent cell–mediated cytotoxicity (ADCC). evaluated in the setting of minimal residual disease
• Complement-dependent cytotoxicity (CDC). and in adjuvant setting.
• Interfering with ligand-receptor interactions, including • Epratuzumab is an anti-CD22 antibody under clinical
down-regulation of receptor expression on the cell trial for CD22 expressing B-cell ALL and NHL.
surface. • Alemtuzumab is an anti-CD52 antibody, being evaluated
• Modification of signaling pathways to produce in children with recurrent ALL expressing CD52.
apoptosis. • Trastuzumab is an anti-HER2 antibody, being evaluated
• Delivery of toxic substances to cancer cells. The in children with osteosarcoma.
antitumor activity of monoclonal antibodies can be • Anti-CD30 antibodies have entered phase I/II evalu
increased by: ation in adults with Hodgkin’s lymphoma and ALCL
– Enhancement of ADCC by priming of effector cells expressing CD30, with some evidence of antitumor
with cytokines like GM-CSF, interleukin (IL)-2 and activity observed.
IL-12; • IGF-1R blocking antibodies are of potential pediatric
– Enhancement of CDC mediated by binding of interest in rhabdomyosa coma and Ewing’s sarcoma.
β-glucan to complement receptor-3 on neutrophils;
– Conjugation with cytotoxic entities such as radio
nuclides, toxins, chemotherapy agents, and Nucleic Acid Based Agents/Antisense Agents
enzymes. Significant progress has been made in the The antisense agents are single-stranded DNA-like
past few years in the area of antibody drug conju molecules that modify expression of specific genes by
gates (ADCs) for the selective delivery of cytotoxic targeting based on complimentary base pairing. Antisense
drugs to tumors. These include SGN-35, an ADC molecules can inhibit production of functional protein by
directed against the CD30-positive malignancies their target mRNAs through several distinctive processes:
such as Hodgkin’s disease and anaplastic large • RNA cleavage by RNase H, an ubiquitous endonuclease
cell lymphoma, and trastuzumab-DM1 which has • Inhibition of translation machinery
shown activity in metastatic breast carcinoma.4 • Alteration in RNA splicing
The toxicities associated with monoclonal antibodies • Degradation of homologous RNA by small interfering
can generally be attributed to the action of the antibody RNA (RNA interference)
on normal cells expressing its target antigen. For example, Fomivirsen was approved by the FDA in 1998 for CMV
skin rash with epidermal growth factor receptor (EGFR) retinitis in AIDS.6 Bcl-2 antisense agent, oblimersen, is
targeted antibodies, severe pain with GD2 ganglioside
a phosphorothioates, that can induce RNase H cleave
targeted antibodies and first dose reaction with CD52 tar-
of BCL-2 mRNA leading to activation of apoptotic
geted alemtuzumab.5
pathways. It is being studied in pediatric population in
neuroblastoma (phase1 trial), as higher BCL-2 expression
Clinical Significance of Monoclonal Antibodies5 is associated with unfavorable histology and N-Myc gene
• Rituximab is an anti-CD20 chimeric IgG1 antibody amplification. Prolonged aPTT and thrombocytopenia are
having cytotoxic effect, both through complement major side effects due to polyanionic backbone structure.
activation and ADCC. Rituximab may also induce
apoptosis through down-modulation of Lyn kinase. Therapies Targeted to Apoptotic Pathways
The later may contribute to the chemosensitizing
activity of rituximab. Rituximab is the first US food The ability of cancer cells to evade apoptosis, provide
and drug administration (FDA) approved monoclonal a survival advantage during tumorigenesis and can
antibody for cancer therapy. Clinical trials evaluating also provide cancer cells with increased resistance
the addition of rituximab to standard chemotherapy to treatment. An obvious approach to enhance the
have documented improved outcome for both indolent effectiveness of cancer therapy is by manipulating the
lymphomas and aggressive lymphomas. It is being dysregulated cancer cell apoptosis pathways to favor cell
studied in children with CD 20 expressing tumors death. The two primary apoptotic pathways, the extrinsic
like Burkitt’s lymphoma, DLBCL, and post-transplant (death receptor) pathway and intrinsic (mitochondrial)
lymphoproliferative disease. pathway, are described in Flow chart 1.
Chapter-50 Biological Response Modifiers 503
Flow chart 1 Extrinsic and intrinsic apoptotic pathways5 Molecules Targeting IAPs
Inhibitors of apoptotic pathways (IAPs) are cytoplasmic
proteins that can inhibit caspases. Survivin, X-IAP, c-IAP1,
and c-IAP2 have been most studied for their association
with cancer. XIAP overexpression appear to be associated
with poor prognosis in children with AML.7 XIAP antisense
molecules have entered clinical evaluation in AML. Survivin
is of particular pediatric interest because of association
between survivin over expression and poor prognosis
in neuroblastoma. Survivin maps to chromosome band
17q25, a region that is often represented by chromosome
gain and poor prognosis in neuroblastoma.8
Survivin antisense molecule (LY2181308) has entered
clinical evaluation. The molecule inhibited tumor growth
in xenograft models and sensitized cancer cells to radiation
therapy and chemotherapy.9
TNF-related Apoptosis Inducing Ligand (TRAIL)

