Disorders of

Platelets
Fellows education
Hala Omer –F3
 Objectives
 Discuss the basic understanding of megakaryopoiesis and
platelet production in the context of childhood platelet
disorders

 Review clinical and laboratory features of the inherited and

acquired platelet disorders
Disorders of Platelets

 Production and life - span

 Morphology and function
 Quantitative disorders
 Thrombocytopenia

 Thrombocytosis

 Qualitative disorders
 Platelet transfusion
Megakaryocyte and Platelet
Development
 CFU-Me (pluripotential hemopoietic
stem cell or hemocytoblast) →
megakaryoblast →
promegakaryocyte →
megakaryocyte
 They are produced primarily by the
liver, kidney, spleen, and bone
marrow.
 The primary signal for
megakaryocyte production is
thrombopoietin or TPO.
 Other molecular signals for
megakaryocyte differentiation
include GM-CSF, IL-3, IL-6, IL-11,
chemokines (SDF-1, FGF-4).[3] and
erythropoietin.
Regulation of Platelet Production

 Thrombopoietin ( TPO ) - primary regulator of megakaryocyte ( MK )

development and platelet number
 Gene for thrombopoietin located on chromosome 3 region q 27-28
 Thrombopoietin receptor, c-mpl, located on hematopoietic stem cells,
megakaryocytes and platelets
 Major site of thrombopoietin production : liver cells
 Thrombopoietin levels
  in marrow failure states e.g. aplastic anemia
 N or slight  in ITP, essential thrombocythemia
  in liver failure

Kaushansky K Blood 2008 ; 111 : 981-986

Platelet Turnover and Aging

 Average platelet lifespan : 7 – 10 days

 Removed from circulation by monocyte – macrophage system
 Approximately 25 – 35% of circulating platelets located in the spleen
 15 – 25% of the daily turnover of all platelets utilized for maintenance of
vascular integrity
 Young ( “reticulated” ) platelets
 contain messenger RNA from their megakaryocyte
precursors (can be detected by labeling with RNA fluorochromes such as thiazole
orange)
 functionally more competent than old platelets
Platelet Survival in Normal Subject, ITP
Patient and Patient with Hypersplenism

J.N. George, in Hemostasis and Thrombosis by Coleman et al ( 2001 )

1/A five-year-old child has thrombocytopenia, and you are seeking its
mechanism. The patient is infused with radiolabelled platelets, and the initial
recovery and subsequent survival of the transfused labeled platelets is
depicted in the figure. Which of the following is the most likely mechanism
for the child’s thrombocytopenia?

A. Sequestration in an enlarged spleen

B. Decreased platelet production due to the reduction in
megakaryocytes
C. Ineffective thrombopoiesis
D. Immune destruction
E. Mechanical destruction
A. Sequestration in an enlarged
spleen
2/ Thrombopoietin (Tpo) is the primary
regulator of megakaryocyte growth and
development. Very high levels of Tpo are seen
which two of the following conditions?
A. immune thrombocytopenic purpura (ITP)
B. liver failure
C. aplastic anemia
D. congenital amegakaryocytic
thrombocytopenia (CAMT)
Answer: C and D

Explanation: Thrombopoietin is constitutively produced

in the liver, hence the decreased levels in patients with
liver failure. Thrombopoietin is either normal or at most
slightly increased in cases with increased
megakaryocyte/platelet mass (e.g. ITP).
Disorders of Platelets

 Production and life - span

 Morphology and function
 Quantitative disorders
 Thrombocytopenia

 Thrombocytosis

 Qualitative disorders
 Platelet transfusion
Platelet Anatomy

 Shape: round and flat discs, with diameter of 1-2 m and volume of
7-9 fL
 Plasma membrane
 Receptor glycoproteins eg GPIIb-IIIa ( IIb3 )
 Surface-connected canalicular system
 Cytoskeleton
 Organelles
 Mitochondria, lysosomes, and peroxisomes
 Alpha granules ( ~ 80 per platelet )
 Dense granules ( 3 - 8 per platelet )
Platelet Anatomy
Resting Platelet Activated Platelet

J. George Lancet 2000 ; 355 :1531-1539

Enumeration of Platelets
• Normal platelet count : 150-400 x 109 /L
• 2.5% of “normals” will have platelet count < 150 x
109 /L
 Electronic
 Not accurate at very low counts ( <10 x 10 9 /L )
 Does not detect megathrombocytes
 Does not detect pseudothrombocytopenia
 Peripheral blood smear
 Number (15-20,000 per mm3/ platelet / high power field)
 Size
 Morphology ( e.g. Gray platelet syndrome )
 Other ( e.g. schistocytes, leukocyte inclusions, leukemia )
 Pseudothrombocytopenia
 In vitro clumping of platelets, resulting in
artificially low platelet count ( frequency ~ Platelet clumping
0.1% of blood counts)
 Most often due to EDTA-dependent antibodies
against platelets ( most IgG, some IgM, IgA )

 Diagnosis
 Suspect when thrombocytopenia is reported in
non-bleeding patient
 Platelet clumps on blood film from EDTA
specimen
 Accurate platelet counts may be obtained by:
 Examination of EDTA blood
at 37°C
 Manual platelet count performed on blood
collected by finger/heel stick into citrate
anticoagulant
Hemostatic Plug Formation
 Platelet Participation in Hemostasis

1. ADHESION / ACTIVATION Platelets bind to subendothelial , VWF/collagen

via the platelet GPIba , and GPIa-IIa receptors

2. AGGREGATION : Platelets aggregate following cross-linking of platelet

activated GPIIb-IIIa by fibrinogen or VWF

3 .PROPOGATION OF THE Activated platelets provide an anionic

coagulation reaction aminophospholipid ( PL ) rich surface for the assembly of
procoagulant enzyme complexes
- Tissue factor – VIIa
- Tenase ( FIXa, FVIIIa, Ca++,PL )
- Prothrombinase ( FXa, FVa, Ca++, PL )

4. CLOT STABILIZATION : - FXIIIa

Platelet Function

ASA

Rao AK J Thromb Haemost 2003 ; 1 :671-681

Assessment of Platelet Function
 Bleeding time
 Platelet aggregation
 Agonists: collagen, arachidonate, epinephrine, ADP, ristocetin
 Glanzmann thrombasthenia ( reduced / normal GPIIb-IIIa )
 no response to collagen, arachidonate, epinephrine, ADP
 response to ristocetin
 Bernard-Soulier syndrome ( reduced / normal GPIb-IX-V )
 normal response to collagen, arachidonate, epinephrine, ADP
 no response to ristocetin
 Automated platelet function tests e.g. PFA-100
 Platelet EM
 Whole mount EM to measure dense granules
 Transmission EM for alpha granules and general morphology
 Flow cytometry
 Measure surface glycoprotein levels ( approximately 80,000 copies of GPIIb-IIIa and 25,000
copies of GPIb-IX-V / resting platelet )
Bleeding Time