Receptor Agonism
By death receptor pathway, TRAIL induces apoptosis in
many cancer cell lines but does not do so for most normal
cells. TRAIL-induced apoptosis does not require p53
Extrinsic pathway is initiated by ligation and clustering function, which could allow TRAIL to remain effective
of members of death receptor superfamily (e.g. tumor against cancer cells that are resistant to chemotherapy
necrosis factor receptor-I, Fas, and the TNF-related and radiation therapy due to loss of p53 function. A
apoptosis inducing ligand receptors TRAIL-R1 and recom binant soluble version of human TRAIL, and
TRAIL-R2) followed by recruitment of adaptor proteins agonistic antibodies directed towards TRAIL receptors
and caspase-8, which can then activate downstream (e.g. mapatumumab) have entered clinical evaluation.
effector caspases leading to apoptosis. The intrinsic Of note, TRAIL induced apoptosis in pediatric-relevent
(mitochondrial) pathway is responsive to internal toxic cancer cell lines including those of Ewings sarcoma,
stimuli (e.g. DNA damage, disruption of microtubules, etc.). rhabdomyosarcoma, and high grade glioma.
Inhibition of Bcl-2 and Bcl-xL function or direct activation
of Bax and Bak in mitochondrial membranes results Therapies Targeted to Extracellular Survival
in the release of cytochrome C and other proapoptotic Signaling Pathways
factors into the cytoplasm. Subsequent formation of
apoptosome in cytoplasm results in production of active Growth factor receptor activation can initiate signaling
caspase-9 and which then activates downstream along multiple intracellular pathways that promote pro
effector caspases. Inhibitors of apoptotic pathways are liferation and survival (Flow chart 2). These pathways
evolutionarily conserved cytoplasmic proteins that can include:
inhibit caspases. • Mitogen activated protein (MAP) kinase pathway or
These pathways can be targeted at Bcl-2 family extracellular signal regulated kinases (ERKs) cascade
proteins, inhibitors of apoptotic pathways (IAPs), and the is initiated when Ras activation leads to Raf membrane
death receptor pathway. recruitment and activation, followed by activation
of MEK and ERK. Activated ERK can translocate to
the nucleus and phosphorylate specific transcription
Inhibition of Bcl-2 Family Proteins
factors.
Oblimersen is a Bcl-2 antisense agent. Gossypol, a natural • Growth factor receptor signaling also leads to PI3K and
product derived from cottonseed extract, is a small molecule AKT activation. PTEN can reduce the extent of AKT
inhibitor of Bcl-2 and Bcl-xL. Preclinical studies demon activation, whereas mutations of PTEN resulting in
strated in vitro and in vivo anticancer activity for this agent. absence of PTEN can lead to constitutive AKT activation.
Flow chart 2 Extracellular survival signaling pathways5 HER3 (ErbB3), and HER4 (ErbB4). Binding of either EGF
or transforming growth factor alpha (TGF-α) to EGFR
activates kinases and initiate signaling cascades.
Small molecule inhibitors (gefitinib and erlotinib) and
monoclonal antibodies (cetuximab) are the EGFR blocking
agents most evaluated clinically. Gefitinib is a well-
tolerated oral EGFR-tyrosine kinase inhibitor, improved
disease-related symptoms and induced radiographic
tumor regressions in patients with NSCLC persisting after
chemotherapy.10 Use of erlotinib in advanced nonsmall
cell lung cancer demonstrated survival advantage.11 A
phase 1 study of gefitinib by COG12 demonstrated that the
drug is well tolerated in pediatric patients at oral doses 150
to 500 mg/m2. Skin rash, anemia, diarrhea, nausea, and
vomiting were common side effects. Preliminary evidence
of activity was noted in Ewing’s sarcoma, CNS tumors and
Wilm’s tumor. Gefitinib was shown to have synergistic
action with topotecan or irinotecan and additive action
with cyclophosphamide in neuroblastoma cell lines.13
Cetuximab was approved by FDA for use in com
AKT plays a central role in promoting survival by bination with irinotecan (or as a single agent if patients
phosphorylating multiple proteins involved in survival/ cannot tolerate irinotecan) for patients with advanced
apoptosis pathways. AKT activates mTOR, which leads EGFR-expressing colorectal cancer that is refractory
to phosphorylation of eukaryotic initiation factor 4E to irinotecan-based chemotherapy.14 A case report by
(eIF4E)-binding protein 1 (4E-BP1) and ribosomal Grisanti S et al15 showed anticancer activity of cetuximab
protein S6 kinase 1 (S6K1). Phosphorylation of 4E- against recurrent and metastatic mucoepidermoid car
BP1 by mTOR frees eIF4E and promotes translation cinoma of salivary gland. The report also revealed inability
of mRNAs with complex secondary structures in their of cetuximab to cross the blood-brain barrier and the
5′-untranslated region (e.g. cyclin D1 and c-Myc). consequent development of CNS metastases during
Activation of S6K1 leads to increased mRNA translation. treatment.
• Growth factor receptor signaling can also lead to Lapatinib is a small molecule reversible tyrosine kinase
NF-κB pathway activation, in part through AKT inhibitor of both EGFR and HER-2. Objective responses
phosphorylation and activation of IkB kinase (IKK), have been observed in patients with HER-2 overexpressing
which leads to IkB phosphorylation and freeing of NF- breast cancer.16
κB to translocate to the nucleus. Trastuzumab is a humanized antibody that targets
• STAT pathway can also promote proliferation and the extracellular domain of HER-2 over expressing breast
survival of following growth factor receptor activation. cancer and newly diagnosed metastatic osteosarcoma.
The importance of these signaling pathways in cancer Trastuzumab is approved by FDA for use as monotherapy
cells is indicated by the variety of activating mutations in patients with HER-2-overexpressing metastatic breast
reported for members of the receptor tyrosine kinase cancer. A concern with the use of trastuzumab is increased
family, including EGFR mutations, translocations resulting incidence of cardiac dysfunction.
in platelet-derived growth factor receptor (PDGFR)
activation, KIT mutations in gastrointestinal stromal KIT and PDGFR Inhibitors
tumors, and FMS like tyrosine kinase-3 (FLT3) mutations
in AML. Activating mutations in cancer cells also occur KIT (CD117) and PDGF receptors, along with FLT3, are
in downstream signaling pathways, as exemplified by members of PDGF receptor subfamily of receptor tyrosine
mutations of PTEN (which results in AKT activation) in kinase. Binding to their ligand activate tyrosine kinase and
many cancer types and mutations of B-Raf in melanoma. subsequent signaling pathways.
Imatinib, sunitinib and masitinib are small molecule
inhibitors of KIT and PDGFR, with imatinib having more
EGFR Inhibitors widely accepted additional activity as bcr/abl kinase
EGFR family includes four members: EGFR/human inhibitor. Imatinib has a well established role in CML
epidermal growth factor receptor 1(HER1), HER2 (ErbB2), and Ph+ ALL with bcr/abl kinase activation. Tyrosine
kinase activating mutations are important as predictors of dasatinib in managing intracranial leukemic disease and
of single agent response to imatinib. Gastrointestinal substantial clinical activity in patients who experience CNS
stromal tumors typically express KIT with gain of function relapse while on imatinib therapy.
mutation and imatinib is effective in blocking signaling
from these mutant receptors. Sunitinib may be active in Mammalian Target of Rapamycin
imatinib resistant GISTs. Masitinib (AB1010), is a novel, (mTOR) Inhibitors
potent and selective tyrosine kinase inhibitor targeting
KIT that is active, orally bioavailable in vivo, and has Mammalian target of rapamycin (mTOR), a serine/
low toxicity.17 PDGFR and KIT signaling may play roles threonine kinase that is ubiquitously expressed in
in the growth and survival of some pediatric cancers.18 mammalian cells, is an important regulator of cell growth
Approximately 60 percent of pediatric AMLs express KIT. and proliferation in response to external factors (e.g.
High grade gliomas, sarcomas and neuroblastoma also growth factors) and nutritional conditions, through its
express PDGF subfamily receptors, but the concentrations downstream effectors, 4EBP1 and S6K. Inappropriate
required for growth inhibition exceeded those required mTOR activation has been implicated in the pathogenesis
for KIT and PDGFR inhibition by 20-fold, suggesting that of numerous tumor types. The largest body of clinical
targets other than KIT and PDGFR may be responsible for experience with mTOR inhibitors is in the solid organ
imatinib’s effect against these cell lines. transplant setting. Rapamycin (sirolimus), temsirolimus,
ridaforolimus, and everolimus are the mTOR inhibitors,
FLT3 Inhibitors which are being studied in cancer patients. Temsirolimus is
a pro-drug, and its primary active metabolite is rapamycin
FLT3 is expressed primarily on hematopoietic and neural (sirolimus). Temsirolimus is approved by the FDA for the
tissues. Activating mutations in FLT3 have been observed treatment of advanced renal cell carcinoma (RCC). It is
in adult and childhood AML, in children with hyperdiploid administered intravenously on a once-weekly schedule.
ALL and in infants with ALL with MLL rearrangements.19 Ridaforolimus is also administered intravenously on
CEP-701, PKC-412, sunitinib and sorafenib are small an intermittent schedule, although an oral formulation
molecule tyrosine kinase inhibitors which already entered is currently being evaluated in sarcoma. Everolimus
clinical evaluation. Although the clinical responses to is an orally available mTOR inhibitor that is typically
FLT3 inhibitors are somewhat encouraging, true clinical administered on a continuous daily schedule or on a
benefit will require additional measures to enhance the weekly schedule in combination regimens. Everolimus
magnitude of these responses, including evaluation of has recently obtained FDA approval for the treatment of
regimens that combine an FLT3 inhibitor either with advanced RCC after failure of treatment with sunitinib or
standard chemotherapy agents or with other cell signaling sorafenib.22 Reversible leukopenia, thrombocytopenia,
inhibitors.20 and dose-dependent hyperlipidemia have been the
principal toxicities associated with rapamycin and evero
Src Family Kinase Inhibitors limus in the transplant setting. Preclinical studies in
pediatric setting have shown activity of rapamycin against
Src family kinases (SFKs) have a critical role in cell adhesion, a number of pediatric malignancies, including ALL, rhab
invasion, proliferation, survival, and angiogenesis during domyosarcoma, osteosarcoma, medulloblastoma, and
tumor development. Dasatinib is an orally available small- Ewing’s sarcoma. Preclinical observations that the combi
molecule multikinase inhibitor. It potently inhibits BCR- nation of rapamycin and tyrosine kinase inhibitors (e.g.
ABL and SFKs, but also inhibits c-KIT, PDGFR, and ephrin FLT3 or Bcr-Abl inhibitors) showed enhanced activity
receptor kinase. Dasatinib is about 300 times more potent in vivo against leukemias provide rationale for exploring
than imatinib in cells expressing unmutated BCR-ABL such combinations in the pediatric setting.23
in vitro, and have good CNS penetration. It effectively inhibits
the growth of leukemic clones harboring all known imatinib-
Histone Deacetylase Inhibitors
resistant BCR-ABL kinase domain point mutations, with the
exception of V299L, T315I, and F317L mutations. Dasatinib Histones are a family of nuclear proteins that interact with
is approved for the treatment of patients with BCR-ABL- DNA, resulting in DNA being wrapped around a core of
positive CML and ALL, resistant or intolerant to imatinib. It histone octamer within the nucleosome. Acetylation of
has been used at doses of 100 mg/m2/day in chronic phase selected lysine residues plays a key role in controlling the
of CML, 140 mg/m2/day in ALL and accelerated/crisis phase function of many proteins, including histones. The level of
of CML in adults, and 60 to 160 mg/m2/day in children. protein acetylation is maintained by the counterbalancing
Porkka et al21 demonstrated promising therapeutic potential actions of histone acetyltransferases (HATs) and histone
deacetylases (HDACs). Histone acetylation alters chroma- inhibit this process and thus interfere with the function of
tin structure and induces a local chromatin environment RAS. One such FTI is tipifarnib (R115777), which is currently
conducive with gene transcription, whereas histone de being evaluated in acute leukemias, juvenile myelomonocytic
acetylation is commonly associated with repression of leukemia, pediatric brain tumors, and neuroblastoma.
transcription. Goemans et al identified T-cell ALL and AML-M5 as the most
Through histone hyperacetylation-mediated changes sensitive subset of pediatric acute leukemia.26 Oral tipifarnib
in chromatin conformation and gene expression, histone is well tolerated in children receiving the drug twice daily for
deacetylase (HDAC) inhibitors induce differentiation, cell 21 days and a continuous dosing schedule at 200 mg/m2/
cycle arrest, apoptosis, growth inhibition and cell death, dose, which is equivalent to the maximum tolerated dose
which are more pronounced in transformed cell-lines (MTD) in adults. The pharmacokinetic profile of tipifarnib in
than in normal cells. Additional anti-cancer effects of children is similar to that in adults.27
HDAC inhibitors include inhibition of migration, invasion
and angiogenesis in vivo. Proteosome Inhibitors
Preclinical data have demonstrated the efficacy of
various HDAC inhibitors as anticancer agents, either as The 26S proteosome regulates the degradation of many
monotherapies or in conjunction with other treatments proteins involved in cell cycle control, apoptosis, and
such as chemotherapy, biologic therapy, or radiation tumor growth. The inhibition of the proteosome by
therapy. Vorinostat and depsipeptide, two actively specific inhibitors, which results in stabilization of tumor
studied HDAC inhibitors, were recently approved by suppressor proteins IkB, p21 and p53, is a viable target for
the FDA for the treatment of refractory cutaneous T-cell antitumor therapy. Most prominently, the proteosome
lymphoma. Other inhibitors, for example, belinostat inhibitor bortezomib was approved by the FDA for the
(PXD101), PCI-24781, ITF2357, MGCD0103, MS-275, treatment of relapsed or refractory multiple myeloma
valproic acid and panobinostat (LBH589) have also in adults, and is presently considered for pediatric
demonstrated therapeutic potential. It is noteworthy that malignancies such as leukemias, lymphomas, neuro
ITF2357 showed significant anti-Hodgkin’s lymphoma blastoma, rhabdomyosarcoma, and Ewing’s sarcoma.
activity. Panobinostat showed consistent antileukemic The first clinical trials by the Children’s Oncology Group
effects. Belinostat appears to be promising for treating low (COG) were conducted with bortezomib for the treatment
malignant potential ovarian tumor. The combination of of refractory solid tumors and refractory leukemia.
demethylating agents, valproic acid, and all transretinoic Bortezomib is well tolerated in children with recurrent or
acid (ATRA) has significant clinical activity in leukemia refractory solid tumors and leukemia. The recommended
and MDS. Epigenetic agents in combination regimens for phase II dose of bortezomib for children was 1.2 mg/m2/
cancer therapy are being actively studied.24 dose, administered as an intravenous bolus twice weekly
Role of valproic acid in infant spinal glioblastoma needs for 2 weeks followed by a 1-week break. Thrombocytopenia
further evaluation as a case report showed decrease in the was dose limiting toxicity.28
size of the tumor and improvement of symptoms with the
use of sorafenib plus valproic acid.25 Preclinical studies have Angiogenesis Inhibitors
shown activity of HDAC inhibitors in some pediatric tumors Although angiogenesis is a complex process involving
including neuroblastoma (with ATRA), medulloblastoma, many factors, VEGF appears to be rate limiting in
Ewings sarcoma and Burkitt’s lymphoma. normal and pathologic blood vessel growth. Therapeutic
approaches to targeting this angiogenic pathway include
Protein Farnesyl Transferase Inhibitors antibodies directed against VEGF, antibodies directed
Ras is an important anticancer target, but intracellular ras against VEGF-R2, small molecule inhibitors of VEGF-R2,
signaling requires its association with the cell membrane, and interference of integrin-matrix interactions. Trials are
which in turn requires a post-translational addition of currently underway to evaluate several antiangiogenesis
farnesyl or geranyl group at carboxy terminal cysteine agents, including SU5416, bevacizumab, TNP-470,
residue, a process known as prenylation. There are two thali
domide, SU6668, ZD4190, ZD6474, and PTK787.
forms of prenylation-farnesylation by farnesyltransferase, Bevacizumab is a monoclonal antibody that inhibits a
and geranylation by geranylgeranyl transferase. Farnesy single isoform of the VEGF ligand, VEGF-A. It has FDA
lation is the dominant class of post-translational modi approval for administration as second line treatment of
fication required for proper intracellular localization metastatic carcinoma of the colon or rectum. It is also
of RAS to the inner surface of the cell membrane. approved in the USA and Europe for the first-line treatment
Farnesyl transferase inhibitors (FTIs) were developed to (in combination with interferon) of advanced RCC.29
Thalidomide has antiangiogenesis and other biologic with Kaposi’s sarcoma with mucocutaneous or asympto
activities. It was approved by FDA for erythema nodosum matic visceral involvement and patients with follicular
leprosum and multiple myeloma. Lenalidomide is active lymphoma have also been shown to benefit from IFN-alpha.
against multiple myeloma and is being tried in childhood Constitutional symptoms are quite common in
refractory solid tumors/brain tumors. Sunitinib and patients receiving IFN therapy, and are likely to occur in
sorafenib are VEGFR small molecule inhibitor which signi 80 percent or more of patients. These typically consist of
ficantly prolonged progression free survival in patients fever, fatigue, headaches, and myalgias. More serious
with advanced RCC. are the neuropsychiatric issues, which include depres
sion (45%), confusion (10%), and mania (1%). Close
Cytokines monitoring of patient mental status or prophylactic
use of antidepressants can reduce the risk for these side
Active immunotherapy with cytokines such as interferons effects. Gastrointestinal side effects, myelosuppression,
(IFNs) and interleukins (ILs) is a form of nonspecific active autoimmune thyroid dysfunction are among the other
immune stimulation. The cytokines have been tested as significant toxic effects.34
therapies for many hematologic and solid neoplasms Interferon-gamma has been shown to enhance DNA
and have demonstrated therapeutic benefits in various fragmentation and cytotoxicity caused by tumor necrosis
cancers. To date, only two cytokines have achieved factor.35 Daily subcutaneous recombinant gamma-IFN
approval for cancer. IL-2 for the treatment of metastatic can be easily administered on an outpatient basis with
melanoma and renal cell carcinoma, and IFN-alpha for minimal local skin toxicity, results in prolonged serum
the adjuvant therapy of stage III melanoma.30 levels, and is associated with immunological changes of
potential antitumor significance.36
Interferons
Interferons (IFNs) are a group of glycoproteins that are
produced by a variety of cells stimulated by viral antigens Interleukins
and mitogens. There are three types of interferons IL-2 is a glycoprotein produced by mature T-lymphocytes
produced by a variety of cells. IFN-alpha is produced by during an immune response after receiving a signal from
macrophages and lymphocytes, IFN-beta is produced an antigen-presenting cell (APC). IL-2 increases HLA-
by fibroblasts and epithelial cells, whereas IFN-gamma restricted cytolytic activity of cytotoxic T-lymphocytes
is produced by CD4+, CD8+, natural killer (NK) cells, and and NK cells. Furthermore, the activation and expansion
lymphokine-activated killer (LAK) cells. IFNs have anti of lymphocyte-activated killer (LAK) cells, which are a
proliferative, immunomodulatory, apoptotic inducing, mixture of NK cells and CD4/CD8 T cells, is responsible
and antiangiogenesis activities.31 for HLA-unrestricted killing of all tumor cell lines. The IL-2
In a cooperative group multi-institutional clinical also have regulatory effect on immune response through
trial, stage III melanoma patients were treated with 1 activation of regulatory T cells. The balance between
year of IFN-alpha-2b. An overall improvement in median
effector T cells and regulatory T cells may be critical for
relapse-free survival from 1 to 1.7 years and in median
influencing the rejection or acceptance of tumors.
overall survival from 2.8 to 3.8 years was reported.32
IL-2 is a promising immunotherapeutic agent for the
Based on initial clinical trials data, IFN-alpha was
treatment of metastatic melanoma, acute myelogenous
approved by the FDA for the treatment of hairy cell leukemia
leukemia, and metastatic renal cell carcinoma. While
(HCL). Despite, the initial enthusiasm, a large number
of patients developed relapse after discontinuation of high-dose IL-2 regimens have shown clinical benefit in
therapy. The introduction of nucleoside analogues, with a the treatment of melanoma and renal cell carcinoma,
complete response rate close to 90 percent, has relegated serious dose-limiting toxicities have limited their clinical
IFN therapy to second-line treatment in patients who have use in a broader group of patients. The toxicity profile of
refractory disease or in those with contraindications to IL-2 is largely associated with a capillary leak syndrome.
nucleoside analogs. In addition, IL-2 can cause constitutional symptoms
Interferon-alpha has also been tested in patients who (e.g. fever, chill, fatigue) and gastrointestinal side effects,
have CML, and preliminary trials suggested that complete pulmonary edema, cardiac arrhythmias, myocarditis,
hematologic responses were possible in more than half of reversible renal and hepatic dysfunction, pruritus,
patients who had CML, with complete cytogenic responses in electrolyte abnormalities, thrombocytopenia, anemia,
nearly 25 percent. Prospective randomized trials documented and coagulopathy. Although early studies with IL-2
the superiority of IFN-alpha over chemotherapy.33 Patients reported a 2 percent mortality rate that was generally
related to gram-positive sepsis, current IL-2 centers that their expression in cancer cells and normal testis), have
routinely use prophylactic antibiotics report no mortality. widespread expression in pediatric cancers including
Low dose and combination regimens have been tried gliomas, medulloblastoma, neuroblastoma and Ewing’s
to reduce the toxicity, but these attempts were mostly sarcoma. The forth group of tumor antigens include
disappointing. The addition of IL-2 to chemotherapeutic mutated forms of normal “self” molecules. For example,
regimens (biochemotherapy) has been associated with breakpoint region of translocation in leukemias represent
overall response rates of up to 60 percent in patients with noval epitope that do not exist in normal tissues and hence,
metastatic melanoma, but this has yet to be translated may be susceptible to immune targeting. Finally, viral
into a confirmed improvement in survival. It remains to antigens also provide potential tumor targets particularly
be determined whether further modifications of IL-2- relevant to EBV associated tumors in children.39
based regimens or the addition of newer agents to IL-2 will In summary, although cancer vaccines have shown
produce better tumor response and survival.37 limited success so far, the barriers to inducing effective
IL-12, IL-15, IL-18, IL-21, IL-23 and GM-CSF are among tumor immunity are rapidly being defined. Future
challenges include investigating whether immunotherapy
the other cytokines under investigation for their antitumor
is most effective as a combinatorial therapy with surgery,
activity.
chemotherapy and/or radiation therapy.30
Tumor Vaccines SUMMARY

Current cancer vaccines aim to improve tumor-antigen Although current treatment approaches in children with
presentation and host T-lymphocyte activation. This is cancer are highly successful, we need new treatment
done by enhancing antigenic peptide-MHC molecule approaches for a significant number of unresponsive and
stability, by restoring costimulatory signals, and by relapsed cases. On the other hand, it is very difficult, rather
amplifying recruitment of the host’s immune effector cells. unethical, to introduce experimental therapy in an already
Some clinical trials used autologous or allogeneic tumor successful standard regimen. Furthermore, effect of a
cells genetically modified to express immunostimulatory new modality is difficult to quantify in a patient receiving
molecules, and some of the trials used tumor cells established treatment regimen or in a heavily pretreated
admixed with nonspecific adjuvant or soluble cytokines patient. Because of these problems, most of the studies
(adjuvant-specific immunotherapy) like BCG, IFN, or have been done in adult cancer patients. Till date, the
GM-CSF. Other approaches have used vectors encoding success of biological therapy is limited but significant. With
immunomodulatory gene products or tumor antigens more and more understanding of molecular pathology,
directly administered in situ in the tumor, with limited biological response modifiers may have more defined role
efficacy. Overall, the toxicity of these approaches is in pediatric cancer treatment.
minimal. Some studies report a rise in the frequency
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Index
Page numbers followed by f refer to figure, t refer to table and fc refer to flowchart.
A Anaplastic large cell Antibody drug conjugates 502