 Methods : Duke, Ivy, Template ( modified Ivy )

 Normal range : typically 2 to 10 minutes
 Usually prolonged if platelets markedly reduced in number or
defective in function
 Problems: poorly reproducible ( influenced by temperature of
puncture site, skin depth, fat and capillary density ) and operator
dependent ( way blood is “blotted” away from incision )
 May have some use in the assessment of selected patients with
inherited platelet or vessel wall disorders
Platelet Aggregation Testing
 Use platelet rich plasma
 Measure change in light transmission
( optical density ) through platelet-rich plasma
 Use epinephrine, ADP, collagen, ristocetin, and arachidonic acid
as agonists
 Subject to artifact and technical variables
 Problems with thrombocytopenia
( <100 x 109 /L )
 Whole blood aggregation testing used in some laboratories
Platelet Aggregation Profiles of Normal
Controls, Glanzmann Thrombasthenia &
Bernard-Soulier Syndrome Patients
ARACHIDONIC
ACID

Shapiro AD Haemophilia 2000 ; 6 (Suppl 1) : 120-127

 Platelet EM
 Alpha granules:
• VWF: plays a major role in blood coagulation
• Fibrinogen: binds to platelet GPIIb/IIIa to agrregate platelets
• PF4: binds heparin and provides neutralization of heparin-
like molecules. This inhibits local ATIII and enhances
coagulation
• Growth factors: PDGF/ TGF-β1
• Coagulation factors: Factor V
• P-selectin: Cell adhesion molecule which promotes platelet
aggregation through platelet-fibrin and platelet-platelet
binding
 Dense granules:
• ADP/ATP: activates platelets causing “recruitment” of
additional platelets
• Serotonin: powerful vasoconstrictor
• Calcium: required for binding of fibrinogen to platelet
GPIIb/IIIa
Platelet EM -
Granules defects examples
Flow cytometry in
Glanzmann
syndrome
Flow cytometry in
Bernard –Soulieur
syndrome
 1/ A 4 year old male with no past medical history of bleeding is referred
because of epistaxis. An ear, nose, and throat doctor ordered a PFA-100
test. The results show prolongation of the PFA closure time with
epinephrine, but not with ADP. Based on these results you suspect he has
recently taken aspirin. The effect of aspirin on platelet function is a result
of which of the following?

 A. Inhibition of the glycoprotein IIb/IIIa receptor

 B. Increased release of prostacyclin
 C. Irreversible inhibition of cyclooxygenase 1
 D. Irreversible inhibition adenosine diphosphate receptors
 E. Reversible inhibition of cyclooxygenase 1
CORRECT :C
 Explanation: Aspirin (ASA) blocks thromboxane A2 synthesis from
arachidonic acid by irreversibly inhibiting cyclooxygenase 1. The
result is impaired platelet aggregation. Because epinephrine is
dependent on thromboxane A2, ASA will influence the PFA-100 with
epinephrine but will be normal with adenosine diphosphate (ADP).
The effects of ASA last the life of the platelet, approximately 7 days to
10 days.

 Nonsteroidal anti-inflammatory medications reversibly inhibit

cyclooxygenase 1. A new class of drugs act by inhibiting the
glycoprotein IIb/IIIa receptor causing platelets to be unable to bind
fibrinogen and aggregate. Dipyridamole causes a release of
prostacyclin, an inhibitor of platelet aggregation. Thienopyridines act
by irreversibly inhibiting ADP receptors, important in inducing platelet
conformational changes and aggregation.
 2/ A sixteen-month-old child has easy bruising and intermittent rectal
bleeding. A CBC, including platelet count, is normal. The bleeding time is
greater than 25 minutes. Platelet aggregation testing shows markedly reduced
or absent responses to adenosine diphosphate, epinephrine, and collagen.
Which of the following tests can best establish a definitive diagnosis?
 A. Platelet electron microscopy
 B. Bone marrow aspirate
 C. Flow cytometry
 D. Measurement of platelet prostaglandin synthesis
 E. Measurement of platelet ATP to ADP ratio
 Answer : Flow cytometry
3/ You are consulted because a child has a platelet count of 21,000/mm3 and requires
elective surgery. He is otherwise well and has no evidence of bleeding including no
bruising or petechiae. He has no history of bleeding in the past with tonsillectomy and
adenoidectomy. The peripheral blood smear shows platelet clumps. What do you
recommend?

 A. Platelet transfusion prior to surgery

 B. Cancellation of surgery until the platelet count rises
 C. Repeat the CBC using a tube containing ACD or citrate
 D. No further evaluation necessary before surgery
 E. Repeat the CBC using a tube containing EDTA
Answer:C

 This is a case of pseudothrombocytopenia. It is due to antibodies that bind to

an antigen only exposed in the presence of EDTA anticoagulant. The antibody
causes clumping in vitro only. The best way to confirm the diagnosis is to
obtain a CBC in a tube containing citrate, ACD, or heparin. Usually a different
anticoagulant will correct the artificially low platelet count.
4/ The anchoring of platelets to the injured vessel wall is facilitated by the
binding of von Willebrand factor to which of the following?
 A. Thrombospondin
 B. Platelet Factor 3
 C. Platelet Factor 4
 D. Glycoprotein 1b/IX
 E. Ristocetin
Correct :D
Disorders of Platelets

 Production and life - span

 Morphology and function
 Quantitative disorders
 Thrombocytopenia

 Thrombocytosis

 Qualitative disorders
 Platelet transfusion
Etiology of Thrombocytopenia
 Decreased platelet production
 Congenital thrombocytopenias e.g. TAR, congenital amegakaryocytic
thrombocytopenia ( CAMT ), Fanconi anemia
 Aplastic anemia
 Marrow infiltration e.g. acute leukemia
 Myelodysplasias
 Infection e.g. HIV, hepatitis C
 Paroxysomal nocturnal hemoglobinuria
 Drugs
 Increased platelet destruction
 IMMUNE
 ITP
 Alloimmune thrombocytopenia
 Heparin-induced thrombocytopenia ( HIT )
 Infection
 Drugs
 NON-IMMUNE
 Disseminated intravascular coagulation ( DIC )
 Kasabach-Merritt syndrome
 TTP / HUS
 Platelet sequestration
 Hypersplenism
Clinical Manifestation of Platelet
Disorders
Primary hemostatic disorder
 Bleeding immediate
 Bleeding into skin and mucous membranes
 Petechiae, purpura, ecchymoses, oropharyngeal bleeding, epistaxis,
gastrointestinal bleeding, hematuria, menorrhagia
 Hemarthroses, intramuscular hematomas uncommon
 Careful personal and family history of abnormal bleeding very important
 Classification of Inherited Thrombocytopenia
According to Platelet Size
DISORDER GENE INHERITANCE

SMALL PLATELETS
 Wiskott Aldrich syndrome WAS X-linked

 X- linked thrombocytopenia WAS X-linked

NORMAL-SIZED PLATELETS
 Thrombocytopenia absent radius ? Autosomal recessive
( TAR )
 Congenital amegakaryocytic C-mpl Autosomal recessive

thrombocytopenia ( CAMT )
 Congenital amegakaryocytic HOX Autosomal dominant
thrombocytopenia with radioulnar
synostosis
 Familial platelet disorder with AMLI Autosomal dominant
predisposition to acute
myelogenous leukemia
Balduini C et al. Haematologica 2002 ; 87 : 860-880
Nurden AT J Thromb Haemostas 2005 ; 3 : 1773-1782
 Inherited Macrothrombocytopenias
DISORDER GENE / ASSOCIATED
PRODUCT ABNORMALITIES

• Bernard-Soulier syndrome GPIBA, GPIBB, GP9

• Velocardiofacial syndrome ?GPIBB( 22q11 ) Cleft palate, cardiac defects,

learning disabilities
• Pseudo von Willebrand disease GPIBA

• X-linked thrombocytopenia and GATA1

dyserythropoiesis + anemia

• MYH9 – related disease MYH9 Leukocyte inclusions, high

tone hearing loss, renal
involvement, cataracts

• Paris – Trousseau type ?FLI1

thrombocytopenia

• Jacobsen’s syndrome ?FLI1 Cardiac defects, mental

retardation
• Gray platelet syndrome NBEAL2

Balduini C et al Haematologica 2002 ; 87 : 860-880

Treatments for bleeding episodes is by
fibrinolytic inhibitors rather than platelets
transfusions
Wiskott-Aldrich Syndrome /
X-Linked Thrombocytopenia ( XLT )
 Sex linked inheritance
 Classic features include
 thrombocytopenia with small platelets
 eczema
 immune deficiency ( T cell defects, early  in
IgM levels )
 defect in WAS gene ( gene product WASP )

Ochs HD Clin Rev Allergy Immunol 2001 ; 20 : 61-86

MYH9-related Disease

 Autosomal dominant Platelet

 Chromosome 22q11-13
 Nonmuscle heavy chain gene 9
( MYH9 )
 Nonmuscle myosin heavy chain
IIA

May-Hegglin anomaly

Drachman JG Blood 2004 ;1003 : 390-398

 MYH9-related Disease
Clinical Features May-Hegglin Sebastian Fechtner Epstein
Anomaly Syndrome Syndrome Syndrome

Macrothrombocytopenia Yes Yes Yes Yes

Leukocyte-inclusions Yes Yes Yes No

( Döhle-like bodies )

Hearing impairment No No Yes Yes

Cataracts No No Yes No

Nephritis No No Yes Yes

Seri M et al Medicine 2003 ; 82 : 203-215

N. Pujol-Moix et al Haematologica 2004 ; 89 : 330-337
 Nonmuscle myosin IIA
immunofluorescence

Neutrophil 5 µm Neutrophil 5 µm

Control Patient
Fanconi Anemia
 autosomal recessive disorder
• defect in DNA damage repair or replication
• phenotypic abnormalities: abnormal facies, short stature, café
au lait and hypopigmented spots, microcephaly, structural renal
abnormalities
• mild/moderate thrombocytopenia or leucopenia often precede
pancytopenia ( median age at diagnosis 6-8 yr )
•  chromosome breaks ( to diepoxybutane or mitomycin C )
•  risk of malignancies ( leukemia, myelodysplasia, hepatic and
solid tumors )
Immune Thrombocytopenia ( ITP )
Clinical Features
CHILDREN ADULTS

Presentation Abrupt Insidious

Preceding Common Uncommon

Infectious Illness ( ~ 2/3 Of Cases )

Spontaneous > 80% < 5%

Remission

Historically chronic ITP is defined as a platelet count of < 150 x 109/l for > 6
months; more useful definition is a platelet count of < 100 X 109/L for > 1 year
Mechanisms of Platelet Destruction
in ITP
 Anti-platelet antibodies
 Usually IgG, directed against platelet membrane antigens ( most
commonly GPIIb-IIIa )
 Antibody-coated platelets destroyed by monocytes / macrophages
in spleen and liver
 Antigen-antibody complexes resulting from viral infection
 Cell-mediated cytotoxicity

Cines DB, Blanchette VS N Engl J Med 2002 ; 346 : 995-1008

Cooper N, Bussel J Journal of Pediatrics 2006 ; 133 : 364-374
Presenting Features in 471 Children with
Newly-Diagnosed ITP
MALE : FEMALE RATIO 223 : 248

PRECEDING INFECTIOUS 64%

ILLNESS

HEMORRHAGIC MANIFESTATIONS
 PURPURA / PETECHIAE 97%
 EPISTAXIS 30%
 HEMATURIA 7%
 INTRACRANIAL HEMORRHAGE ~ 0.2% Cines & Blanchette, N Engl J Med
2002; 346:995-1008

Blanchette V Thrombosis and Hemostasis 2003 ; 29(6) : 605-617

Lilleyman J Arch Dis Child 1994 ; 71 : 251-253
Initial Management of Children with
Newly-Diagnosed ITP