lymphoma 452, 453, 457 Antidiuretic hormone 464
ABO incompatibility 50, 364, 366, 367 Androgen 244 Antigen presenting cells 475, 501, 507
Absolute neutrophil count 248 deficiency 159 Antilymphocyte globulin 156
Acanthocytes 97f Anemia 24, 29, 45, 49, 53, 54, 90t, 91fc, Antimicrobials 345
Acetylsalicylic acid 337f 117, 149, 156, 158, 194, 233, 244, 322 Antiphospholipid antibody
Acidified serum lysis test 243 aplastic 241, 247, 248, 250, 252, syndrome 350
Acquired disorders 276 321f, 486 Antiplasmin 43
Activated protein C 10, 12 associated with endocrine Antiplatelet antibody 322
Acute graft versus host disease 483 disorders 158 Antiplatelet drugs 353
Acute lymphoblastic leukemia chronic 365 Antithrombin 42, 70, 348
cytogenetics of 397 classification of 87, 91f Antithrombotic agents 352
management of 402 hemolytic 49, 97, 97f, 227 Anti-thymocyte globulin 250, 476
pediatric 395 hypochromic microcytic 108fc Aplastic anemia
Acute myeloid leukemia, in newborn 45, 46, 49 acquired 247
pediatric 408, 412 incidence of 102t classification of 248t
Adenosine diphosphate 10, 42 macrocytic 93fc, 94 Appendicitis, hematology of 260t
Adenosylcobalamin 130 mediterranean 163 Apt test 72
Adrenal insufficiency 159 megaloblastic 126, 127, 138, 140 Arachidonic acid 341, 343
Adrenaline 343 microcytic hypochromic 92f, 93fc, 109, Arterial blood gases 61
Age-specific blood cell indexes 88t 109f, 110f Arthroscopic synovectomy 292
Alder anomaly 268 mild 106 Arthrotomy, conventional 292
Alder granulation 268t neonatal 54fc Asphyxia, intrapartum 59
Alemtuzumab 498 nonphysiologic 46 Aspirin-like defects 337
Allele-specific oligonucleotide 208 normocytic normochromic 94fc Autoimmune disease 266
Allergic reaction 385 nutritional 100 Autoimmune disorder 338
Allogenic stem cell transplant 426 of chronic disease 149, 150 Autoimmune hemolytic anemia,
Alloimmune neutropenia 265, 265t causes of 149t classification of 228
Alloimmune thrombocytopenia, of chronic renal insufficiency 154 Autoimmune thrombocytopenia,
neonatal 79 of infancy 29 neonatal 79
Alloimmunization 388 of liver disease 157f Autologous transplants 479
Alpha interferon 327 of prematurity 29-31, 31t, 33, 34, 34t, 45 Automated hematology analyzer 60
Alpha naphthyl acetate 410 management of 31 Autosomal recessive disorders 7, 277
Alpha naphthyl butyrate 410 pernicious 131, 132f Autosplenectomy 197
Alpha thalassemias 204 physiologic classification of 88t Avascular necrosis 197
Alport’s syndrome 269 prevention of 55 Azathioprine 315
Alternative thrombin inhibitors 353 severe aplastic 248
Amegakaryocytic thrombocytopenia,
congenital 82, 256
sickle cell 191-192, 195f B
sideroblastic 97f, 269
American Academy of Pediatrics 66 Angiogenesis 43 Bacterial infections 261t, 381
American Academy Recommendation 66 inhibitors 506 babesiosis 382
Amplification refractory mutation Anorexia nervosa 160 leishmaniasis 382
system 207, 208 Antibody dependent cellular malaria 381
Amylophagia 106 cytotoxicity 497 microfilariasis 382
512 Textbook of Pediatric Hematology and Hemato-Oncology
syphilis 381 Casitas B lymphoma 431 Combination therapy 179