 OBSERVATION
 87% of 55 children with acute ITP, 51 managed without therapy, in remission at 6
months ( Dickerhoff 2002 )
 IVIG
 time to reach platelet count of > 20 x 109 /L faster in children treated with IVIG or
oral corticosteroids versus no therapy.
IVIG 0.8g/kg x 1 equivalent to IVIG 1g/kg x 2 ( Blanchette 1993; Blanchette 1994 )
 ORAL PREDNISONE 4 mg/kg/d x 4 days
 20/24 ( 83% ) of children had platelet counts > 20 x 109 /L at 48 hr following the start
of therapy ( Carcao 1998 )
 IV ANTI-D
 75 mg/kg results in a significantly faster increase in platelet count than 50 mg/kg
( Newman 2001; Tarantino 2006 )
Indications for Bone Marrow Aspirate and
Biopsy in Children with ITP
 Patients with lassitude, protracted fever, bone or joint pain
 Presence of additional cytopenias
 Rule out marrow infiltration ( e.g. leukemia, solid tumors, storage
diseases )
 Rule out marrow failure ( e.g. aplastic anemia, myelodysplastic syndrome
)
 Unexplained macrocytosis
 Prior to splenectomy ( if not already done )
 ? Prior to steroid therapy
 Chronic ITP
 Approximately 20 - 25% of an unselected group of
children with primary acute ITP will develop chronic
ITP if defined as a platelet count of < 150 x 109/ L at
6 months following initial diagnosis
However…
 The frequency of children with symptomatic , severe
chronic ITP ( defined as a platelet count of < 20 x
109 /L at 1 year following presentation ) is much
lower and of the order of 5%
Management of Children with
Chronic ITP
 Corticosteriods
 IVIG
 IV anti-D
 Rituximab ( 31% response rate, Bennett 2006)
 Other single agent therapy e.g. azathioprine
 Combination therapy ( vincristine / methylprednisolone /
cyclosporine; 70% response rate, Williams 2003 )
 Splenectomy

Bennett CM Blood 2006 ; 107 : 2639-2642

Williams J Pediatr Hem/Onc 2003 ; 25 : 232-235
Blanchette V J Pediatr Hem/Onc 2003 ; 25 : S28-33
Guidelines for Splenectomy
in Children with ITP
“ Persistence of disease for 12 months after diagnosis with bleeding
symptoms and a platelet count < 10,000 ( ages 3 to 12 years ) or
10,000 – 30,000 with bleeding symptoms ( ages 8 to 12 yrs )… ”
ASH ITP Guidelines Panel, 1996

“ Splenectomy is rarely indicated in childhood ITP.

It is occasionally justified for life-threatening bleeding and for
children with chronic unremitting and severe ITP whose disease has
been present for more than
12-24 months with demonstrable impairment of their quality of
life… ” British Committee for Standards in Haematology General
Haematology Task Force, 2003

George J et al Blood 1996 ; 88 : 3-40

Provan D et al Brit J Haematol 2003 ; 120 : 574-596
Blood smear showing a red blood Nuclear Medicine scan showing an
cell with a Howell – Jolly Body accessory spleen
If ITP recurs and is persistent following an initial complete response to
splenectomy, how will physicians suspect and then confirm presence of an
accessory spleen?

- Absence of Howell-Jolly bodies in red blood cells on a peripheral blood

smear should raise the suspicion of an accessory spleen in this clinical setting.

- A nuclear medicine radioisotope tagged autologous RBC scan can be used

to document presence of an accessory spleen(s).
· NPlate (AMG 531)
Lancet 2008; 371:395
· Promacta ( Eltrombopag )
N Engl J Med 2007; 357:2237
Emergency Management of Organ or
Life-threatening Hemorrhage
in Children with ITP
 Platelet transfusion ( larger than usual dose by IV push )
 IV methylprednisolone 30 mg/kg ( max 1 gram ) over 20
minutes ( repeat daily up to x 3 as clinically indicated )
 IVIG 1 gram/kg over 4 – 6 hours ( repeat daily up to x 5
as clinically indicated )
 + emergency splenectomy

van Hoff J et al. J. Pediatr 1988 ; 113 : 563-566

Autoimmune Lymphoproliferative
Syndrome ( ALPS )
 Abnormalities of programmed cell death (apoptosis) due to mutations in the Fas, Fas
ligand or caspase 8 / 10 genes
 Clinical Factors
 adenopathy ( especially cervical/axillary )
 hepatosplenomegaly
  risk of lymphoma
 Laboratory Features
 immune cytopenias ( ITP, neutropenia, hemolytic anemia ) *
 hyperglobulinemia
  double negative ( CD4 / CD8 ) T cells
  IL – 10
 Treatment
 consider trial of mycophenylate mofetil ( MMF )
* Other causes of combined autoimmune cytopenias include common variable
immunodeficiency ( CVID ) and SLE-associated ITP
Worth et al Brit J Haematol 2006 ; 133 : 124-140
 You are examining a 7 month old male infant who is admitted to the hospital
with pneumonia. You notice that he has eczema on his face and scattered
petechiae on his trunk. CBC is normal with the exception of a platelet count
of 17,000/mm3 with small platelets on the peripheral blood smear. What is
the primary defect in this disorder?

 A. Mutation affecting the thrombopoietin receptor

 B. Mutation affecting the WASP protein
 C. Autoantibody against platelet membrane receptors
 D. Mutation in the HOX gene
 E. Abnormal binding of von Willebrand factor to the platelets
 Correct :B
 Explanation: This is child has Wiskott- Aldrich Syndrome (WAS), an X-
linked condition associated with thrombocytopenia, eczema, and
immunodeficiency. It is caused by a mutation in the WASP gene and should
be considered in any male with thrombocytopenia and small platelets. X-
linked thrombocytopenia (XLT) is also caused by a mutation involving
WASP, but results only in thrombocytopenia without the additional
complications of WAS.

 Congenital amegakaryocytic thrombocytopenia is caused by mutations

affecting the thrombopoietin receptor, immune thrombocytopenia is
associated with antibodies directed against platelet membrane receptors, and
Type 2B von Willebrand disease (vWD) and platelet-type vWD result from
enhanced binding of von Willebrand factor to the platelets. Mutations in the
HOX gene have recently been identified in patients with amegakarocytic
thrombocytopenia and radial-ulnar synostosis (ATRUS).
 You are asked to see a 10-year-old boy with a diagnosis of autoimmune
lymphoproliferative syndrome (ALPS). His past medical history includes a
diagnosis of ITP starting at age 6 years plus fluctuating cervical and axillary
adenopathy and one episode of clinically significant autoimmune hemolytic
anemia at age 8 years. His major problem is clinically persistent severe
thrombocytopenia that responds only transiently to IVIG or corticosteroid
therapy. His CBC shows a hemoglobin of 120 g/L, total WBC 7,000 /µL,
absolute neutrophil count 750 /µL and platelet count 15,000 /µL. Which one of
the following three therapeutic interventions is preferred in this case?
 A. Splenectomy
 B. Mycophenolate mofetil (MMF)
 C. Combination chemotherapy
 Answer: B
 Explanation: A trial of oral mycophenolate mofetil (MMF) is recommended in
this case. Splenectomy should be avoided because of the significant risk of
overwhelming post-splenectomy sepsis. Monotherapy with MMF is preferred
to combination chemotherapy because of the anticipated favorable response to
MMF in cases of ALPS.

Supplementary Question

 (a) List two characteristic physical findings in patients with the autoimmune
lymphoproliferative syndrome (ALPS)
 (b) What is the most common mutation leading to ALPS
 (c) What is the diagnostic flow cytometric abnormality found in ALPS cases?
 (a) List two characteristic physical findings in patients with the autoimmune
lymphoproliferative syndrome (ALPS)
- hepatomegaly
- splenomegaly
- lymphadenopathy (especially in the cervical chain)

 (b) What is the most common mutation leading to ALPS

- mutations in the Fas receptor

 (c) What is the diagnostic flow cytometric abnormality found in ALPS cases?
- An increase in double negative  T cells (CD4 negative /
CD8 negative)
Which one of the following statements is false (incorrect) for the condition
congenital amegakaryocytic thrombocytopenia (CAMT)?
 A. Severe thrombocytopenia is evident from birth.
 B. A marked reduction in the number of megakaryocytes is seen in a
bone marrow aspirate/biopsy.
 C. Improvement in the thrombocytopenia occurs during the first few
years of life.
 D. Mutations in the thrombopoietin receptor can be detected in most
cases.
 Answer: C
 Explanation:Severe thrombocytopenia in children with
congenital amegakaryocytic thrombocytopenia is persistent
throughout life as contrasted to children with thrombocytopenia
absent radius (TAR) syndrome in whom thrombocytopenia
becomes less severe during the first years of life. Many
children with amegakaryocytic thrombocytopenic develop
pancytopenia by the second decade of life.
 A 3 year old boy is admitted with the sudden onset of bruising, a generalized
petechial rash and a history of intermittent epistaxis for 2 days. Ten days before
admission the child was treated for an upper respiratory tract infection. Physical
examination was unremarkable apart from the skin findings. A hemoglobin
level was 100 g/L, total WBC and differential count normal and platelet count 2
x 109/L. The blood group was O Rhesus positive. Which one of the following
management options is contraindicated in this particular case?

 a) oral prednisone 4 mg/kg/day

 b) IV anti-D 75 µg/kg
 c) IVIG 1 gram/kg x 1
 d) close observation in hospital
 Correct:B

 The history of epistaxis and a low hemoglobin level exclude intravenous

anti-D as a first choice option because of the obligatory fall in
hemoglobin that will be induced by this therapy.

Supplementary Question

 List three (3) relative contraindications to use of IV anti-D as a platelet

enhancing strategy in Rhesus positive children/adolescents with
autoimmune thrombocytopenia purpura (ITP), no prior exposure to anti-
D and clinically significant thrombocytopenia (platelet count < 20 x
109/L):
 - Prior splenectomy
 - Co-existing clinically significant anemia (hemoglobin level
< 100 g/L)
 - Positive direct Coombs test with evidence of active
hemolysis
 4/You are asked to see an 8 year old girl who parents tell you that an older
sibling died of complications from bone marrow failure and a diagnosis of
Fanconi anemia some three years previously. On examination the girl has
phenotypic features consistent with a diagnosis of Fanconi anemia. She is
short with typical facies and presence of pigmentation and café au lait spots on
her trunk. She is missing one digit on her hands. Prior to this presentation she
has been well without a history of serious infections. She has been noted
recently to have some peripheral bruising and occasional nose bleeds. Which
of the following hematologic manifestations is least likely in this particular
case?

 a) thrombocytopenia
 b) neutropenia
 c) macrocytosis
 d) pancytopenia
 e) an increased hemoglobin F level
pancytopenia
 5/ You are referred a 3 month old male because of persistent severe thrombocytopenia
documented in the neonatal period. The infant is well, not dysmorphic and a detailed
physical examination is normal. A CBC shows a platelet count of 10 x 109/L with a normal
hemoglobin level, WBC and white blood cell differential count. The platelets appear normal
on examination of a blood smear. A bone marrow aspirate/biopsy shows a marked decrease
in the numbers of megakaryocytes with normal erythropoiesis and granulopoiesis. There is
a normal response to transfusion with random donor platelets.
 (a) What is the most likely diagnosis in this case?
 (b) What is the underlying cause for this disorder?
 (c) What is the expected natural history of the disorder?
 (d) What is the most effective long-term treatment in such cases?
 (a) What is the most likely diagnosis in this case?
 - Congenital amegakaryocytic thrombocytopenia (CAMT)


 (b) What is the underlying cause for this disorder?

 - Mutations in the thrombopoietin receptor, c-mpl


 (c) What is the expected natural history of the disorder?

 - Progression to pancytopenia within the first two decades of life

 (d) What is the most effective long-term treatment in such cases?

 - HLA matched stem cell transplantation
 6/The MYH9 diseases (the May Hegglin anomaly and the
Sebastian/Fechtner/Epstein syndromes) are caused by mutations in the
gene for the non-muscle myosin heavy chain IIA.

 (a) List the two most important abnormalities seen on peripheral

blood smears of patients with these disorders:

 (b) Patients with these hematologic findings should be screened for

which of the following?
 a) List the two most important abnormalities seen on
peripheral blood smears of patients with these disorders:
 - giant platelets -
Döhle-like bodies in neutrophils

 (b) Patients with these hematologic findings should be

screened for which of the following?
 1. cataracts*
 2. high-tone deafness*
 3. nephritis*
7/ Large platelets are not a feature of which one of the following
disorders:
 A. May-Hegglin anomaly (MYH9-associated
thrombocytopenia)
 B. Gray platelet syndrome
 C. Wiskott-Aldrich syndrome
 D. Bernard-Soulier syndrome
Answer: C

 Explanation: Small, pin-point platelets on a blood smear are

characteristic of thrombocytopenia seen in cases of Wiskott-
Aldrich syndrome. In all of the other conditions cited platelets are
large (macrothrombocytopenia).
8/ A 3-year-old boy recently had several ear infections. Physical exam shows mild
eczema, petichiae, and resolving otitis media. The CBC is normal except for a platelet
count of 37 and MPV of 6. The following is NOT true:
a) This patient has an existing immunodeficiency.
b) This patient is at risk of developing lymphoma.
c) This patient has ITP and should receive IVIG.
d) This patient has an X-linked syndrome.
e) This patient may need a bone marrow transplantation
Correct :D
9/ The laboratory finding most likely associated with ITP is:
a) Decreased megakaryocytes in BM.
b) b) Fragmented cells on peripheral smear.
c) c) Increased MPV.
d) d) Normocytic normochromic anemia
e) e) Prolonged PT.
Correct:c
10/ Which of the following best describes the mechanism of action common to
treatment of childhood ITP with corticosteroids, IVIG, and anti-D? a)
Suppression of anti-platelet antibody synthesis.
b) Inhibition of Fc receptors on macrophages.
c) Activation of suppressor T cells.
d) Unbinding of the anti-platelet antibody from the platelet surface.
e) Inhibition of cytokine release
Correct:b
11/ You are consulted to see a patient in the newborn nursery because of
petichea and bruising. You find the following signs