toxoplasmosis 382 Cell Combined modality therapy 443
trypanosomal infection 382 death 260 Common hereditary coagulation
Basic fibroblast growth factor 43 megaloblastic 96f disorders 296
Basophilic stippling 95f membrane 333f Complement dependent cytotoxicity 502
Battered baby syndrome 284f Cellulose acetate electrophoresis 171f Complete blood count 48, 89, 155, 168,
B-cell lymphoma 457 Central nervous system 60, 398, 399, 402- 247, 396, 421
Bernard-Soulier syndrome 81, 333, 336 404, 433, 452, 459, 463, 465 Complete cytogenetic response 425
Bevacizumab 498 Central venous pressure 53 Complete remission 402, 441, 445
Biological response modifiers 501 Cephalhematoma 74, 74f, 277f Compound heterozygotes 192
classification of 501 Cerebral folate transport across Computed tomography 464
Blast crisis 420, 428 choroid plexus 135 Concurrent inflammation 239
Bleeding Cerebral venous thrombosis 241 Confirmatory tests 73, 109, 109t
disorder 70, 73, 277f, 278 Chediak-Higashi syndrome 270 Continuation chemotherapy 405
management of 314 Chelation therapy 175, 201 Conventional therapy 145
neonate 68, 70, 74, 75 Chemotherapeutic drugs 345 Cord blood, hemoglobin
time 43, 281, 300 Chemotherapy 456 concentration of 23t
Blood circulation, onset of 4 intensive 435 Cord clamping 59
Blood coagulation low dose conventional 434 Corn-soya-milk preparation 120
cell based model of 11 regimens 444 Corticosteroid therapy 323
inhibitors 12 Chest syndrome, acute 197 Cotswold’s revision of Ann Arbor staging
physiology of 10 Children’s Cancer Group 403, 446 classification 443t
theories of 11 Children’s Oncology Group 401-403, Cryohydrocytosis 218
Blood loss,obstetric causes of 46 455, 506 Cryoprecipitate 294, 302, 315
Blood transfusion 32, 234, 240 Chimeric antigen receptors 494 Cubulin 132
issues in 32 Cholecystitis 199 Customized traction system 290f
noninfectious hazards of 384, 385 Cholestatic disorders 66 Cyclic neutropenia 262
role of 31 Chorionic villus sampling 207 Cyclophosphamide 315
Blood urea 60 Chromium chloride 144 Cyclosporine 250, 251, 315
Blood volume 24 Chromosomal anomalies 81 Cystic fibrosis, atypical 269
Bone disease 172, 182 Chromosomal disorders 58 Cytogenetics 400, 422
Bone marrow 395, 396, 421t, 433, 452, Chromosomal translocations 397, 400 Cytokines 507
464, 479 Circumferential microtubular system and Cytomegalovirus 32, 80, 249, 369, 376,
aspiration 157, 248, 321f, 322 microfilaments 332 380, 434
evaluation 111 Cirrhosis 156 Cytoplasmic anomalies 268
examination 112, 154, 233, 242, 322 Citrate toxicity 388 Cytotoxic drug therapy 315
failure syndromes, inherited 255 Clot retraction 281
harvesting 480f Clotting tests 339 D
transplantation 201, 479, 482, 482f CNS disease, treatment of 467
trephine biopsy 248 Coagulation Dactylitis 196, 196f
Bone mineral density 172 acquired inhibitors of 311-314 Daycare transfusion center 175
Brain neurotransmitters 38 cascade 11fc, 312 Deep vein thrombosis 352
Breastfeeding, protection and further aggregation and Deferiprone, side effects of 177
promotion of 116 activation of 17 Dehydration 350
British Committee for Standards in inhibitors, concentrates of 359 Dense granules 333
Hematology 332 intravascular 278f Dense tubular system 332
Burkitt lymphoma 452 proteins 41 Dermal and epididymal veins,
Burns 95f system 41 thrombosis of 241
Burr cells 157 Cobalamin 128, 135, 142, 142t Desferal infusion pumps 176f
Busulfan-cyclophosphamide 482 absorption and transport of 129f Desferrioxamine 176
deficiency 141 toxicity of 177
Desferrithiocin 178
C development of 130
intracellular metabolism of 130f Desmopressin acetate 300
Cancer 158 prophylaxis with 145 Diabetes insipidus 463
Cardiopulmonary system 60 Cold agglutinin disease 231 Diamond-Blackfan anemia 256
Cardiovascular disease 448 Cold centrifuge 174f Dichlorophenol indophenol 222
Carnitine deficiency 268 Collagen ADP 340 Diet containing low iron 104
Index 513
Dilute Russel vipor venom test 283 Extrinsic and intrinsic apoptotic G
Diphyllobothrium latum 132 pathways 503fc
Direct anti-globulin test 386, 388 Gall stones 180
Gamma carboxylase 12
Disease directed therapy 476 F Gamma glutamyl carboxylase 64, 74
Disseminated intravascular
coagulation 61, 70, 78t, 80, 98, 350, Familial hemophagocytic Gamma thalassemia syndrome 53
356, 356fc, 357f, 363, 367, 367t lymphohistiocytosis 470-472, 476 Gastrointestinal bleeds 291
Divalent metal transporter 151 Familial neutrophilia 258 Gastrointestinal surgery 105
Dohle bodies 95, 96f, 269 Familial thrombophilia 350 Gastrointestinal system 60, 464
Down syndrome 413 Fanconi anemia 82, 255 Gemtuzumab ozogamicin 413, 497
Drug immune neutropenia 266 Farnesyltransferase inhibitors 413, 434, Gender-adapted chemotherapy 444
Drug induced immune hemolytic 435, 506 Gene inheritance of 166
anemia 229, 232 Febrile neutrophilic dermatosis, Gene therapeutics, procedure of 492
Drug induced platelet function acute 258 Gene therapy 182, 491, 492f, 493
defects 338 Febrile nonhemolytic transfusion Genetic syndromes 263t, 264t
Dyskeratosis congenita 256 reactions 384, 385, 387, 390 Germinal centre 440
Dysprothrombinemia 304 Fechtner’s syndrome 269 Giant granulation 270
Felty’s syndrome 266 Gibbon ape leukemia virus 493
Femoral head, avascular necrosis of 197 Glanzmann’s thrombasthenia 19f, 278,
E 279f, 333, 336, 337
Fetal erythropoiesis 58
Eculizumab 244 Fetal hemostatic system 41 Glucocorticoids 235
Ehler-Danlos syndrome 72, 73 Fetal latent iron deficiency 38 Glucose 6 phosphate dehydrogenase
Elastic modulus 346 Fetomaternal hemorrhage, chronic deficiency 52, 98, 219
Electrocardiogram 178 causes of 47 Glutathione 219, 221
Electronic methods, advantages of 48 Fetoplacental hemorrhage, causes of 47 Glycogen granules 95f
Elliptocytes 95f, 217f Fibrin degradation products 358 Glycolytic pathway, enzymes of 223t
Elliptocytosis, hereditary 216, 217 Fibrin sealant 294 Glycoprotein 296, 298
Endocrine dysfunction 172 Fibrin stabilizing factor specific acute antibody assay 322
Endocrine system 464 deficiency 307, 308 Glycosyl phosphatidyl inositol 238
Endothelial protein C receptor 12 Fibrinogen deficiencies 303 Graft versus host disease 252, 365, 385,
Enzyme linked immunosorbent 306 Fibrinogen degradation products 338 483, 487, 494
Epidermal growth factor receptor 502 Fibrinogen split products 80 grading of 484t
Epinephrine 343 Fibrinolytic activity 69 Granule 333f
Epithelial cells 106 Fibrinolytic pathway 13 exocytosis 471f
Epratuzumab 497 Fibrinolytic system 43 release assay 475
Epstein-Barr virus 369, 376, 380, 434, Fibrosis, hepatic 485 Granulocyte 268, 270, 271, 369
439, 452 Flow cytometry 345, 397 colony stimulating factor 263
Erythroblasts 259 Fludeoxyglucose f18 465 cytoplasm, anomalies of 268t
Erythrocytapheresis 201 Fluorescence in situ hybridization macrophage colony-stimulating
Erythrocyte 259 technique 422, 453 factor 498
sedimentation rate 149 Folate nuclei, abnormalities of 271t
Erythropoiesis and intrinsic hematologic disease 138 Gray platelet syndrome 81, 339
hepatic 4 deficiency 141t Gray staining bodies 270
ontogeny of 3 development of 137 Growth retardation 107
Erythropoietin 29, 30, 33, 182 homeostasis, regulation of 135 Guanosine triphosphate 432
current status of 33 receptors 135
early versus late 33 recommended daily allowance of 134t H
recombinant 33, 55, 155 renal retention of 135
resistance 156 structure 133f Haemophilus influenzae 197, 233, 236
Ethinyl estradiol and levonorgestrel 302 Folic acid, prophylaxis with 145 Hairy cell leukemia 507
Ethylene diamine tetra-acetic acid 281 Follicular lymphoma 453 Ham test 243
Euglobulin clot lysis time 283 Food and Drug Administration 200, 372, Hand-foot syndrome 196, 196f
Evan’s syndrome 266 497, 502 HBV detection 373
Extracellular signal regulated kinases 503 Food stability 134 HCV detection 373
Extracellular survival signaling Free erythrocyte protoporphyrin 109, 111 Heinz bodies 52f
pathways 503, 504fc Fresh frozen plasma 294, 304, 358, Hematinics, deficiency of 267
Extracorporeal membrane 363, 370 Hematological malignancies, gene
oxygenation 365 Frozen cells 369 therapy for 494
Hematoma 319 Hemorrhagic telangiectasia 73 immunodeficiency virus 180, 376,