• The following is true about this condition EXCEPT:

 a) Autosomal recessive.
 b) Absent megakaryocyte on bone marrow aspiration.
 c) Bloody diarrhea and petichae are the most common complications.
 d) Thrombocytopenia becomes worse after the first year of life. e) Other
skeletal abnormalities are common.
Correct:D
12/3 year old girl presents with petichiae 2 weeks after a URTI with
evidence of lymphadenopathy and splenomegally. Blood count shows
platelets of 15, WBC of 60 and Hemoglobin 8. What test would you do to
confirm the diagnosis?
 a) Bone Marrow aspiration
 b) Abdominal ultrasound
 c) Lymph node biopsy
 d) EBV Serology
 e) EBV PCR
Correct :A
Classification of Neonatal
Thrombocytopenia

• EARLY ONSET < 72 Hours

• LATE ONSET > 72 Hours

• Sepsis
• Nectroizing enterocolitis ( NEC )

Roberts I Blood Reviews 2008 ; 22 : 173-186

Aetiology Of Early Onset Neonatal
Thrombocytopenia ( < 72 Hours )
COMMON
• placental insufficiency
• ( maternal pre-eclampsia , intrauterine growth
retardation )
• perinatal asphyxia
RELATIVELY COMMON
• infection
- acquired( eg group B streptococcal infection )
- congenital ( CMV , toxoplasmosis , HIV )
• disseminated intravascular coagulation ( DIC )
• Immune
- alloimmune
- autoimmune ( maternal ITP , SLE )
NEONATAL ALLOIMMUNE THROMBOCYTOPENIA
Neonatal Alloimmune Thrombocytopenia

History
 Isolated unexplained thrombocytopenia in an otherwise well infant; many
cases firstborn
 Normal platelet count in mother with no history of ITP / SLE
 Response to random donor platelet transfusion typically poor

Laboratory
 Confirmation of anti-platelet alloantibodies in mother
 Documentation of fetomaternal incompatibility for a platelet-specific antigen
[ most often HPA-1a ( PlA1 ) in a Caucasian population ]
Treatment
 Treatment of choice antigen negative platelets e.g. typed random donor or
maternal platelets; while awaiting compatible platelets consider trial of
random donor platelets + IVIG
Maternal ITP / Neonatal
Autoimmune Thrombocytopenia
MATERNAL
 Distinguish from gestational thrombocytopenia
 Mild thrombocytopenia ( platelet count 75-150 x 10 9 /L )
 Clinically insignificant
 Distinguish primary vs secondary ITP e.g. SLE associated ITP
 Treat based on maternal indications; avoid splenectomy if possible

NEONATAL ITP
 No correlation between maternal and neonatal platelet counts
 Intracranial hemorrhage rare ( < 1% ); screening head ultrasound recommended
 platelet count Nadir at 3 to 4 days
 IVIG 1 g/kg x 1-2 days + corticosteriods for clinically significant thrombocytopenia + active
bleeding
 Platelet transfusion only in organ / life-threatening situations
 Heparin Induced Thrombocytopenia
( HIT )
 Common in adults ( incidence 1-2% ) - less in children
 Hypercoagulable state - venous or arterial thrombosis
 Mediated by IgG antibodies that reacts with heparin-platelet factor 4 ( PF-4 ) complexes
leading to platelet activation via the platelet FcIIa receptor
 Develops 5-10 days after heparin exposure or within 2 days with previous heparin
exposure
 More common with unfractionated than with LMW heparin ( enoxaparin or
fondaparinus )
 CONSIDER HIT if drop in platelet count by 50% or <100 x109 /L while exposed to heparin
 HIT assay: serotonin release, ELISA heparin-PF-4
 Management: STOP ALL HEPARIN, use alternative anticoagulant ( lepirudin,
argatroban, danaparoid )

Arepally GM et al. N Engl J Med 2006 ; 355 : 809-817

Kelpon JG and Warkentinte Blood 2008 ; 112 : 2607-2616
Thrombocytopenia Associated with Disseminated
Intravascular Coagulation ( DIC )

Usually unwell patient with underlying illness

 Trauma ( esp. head injury )
 Severe infections with septicemia
 Obstetrical catastrophes ( e.g. placental abruption, fetal demise, amniotic fluid embolism )
 Malignancies
 Hypovolemic shock
 Hypoxia
Consumptive coagulopathy due to activation of thrombin
 Progressive decrease in platelet count - activation and clearance
 Consumption of clotting factors - increased PTT and PT / INR
 Consumption of fibrinogen
 Increased D-dimer - secondary lysis of fibrin
 Increased TCT - decreased fibrinogen; inhibition by FDPs
Fragmentation of cells by fibrin strands in microvasculature
 Schistocytes
Kasabach-Merritt Syndrome

Localized Intravasular Consumption In Hemangioma(s)

 Kaposiform hemangioendothelioma
 Tufted angioma
 Infantile hemangioma ( rare )
Lesion(s)
 Cutaneous lesions often dark red/purple ( vs bright red lesions of
strawberry hemangioma )
 May be multiple / visceral ( infantile hemangiomas ); Kaposi
hemangioendotheliomas generally solitary lesions
 Diagnosis: imaging ( ultrasound / MRI ) for extent of lesion or internal
lesions
Hematologic Abnormalities
 Thrombocytopenia
  Fibrinogen,  FDP’s / D-dimer
 Microangiopathic hemolytic anemia

Hall G Brit J Haematol 2001 ; 112 : 851-862

Management of
Kasabach-Merritt Syndrome
Specific
 Corticosteroids (prednisone (3-5 mg/kg/d)
 Vincristine ( 1-1.5 mg/m2 or 0.05 mg/kg IV weekly )
 a-interferon ( 3 million units/m2/d )
 Embolization
 Antifibrinolytic agents ( tranexamic acid / ε-
aminocaproic acid )
Supportive
 Fibrinogen ( FFP, fibrinogen concentrates )
 Platelet transfusion
Thrombotic
Thrombocytopenic Purpura ( TTP )
Diagnostic pentad
 Thrombocytopenia *
 Microangiopathic hemolytic anemia*
 Neurologic abnormalities
 Renal failure
 Fever