scrotal 291f Hemostasis 379, 440
Hematopoiesis 241, 434 analysis system 346 leukocyte antigen 481
development of 3 developmental aspects of 41 T-cell leukemia virus 369, 376, 379
Hematopoietic cell transplantation 447 mechanism of 68, 70, 71 thrombopoietin, recombinant 328
Hematopoietic cytokines 5 neonatal 73 Humeral head, avascular necrosis of 197
Hematopoietic stem cell primary 68, 296 Hydroxybenzyl-ethylenediamine-diacetic
collection of 481 secondary 297 acid 178
transplantation 250, 252, 404, 405, types of 296 Hydroxyurea 181, 423
408, 411, 434, 436, 467, 481t Hemostatic defects, primary and therapy 200
Heme iron 103 secondary 334t Hyperkalemia 388
Hemochromatosis, neonatal 269 Hemostatic disorders 280t Hyperparathyroidism 160
Hemoglobin 23, 87, 121, 154, 165, 431f Hemostatic system, development of 64 Hyperphosphatidylcholine hemolytic
A 194 Hemotopoietic stem cells, anemia, hereditary 217
electrophoresis 193 graft types in 479 Hypersplenism 180, 368
ontogeny of 6 Henoch-Schönlein purpura 279f Hypertension, pulmonary 199
production, physiology of 87 Heparin Hyperthyroidism 159
solubility test 193 low molecular weight 352 Hypertransfusion 59
Hemoglobinopathies 53, 204 unfractionated 352 Hyperviscosity syndrome 57, 59
gene therapy for 493 Hepatitis Hypodiploidy 397, 400
inheritance of 205 A virus 378 Hypopituitarism 159
Hemoglobinuria 240 B 376 Hypoprothrombinemia 304
Hemolysis 192, 244 immunoglobulin 377 Hypothermia 387
chronic 54 virus 376 Hypothyroidism 158
immune mediated 385, 386 C 179, 377 Hypoxia 79
intravascular 239, 240 virus 377, 378
Hemolytic anemia D 378 I
acute 220 virus 378
autoimmune 51, 227, 228, 234, 235 G 378 Ichthyosis 268
Hemolytic disease 50 virus 378 Idiopathic thrombocytopenic
causes of 50 viral 376-379 purpura 229t, 368
Hemolytic transfusion reaction 388 Hereditary elliptocytosis, types of 217 Imerslund-Gräsbeck syndrome 132
Hemolytic uremic syndrome 98, 367 Hereditary platelet function defects, Immune suppressive therapy 250
Hemophagocytic lymphohistiocytosis 82, classification of 335 Immune thrombocytopenic
470, 471, 474, 474t, 475, 476 Heritable platelet defects 344t purpura 276, 279f, 318, 497
management of 475 Heterogeneous nuclear Immunization 240, 253
secondary 471 ribonucleoprotein-e1 135 Immunoadsorption 315
Hemophilia 278f, 285-287 Heterozygosity, loss of 432 Immunodeficiencies,
A 286 Histocompatibility complex, gene therapy for 493
acquired 311 major 470, 501 Immunoglobulins 315
B 287 Histone acetyltransferases 505 Immunoradiometric assay 306
C 307 Histone deacetylase inhibitors 201, 413, Immunosuppressive therapy 236, 250
gene therapy for 493 505, 506 Inactivated poliovirus vaccine 250
severe 288f, 289f Hodgkin lymphoma 439, 440, 441, 441t, Inadequate erythropoiesis 158
surgery in 293 443, 444, 445t, 446, 446t, 447, 448t Indian Council of Medical Research 36
treatment of 286 pediatric 439-443, 444t Induced pluripotent stem cells 493
Hemophilic arthropathy 287, 288, 292 Home therapy programs 288 Infantile genetic agranulocytosis 263
Hemorrhage 46, 48, 49 Howell-Jolly bodies 96f Inferior vena cava thrombosis 241
adrenal 49 Human Inherited bone marrow failure syndrome,
causes of 46 epidermal growth factor classification of 255t
chronic 54 receptor 498, 504 Inherited disorders 275
fetofetal 47 erythrocyte membrane proteins, International PNH Interest Group 242t
fetomaternal 46, 47, 105 major 213t International Reference Method 339
incidence of 46 erythropoietin, recombinant 155 International Society of Thrombosis and
intracranial 323 globin chains, chromosome map of 6f Hemostasis 356
splenic 49 herpes virus 376, 380 Intrathecal methotrexate 476
subconjunctival 277f infection 380 Intrauterine growth restriction 58, 78
Index 515
Intravenous immunoglobulin 325 L Mean platelet volume 81, 320, 321

Intravenous iron 115 Mediastinal mass 441
Intravenous pulse methylprednisolone Lactate dehydrogenase 248, 398, 453 Medicinal iron, supplementation of 119
pulse therapy 324 Langerhans cell histiocytosis 462, Megakaryocytes 321
Iron 156 465-467 Megaloblastic anemia
across placenta, transport of 104 treatment of 466 causes of 127t
chelation therapy 176 Large for gestational age 58, 60 syndrome 145
content of food articles 117t Late onset sepsis 80 Megaloblastosis 140t
deficiency Latent deficiency 36 treatment of 144
anemia 36, 100, 101, 105, 108, 110, Latent membrane protein 440 Methotrexate 456
112, 150 Lazy leukocyte syndrome 263 Methylcobalamin 130
causes of 104 Leg ulcer 180 Methylfolate trap 137
development of 36 Leukemia 321f, 399t, 486 Methylmalonic acid 143
molecular genetics of 110 associated phenotypes 397 Methyltetrahydrofolate reductase 351
placenta in 37 Leukemic stem cells 408 Minimal residual disease 401, 404,
stage 105 Leukocyte 259, 260, 260t 405, 410
stages of 105 adhesion deficiency 434 Minkowski-Chauffard syndrome 214
dextran complex 115 alkaline phosphatase 421 Mitogen activated protein 503
folic acid 113 count 154, 322 Monitoring therapy 153
fortification 118 abnormal 322 Monoclonal antibody therapy 496,
malabsorption of 104 Leukodepleted blood components 369 499, 502
metabolism, abnormal 151 Leukodepletion, method of 369 Monocytes 270
overload 175, 390 Lipopolysaccharides 239 Mosquito bite 72f
Liver function test 154, 172 Mucocutaneous bleeds 319f
replacement therapy 244
Liver iron concentration 170 Mucosa, normal 132f
sources of 102
LMWH, administration of 354t Mucositis 482
status in pregnancy 37
Lupus anticoagulant 283, 313 Mucous membrane bleeds 292
studies 170
Lymph node 443t, 463 Multiagent induction therapy 400
supplementation 33, 120
Lymphadenopathy 441 Multidrug resistance-associated
therapy 113, 153
Lymphoblastic leukemia 395, 396, 396t, protein 134
oral 113
398, 402, 403, 408, 409, 486 Multiparametric flow cytometry 401
parenteral 115
Lymphocyte depletion 441t Multisystem disease 463
transfer, regulation of 107
Lymphoma Muscle
transport 37, 104 bleeds 290
Isolated neutropenia, chronic 263t, 264t lymphoblastic 452
marginal zone 453 hematoma 291f
Lymphoproliferative disease 228 Mutations, diversity of 166
J Mycoplasma pneumoniae 233
Myelodysplastic syndrome 242t, 248
Jaundice, neonatal 220, 221 M Myeloid antigen expression 399
JMML, management of 434
Macrocytosis 140t Myeloid leukemia
Jordan’s anomaly 268
Macrophage activation acute 239t, 396, 408, 409t, 413, 414,
Jude’s staging system for childhood
syndrome 474, 476 486, 487, 497
NHL 454t
Maintenance therapy 403, 411 chronic 337, 419-423, 430
Juvenile myelomonocytic Myeloid neoplasms,
leukemia 422, 430, 431, 433, Malaria 191
hypothesis 220 current classification of 409t
Malignancy 350 Myelokathexis 264
K Marrow hypoplasia 269 Myelomonocytic leukemia, chronic 430
Marrow neutrophils, Myeloperoxidase 397, 409
Kallekrein-Kinin system 12
multinuclearity of 264 Myeloproliferative disorders 95f
Kasabach-Merritt syndrome 80, 280f
Marrow transplantation 266 Myeloproliferative syndromes 338
Kelfer capsules 177f
Kidney function tests 60 Massive transfusions 367
Kleihauer-Betke’s test47f Maternofetal transfusion 59 N
Knee joint Maximum tolerated dose 506
Naked-eye single tube red cell osmotic
bleeding in 277f May-Hegglin anomaly 81, 269
fragility test 169
chronic synovitis of 288f Mean cell hemoglobin concentration 169
National Family Health Survey 37
Koilonychia 106f Mean corpuscular volume 25, 89, 138,
National Nutritional Anemia Control
Kostmann syndrome 256, 263 138t, 248
Program 113, 119
Natural killer cell 473 O Pituitary gland 463