* in clinical practice thrombocytopenia, schistocytosis and an

elevated LDH are sufficient to suggest the diagnosis
George JN N Engl J Med 2006 ; 354 : 1927-1935
Schistocytes in TTP
Pathophysiology of TTP

ADAMTS13 = a disintegrin and metalloprotease with

eight thrombospondin-1-like domains
Management of Idiopathic TTP

 aggressive plasma exchange using plasma as replacement

therapy*

 for relapsed idiopathic cases, especially those with severe

ADAMTS13 deficiency ( activity < 5% ), consider trial of
immunosuppressive therapy ( Rituximab )

* no definitive evidence that cryopoor supernatant is superior to FFP

Sadler JE Blood 2008 ; 112 : 11-18
Hemolytic-Uremic Syndrome

 Infants and young children

 Preceding colitis with bloody diarrhea
 Usually due to E. coli verotoxin ( serotype 0157:H7 )
 Renal failure primary manifestation
 CNS and other organ involvement in some cases
 Normal levels of VWF-cleaving protease
 Management
 Dialysis and other supportive care
 Plasmapheresis usually not required
1/A newborn term infant is found to have bruising and petechiae on his limbs on
the fourth day of life. He is not dysmorphic and his liver and spleen are not
enlarged. The newborn infant is well. His CBC shows a hemoglobin level of 150
g/L, total WBC 8,000 /µL and platelet count 5,000 /µL. A blood smear confirms the
very low platelet count and is otherwise normal. His mother had a history of
chronic ITP that required splenectomy when she was a teenager. Her platelet count
is 250,000 /µL. What is the likely diagnosis in this case?
 A. Neonatal autoimmune thrombocytopenia
 B. Neonatal alloimmune thrombocytopenia
 C. Disseminated intravascular coagulation (DIC)
 D. Kasabach-Merritt syndrome
 Answer: A
 Explanation: Infants of mothers with ITP who have been splenectomized may
have transient neonatal ITP even though the maternal count is normal. This
reflects passage of circulating platelet autoantibodies from the mother to the
infant during pregnancy. The platelet count in such infants is often at its
lowest level a few days following birth.

Supplementary Question
 What is the recommended treatment in such a case?
 A. IVIG
 B. Exchange transfusion to remove anti-platelet antibodies
 C. Transfusion with random donor platelets
 D. IV vincristine
 Answer: A
 Explanation: Infants with neonatal ITP generally respond well to high dose
immunoglobulin-G (IVIG) therapy with or without concomitant
corticosteriods. The response to random donor platelets in such a case would
likely be poor, and exchange transfusions to remove platelet autoantibodies
are an invasive procedure in the newborn. IV vincristine is not indicated in
such a case.
 2/ A term male infant is found unexpectedly to have bruising on the
trunk and a petechial rash on the face and neck. Physical examination is
otherwise normal and the baby appears well. There are no family
members with thrombocytopenia and the parents are Caucasian. A
maternal platelet count is normal. The baby’s CBC is unremarkable
apart from a platelet count of 10 x 109/L. The important immediate step
is:

 a) transfusion of 1 unit of random donor platelets

 b) arrangement of HPA-1a (PlA1) platelet antigen and antibody testing
on the parents
 c) an ultrasound of the baby’s head to exclude intracranial hemorrhage
 d) collection of maternal platelets for urgent viral testing and
transfusion as soon as possible.
Correct:D

 The case is a likely one of neonatal alloimmune

thrombocytopenia due to fetomaternal incompatibility for
the HPA-1a (PlA1) platelet-specific antigen. The definitive
therapy in this situation is compatible PlA1 antigen-negative
platelets harvested from the mother or a known PlA1
antigen donor.
 3/You are asked to see a 6 year old girl who underwent open heart
surgery 7 days previously and who developed moderate
thrombocytopenia (platelet count 50 x 109/L) over a period of 3 days.
She is clinically stable with no evidence of infection. Her platelet
count before surgery was normal. Fibrinogen and D-dimer tests are
normal. The most appropriate immediate next step is:

 a) bone marrow aspirate

 b) request heparin-associated antibody testing
 c) stop all heparin in IV lines, flushes
 d) measure a reticulated platelet count
Correct :c

 This is a possible case of heparin-associated

thrombocytopenia. If the child is receiving heparin, and
especially unfractionated heparin, all heparin must be
immediately discontinued to avoid the chance of
thrombosis. Heparin associated antibody testing should be
ordered as a secondary measure.
 4/A 25 year old pregnant woman with a past history of immune thrombocytopenic purpura
(ITP) treated successfully with splenectomy is due to deliver vaginally in 2 weeks. Her
platelet count is normal. You are asked to advise about the type of delivery and immediate
post-natal care of her baby. Her obstetrician states that there is no obstetric contraindication
to a vaginal delivery. The correct advice is:

 a) obtain a scalp vein blood sample for platelet count determination at the time of delivery
and deliver the baby by C-section if the fetal platelet count is < 50 x 109/L
 b) proceed to an elective C-section
 c) perform in-utero percutaneous umbilical vessel blood sampling before delivery with
measurement of the fetal platelet count and advise delivery by C-section if the platelet count
is < 50 x 109/L
 d) advise a controlled vaginal delivery with measurement of the infant’s platelet count shortly
after delivery
Correct:D

 The current recommendation for management of a pregnant

woman with ITP is conservative. It is important to
recognize that women with a history of ITP treated
successfully with splenectomy may have circulating platelet
auto- antibodies and deliver infants with neonatal ITP and
clinically significant thrombocytopenia.
 5/ A 10 year old boy is referred with a well documented history of recurrent
episodes of severe thrombocytopenia and anemia that have generally followed
transient viral infections, and that have responded promptly to infusions with fresh
frozen plasma. Blood smears during “attacks” were reported to show schistocytes,
polychromasia and markedly reduced numbers of platelets.

 (a) What is the likely diagnosis in this case?