Necrotizing enterocolitis 60, 78, 367 Placenta, accidental incision of 47
Neonatal Ontogeny and hematopoiesis, cytokine Placental growth factor 59
anemia, management of 53 regulation of 5 Placental transport 135
hemostasis, normal 68 Oral cavity 463 Plasma 294
infections 80 Oral chelator 177 glycocalicin 322
intensive care unit 30 Oral iron therapy, side effects of 114 Plasmapheresis 315
polycythemia, incidence of 57 Osteopenia 172, 179, 182 Plasminogen activator inhibitor 43
thrombocytopenia management of 179t Plasmodium falciparum 220
causes of 78, 78t, 79 prevention of 179 Platelets 42, 249
patterns of 78 Osteoporosis 172, 179, 182 activating factor 10
Nephrotic syndrome 350 management of 179t aggregation response 282t
Neutral lipid prevention of 179 aggregation studies 283
storage disease 268 Overhydrated hereditary clumping 15f
vacuoles of 268 stomatocytosis 217 concentrates, transfusion of 358
Neutropenia 262-264, 266 consumption of 80
antibody induced 265 P contractile force 346
autoimmune 265, 265t count 61, 154, 157, 281, 320f, 321f
chronic 263t Packed red blood cell 249, 363, 365, 369 defects 334
Neutrophil 259 Pain derived growth factor 333, 423, 504
benign disorders of 258 abdominal 196, 260t disorders 73, 339
nuclei, hypersegmentation of 271, 271t relief of 289 distribution width 321
specific granule deficiency 269 Paper electrophoresis 171f dysfunction 334, 368
Newborn sickle cell disease screening 193 Pappenheimer bodies 97f flow cytometry 18f
Nicotinamide adenine dinucleotide Parahemophilia 305 function
phosphate 219 Paraprotein disorders 338 analyzer 281, 340
Nitric oxide 43, 201 Paroxymal nocturnal hemoglobinuria, assays 340
Nocturnal hemolysis 239 molecular genetics of 238 defects 334, 337, 339, 340
Nodular lymphocyte 441 Paroxysmal cold hemoglobinuria 231, 232 disorders 332, 335fc, 342f
Nodular sclerosis 441t Paroxysmal nocturnal global screening tests of 339
Nonbioavailable dietary iron 104 hemoglobinuria 238, 248t, 337 granules 16t, 332
Nonhemolytic anemia, treatment of 245 Partial exchange transfusion 61 increased consumption of 367
Nonhemolytic febrile transfusion Partial thromboplastin time, light transmission aggregometry 340
reactions 365, 369 activated 12, 69, 282, 313, 351 morphology of 333f
Non-Hodgkin lymphoma 396t, 451, Parvovirus B19 381 neutralization procedure 283
452, 455 Pearson’s syndrome 256 poor plasma 341
Nonimmune mediated hemolysis 386 Pediatric AML, treatment of 411 rich plasma 19f
Noninvasive prenatal diagnosis 211 Pediatric Hodgkin lymphoma, structure 332
Nonreplacement therapy 302 treatment of 446 transfusions 366, 368
Non-specific esterase 397, 410 Pelger-Huet anomaly 271 types of 366
Nonspherocytic hemolytic anemia, Pentameric IgM antibodies 231 Pneumococcus polysaccharide 263
chronic 220, 222 Pentose phosphate pathway 219f Pneumocystis carinii pneumonia 250
Nonsteroidal anti-inflammatory Perinatal sepsis 80 Pneumonitis, interstitial 484
drugs 286, 289 Peripheral blood PNH cells, types of 239t
Noonan syndrome 431, 432 film 154 Polycythemia 57, 59, 59t, 62
Normal hematological values 23 stem cells 480 primary 58
Normal platelet rich plasma sample 18f Peripheral T-cell lymphoma 453 secondary 59
Normoblast 169f Peroxidase deficiency and vera 337
Normocytic normochromic RBC 25f monocytes 270 Polymerase chain reaction 222, 373
Novel erythropoiesis-stimulating Persistent recurrent bleeding 276f Polymorphonuclear neutrophils 138t
protein 156 Pesaro Thalassemia Risk Classification 485 Pomalidomide 201
Nucleic acid amplification testing 372 PET scan, emerging role of 453 Portal system, veins of 240
Nucleophosfomin mutations 410 Petechiae 319 Postremission therapy 405, 411
Nucleotides, measurement of 346 Philadelphia chromosome 397, 400, 419, Post-thrombotic syndrome 355
Nutrition 128 420f Post-transfusion purpura 385, 389
education and dietary Phlebotomy 62 Post-traumatic stress disorder 405
modification 117 Phosphodiesterase activity, Precursor T-lymphoblastic
Nutritional deficiency 130 inhibition of 338 lymphoma 456
Index 517
Primitive neuroectodermal tumor 487 Reduced folate carrier 401 gene mutations, types of 190
Promyelocytic leukemia, acute 409, 414 Reed-Sternberg cells 440 syndromes 192
Prophylactic therapy 289, 290 Refractory disease 447 trait 192
Prophylaxis Refractory episodes 198 Sickling test 193
intermittent 290 Renal cell carcinoma 505 method of 193
primary 290 Renal damage, chronic 240 Single donor platelet 366
secondary 290 Renal disease 199 Single nucleotide polymorphisms 211
Prostaglandin pathways, Renal dysfunction 240 Single system disease 463
inhibition of 338 Renal failure 157f Sinusoidal obstruction syndrome 483
Protein farnesyl transferase acute 240 Skin ulcers 198
inhibitors 506 chronic 240 Small for gestational age 58, 60
Protein tyrosine phosphatase 433 Renal replacement therapy 156 Soluble plasma transferrin
Proteosome inhibitors 506 Renal system 60 receptor 110, 112
Prothrombin complex concentrates, Renal transplantation 156 Spherocytes 52f, 94f, 215f
activated 314 Respiratory distress syndrome, Spherocytosis, hereditary 51, 52f, 213,
Prothrombin deficiency 304 acute 369, 499 214, 214t, 215, 216
Prothrombin time 66, 281, 300, 313, 351 Restriction fragment length Splanchnic vein thrombosis 241, 494
Proton-coupled folate transporter 134 polymorphism 210 Splenectomy 180f, 216, 236, 245, 326
Proximal renal tubular acidosis 240 Reticular dysgenesis 256 ITP, indication of 326
Pseudo-Chediak-Higashi Reticulocyte 96f, 169f, 193f Splenic vein thrombosis 241
granulation 270 count 25, 26f, 154, 157, 169 Standard deviation 109, 141
Pseudotumors 292, 293f Retinal vein thrombosis 241 Staphylococcus aureus 249
Purpura fulminans, neonatal 354 Retinopathy 199 Stem cell
Pyridoxal isonicotinoyl hydrazone 178 Retroviral infection 379 infusion 482
Pyrophosphorolysis-activated Reverse dot blot analysis 208 transplantation 181, 459
polymerization 211 Reverse transcription polymerase chain current status of 411
Pyruvate kinase 224 reaction 422 sources of 479, 480t
deficiency 52, 224, 224t Revolutions per minute 60 Steroids 315, 323
Rh-compatibility 364, 366, 367 dose of 324
Q Rh-isoimmunization 50 Stomatocytes 95f, 217f
Rheumatoid arthritis 266 Stomatocytosis
Qualitative platelet Ristocetin induced platelet dehydrated 217
defects 279f aggregation 300 hereditary 217, 218
disorders 73, 281 Rituximab 236, 327, 497 Storage iron depletion 105
Quantitative defect 303 Romanowsky-stained blood films, Storage pool defects 336, 337
bacteria in 262t Streptococcus pneumoniae 197
R Russell viper venom 307 Stuttering episodes 198
Sucrose lysis test 243
Radiation therapy 445
intensity modulated 445 S Suicide gene therapy 494
Superconducting quantum interference
technique 445 S-adenosyl-methionine 137 device 171
volume 445 Schilling test 143 Superior vena cava syndrome 453
Radioimmunoassay 112 Screening tests 72, 73t, 108, 111 Supportive therapy 201, 476
Radiotherapy 445, 457 Sebastian platelet syndrome 269 Sweet’s syndrome 258
Radioulnar synostosis 82 Sepsis 350 Synovectomy 292
Random donor platelet 366 hematologic scoring system for 261t types of 292
Rapamycin inhibitors, Serum ferritin 111, 112 Synovitis
mammalian target of 505 Serum iron 112, 155, 243 chronic 288, 289f
Rapid plasma reagin 381 Shock 53 subacute 288
Rare coagulation disorders 303 Shortened erythrocyte survival 150, 158 Systemic lupus erythematosus 228, 229,
Red blood cell 25, 30, 87, 150, 213, 219, Shwachman syndrome 269 319
232, 385 Sickle cell 95f Systemic thrombolytic agents,
agglutination of 504 anemia administration of 354t
deformity 96f genetics of 192
dehydration, prevention of 201 management of 194, 197
distribution width 142, 168, 215, 248 T
disease 190, 191, 194, 195f, 200,
enzymopathy 219 205, 211 Target cell 97f, 169f
membrane disorders 213 homozygous 192 T-cell immunophenotype 452
T-cell lymphomas 456 Transcription mediated amplification 373 Vascular access devices 349
Template bleeding time 340 Transferrin receptors 110, 112 Vascular cell adhesion molecule 201
Terminal deoxynucleotidyl Transferrin saturation 111, 112 Vascular endothelial growth
transferase 452 Transforming growth factor alpha 504 factor 43, 59, 498
Thalassemia 163, 350 Transfusion 153, 200 Vaso-occlusive crisis 195
belt 164 acute hazards of 385 Veins, hepatic 240
inheritance of 167f adequacy of 175 Venacaval interruption 353
intermedia 168, 170f advances in therapy 175 Veno-occlusive disease 483
leg ulcer in 180f associated graft versus host Venous thromboembolism 348, 351
major 168, 170f, 350 disease 369, 389, 390 treatment of 353
management of 168, 173 delayed hazards of 388 Vessel wall 43, 68
minor 169 rate of 175 Viral infection 261t, 266
outdoor center 174 reactions, acute 385 Virus associated hemophagocytic
prenatal diagnosis protocol 207fc related acute lung injury 371, 387 syndrome 470
syndrome 163-165, 204 related immunomodulation 389, 390 Vitamin
trait 167 support 249 B12 128
Therapeutic test 111 therapy 173, 200 absorption and transport of 128
Therapy, recommended durations of 353 chronic 244 recommended daily
Thrombin complications of 175 allowance of 128t
activable fibrinolytic inhibitor 10, 13 initiation of 174 D receptor 173
clotting time 283 transmitted cytomegalovirus 376 K 66, 73
generation time 346 transmitted infections 179, 372, 376 antagonist 352, 353
receptor activating peptides 341 transmitted virus 376 cycle 65
regulation of 42 types of 174 deficiency 48, 64, 65, 73, 74f
time 300, 303 Transient deficiency of coagulation epoxide reductase 12, 65
Thrombocytopenia 79-81, 256, 335, 367 factors, exaggeration of 70 biology of 64, 73
congenital 82 Transient myeloproliferative disorder 413 chemical structure of 65
inherited 80-82 Transient neutropenia 260 Volkmann’s ischemic contracture 290f
neonatal 77, 78 Transplant related mortality 447 von Willebrand disease 276, 279f,
Thromboelastography 340 Trephine biopsy, role of 398 296-300, 301t, 302, 303, 332, 336
Thromboembolic disease, Tumor biology 399 von Willebrand factor 10, 41, 69, 296, 297,
management of 245 Tumor lysis syndrome 395, 455 302, 315, 348
Thrombolytic drugs 345 Tumor suppressor gene 432 von Willebrand syndrome 302
Thrombopoietin 77, 328 Tumor vaccines 508
Thrombosis 240, 241 Twin-to-twin transfusion syndrome 47, 59 W
acute onset of 240 Tyrosine kinase inhibitors 423
neonatal 349 Warfarin, administration of 354t
pathophysiology of 240 U Washed cells 369
pediatric 348, 354 WBC filter 369
Thrombotic thrombocytopenic purpura Ulcers, unhealed 198, 198f Wells-Brookfield cone-plate
367 Umbilical cord 47 microviscometer 57
congenital 78t blood stem cells 480 West nile virus 374
Thromboxane 333 stem transplantation 181 White blood cell 249, 399, 402, 433
Tissue factor pathway inhibitor 10, 12, Umbilical vessels, treatment of 26 Whole blood 346, 364, 365
70, 357, 359 Unfractionated heparin, Whole genome sequencing 211
Tissue plasminogen activator 13, 43, administration of 354t Wiskott-Aldrich syndrome 71f, 81, 280f
345, 348 Uremia 338 Wolman’s disease 269
TKI, toxicity of 426 Uridine diphosphoglucoronate World Health Organization 100, 101, 399,
Toll-like receptor 471 glucoronosyltransferase-1 gene 430, 451
Topical thrombin, use of 302 promoter 222
Total body irradiation 436, 482 Urinary tract X
Total iron binding capacity 105, 111, 112, bleeding 291
149, 170 infections 100 Xerocytosis, hereditary 217
Total leukocyte count 157 Urine hemosiderin 242 X-linked inheritance 71
Total tissue factor pathway inhibitor 42 X-linked recessive pattern 277
Toxic granulation 95f
V
Tranexamic acid 294 Y
Transcobalamin 129 Vaccinations 240
Transcranial Doppler 197 Valproate 413 Yersinia enterocolitica 381

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Textbook of

Pediatric Hematology and

Mamta Vijay Manglani Anupam Sachdeva

The Health Sciences Publisher

Jaypee Brothers Medical Publishers (P) Ltd.

Inquiries for bulk sales may be solicited at: [email protected]

Aditya Kumar Gupta Anand Deshpande

Amol Dongre Aparna Vijayaraghavan

AP Dubey Brijesh Arora

Jagdish Chandra Malobika Bhattacharya

Mukesh M Desai MD Nirmalya D Pradhan

Neha Vilas Dighe Pooja Balasubramanian

Nirav Thacker Priti Desai

PS Patil MD FIAP Rashmi Dalvi

Rajesh B Sawant MD Path Ratna Sharma

Rajiv Kumar Bansal Renu Saxena

Raj Warrier MD FAAP FIAP Revathi Raj

Satya P Yadav Shripad Banavali

Shanaz Khodaiji Soundarya M

Sunil Udgire FNB Vineeta Gupta

Section 2 NEONATAL HEMATOLOGY

4. Variation in the RBC Parameters in the Newborn 23

Section 3 RBC AND WBC DISORDERS

13. Introduction and Classification of Anemias in Children 87

15. Megaloblastic Anemia 126

Section 4 BLEEDING DISORDERS

27. Approach to a Bleeding Child 275

Section 5 TRANSFUSION MEDICINE

35. Blood Components in Pediatric Practice 363

39. Pediatric Acute Lymphoblastic Leukemia 395

48. Gene Therapy 491

DEVELOPMENT OF HEMATOPOIESIS vasculature.1 Initially, clusters of mesodermal cells called

I (ζ2γ2)—and later in the development hemoglobin

Transition from Yolk Sac to Hepatic

cells of liver being erythroid precursors. These are

Bone Marrow Phase (Table 1)

CYTOKINE REGULATION OF ONTOGENY

Hematopoietic Cytokines, Transcription

Commitment to Lymphoid and

Fig. 8 Production of globin chains during the fetal and neonatal

proceeds from embryonic, fetal and then adult life. In

Fig. 6 Transcriptional regulation of common myeloid precursor

Table 2 Hemoglobins in embryo fetus and adult life8

Table 3 Hematologic values of normal fetuses

REFERENCES 6. Schechter AN. Hemoglobin research and the origins of

PLATELET ACTIVATION 4), resulting in the release of adenosine diphosphate

THEORIES OF BLOOD COAGULATION Flow chart 1 The coagulation cascade

Morawitz in 1905 put forward the first theory of blood

STRUCTURE AND ULTRASTRUCTURE

Transmission electron microscope shows detailed Function of Platelets

Fig. 4B Platelet rich plasma (PRP) of Glanzmann’s thrombasthenia sample

shows increase during first few hours of life and reaches

RED CELL COUNT AND RED CELL INDICES7,8

Red Blood Cell Count Fig. 1 Normocytic normochromic RBC in newborn

age. Diagnosis of anemia can be missed by evaluating

Table 3 Signs and symptoms of anemia of prematurity Prevention Strategies

of 2,3 diphosphoglycerate. Therefore considering the Hb Issues in Blood Transfusion11,12

Table 4 Transfusion guidelines

transfusion alternative? The answer may be yes for

of plasminogen along with physio logical deficiency of • Amegakaryocytic thrombocytopenic purpura`

Table 8 Interpretation of serum levels62

PATHOGENESIS Darbepoetin (also called novel erythropoiesis-stimula