 (a) What is the underlying defect in the disorder?
 (b) List two other conditions in which a similar blood smear may be seen?
 (a) What is the likely diagnosis in this case? -
Congenital (familial) thrombotic thrombocytopenic purpura (TTP)

 (a) What is the underlying defect in the disorder? -

Deficiency of the plasma vWF cleaving metalloprotease, ADAMTS13

 (b) List two other conditions in which a similar blood smear may be seen?
 Hemolytic uremic syndrome (HUS)
 Microangiopathic hemolytic anemia in patients with an artificial cardiac
valve/VSD patch (“Waring-blender” phenomenon
6/ Which of the following causes of neonatal thrombocytopenia
carries the greatest risk of Intracranial hemorrhage:
 a) Congenital rubella.
 b) Down syndrome.
 c) Maternal ITP.
 d) Maternal SLE.
 e) Neonatal alloimmune thrombocytopenia
Correct :e
7/A newborn term infant is covered with petechiae. Platelet count is 5. The
remainder of the CBC is normal. The pregnancy, labor, and delivery were
uncomplicated, and the mother’s platelet count is 370. a. THE MOST LIKELY
DIAGNOSIS IS
 a) Neonatal autoimmune thrombocytopenia.
 b) Neonatal alloimmune thrombocytopenia.
 c) Congenital (TORCH) infection.
 d) False result
 e) Kassabach-Merrit syndrome b.
b.WHICH OF THE FOLLOWING IS THE MOST APPROPRIATE THERAPY
FOR THIS INFANT?
 a) IVIG
 b) Platelet transfusion from a random donor.
 c) Platelet transfusion from the father.
 d) Corticosteriods
 e) Observation; no drug therapy necessary.
. A correct. b
B correct. b
Disorders of Platelets

 Production and life - span

 Morphology and function
 Quantitative disorders
 Thrombocytopenia

 Thrombocytosis

 Qualitative disorders
 Platelet transfusion
Essential Thrombocythemia
 Primary myeloproliferative disorder
 Uncommon in childhood
 Clinical features
 Splenomegaly
 Bleeding
 Thrombosis
 Diagnosis
 Platelet count typically 1,000,000 to 4,000,000 per mm3
 Platelet aggregation abnormal
 No underlying conditions causing “reactive” thrombocytosis
Secondary Thrombocytosis
 Inflammation
 Acute and chronic infection
 Connective tissue disease
 Malignancy
 Kawasaki syndrome
 Iron deficiency
 Marrow recovery
 Sickle cell disease
 Post-splenectomy
 Young infants (especially premature)
Disorders of Platelets

 Production and life - span

 Morphology and function
 Quantitative disorders
 Thrombocytopenia

 Thrombocytosis

 Qualitative disorders
 Platelet transfusion
Glanzmann Thrombasthenia

 Autosomal recessive inheritance ( high rate of consanguinity )

 Severe mucocutaneous bleeding starting in infancy
 Deficiency or abnormality of GPIIb-IIIa ( platelet aIIbb3
integrin )
 Normal platelet count and morphology
 Absent platelet aggregation in response to ADP, epinephrine,
collagen
 Normal ristocetin-induced platelet agglutination
Bernard-Soulier Syndrome
 Autosomal recessive inheritance (consanguinity frequent)
 Deficiency or abnormality of GPIb, GPIb, GPIX
 Prolonged bleeding time
 Normal platelet aggregation in response to ADP, epinephrine,
and collagen
 Abnormal or absent agglutination in response to ristocetin
Bernard-Soulier Syndrome

Platelet

Platelet
Milder Inherited Platelet Defects
 Sites of defects
 Membrane receptor ( collagen, ADP or thromboxane A2 )
 Signal transduction apparatus
 Prostaglandin generation mechanism ( cyclo-oxygenase or thromboxane
synthetase )
 Storage granules ( “storage pool disease” )
 Clinical features
 Mild mucocutaneous bleeding
 Variable and often ill-defined inheritance patterns
 Some disorders feature other abnormalities
 Hermansky-Pudlak ( occulocutaneous albinism )
 Chediak-Higashi syndrome
Milder Inherited Platelet Defects
 Normal platelet count and morphology by light microscopy
 Prolonged bleeding time ( variable )
 PFA-100 closure time typically abnormal with
collagen/epinephrine cartridge but normal with collagen / ADP
 Defective but not absent platelet aggregation in response to
ADP, epinephrine and collagen ( reduced or absent
second wave )
 Abnormal platelet EM and platelet ATP secretion in storage
pool disease ( dense granule deficiency )
Gray Platelet Syndrome
 Autosomal recessive ( dominant in 1 family )
 a-granule storage pool deficiency
 Mild bleeding disorder
 Bleeding time normal-prolonged
 Large, agranular, gray, platelets
 Severe deficiency of a-granule proteins
 Associated with myelofibrosis and splenomegaly
 Treatment: •Platelet transfusion

•Desmopressin

•Splenectomy
Gray Platelet Syndrome

Platelet
Platelet
Kahr et al, Nat Genet. 2011;43(8):738-740
Defects in the Interaction Between
von Willebrand Factor and Platelet
Surface GPIb-IX-V
Loss of GPIb-IX-V
 Bernard-Soulier syndrome

Gain of function ( enhanced interaction between

GPIb-IX-V and VWF )
 Type 2B von Willebrand disease ( VWF mutation )
 Platelet-type von Willebrand disease ( “Pseudo” VWD ) ( GPIb mutation )
 Clinical picture:
 Thrombocytopenia, especially with pregnancy and stress
 Variably reduced VWF level;  in high-molecular weight VWF multimers
 Increased ristocetin-induced platelet aggregation ( using 0.5 mg/ml ristocetin )
1/ You are asked to see a 3 year old boy because of a history of severe, recurrent epistaxis.
The child’s parents are consanguineous. A CBC shows a hemoglobin of 100 g/L, MCV 62 fL,
WBC 7.5 x 109/L and platelet count 90 x 109/L. Large platelets are noted on the peripheral
blood smear. Platelet aggregation testing is reported as showing absence of response to
ristocetin.

 (a) What is the most likely diagnosis in this case?

 (b) What is the molecular basis for the disorder?

 (c) List two other hereditary macrothrombocytopenias caused by mutations in the same
genes:
 (a) What is the most likely diagnosis in this case?
 - Bernard-Soulier syndrome

 (b) What is the molecular basis for the disorder?

 - mutations in the genes for GP1b/IX

 (c) List two other hereditary macrothrombocytopenias caused by

mutations in the same genes:
- pseudo or platelet type von Willebrand disease -
velocardiofacial syndrome
2/You are referred a 5 year old girl with a history of excessive bruising. Her parents
were born in Puerto Rico. On physical examination she is found to have
occulocutaneous albinism, rotatory mystagmous and bruises on her legs and arms.
She is not dysmorphic and her CBC is normal with normal appearing platelets on a
peripheral blood smear.
 (a) What is the likely diagnosis in this case?
 (b) What abnormalities would you expect on platelet aggregation testing?
 (c) What is the platelet defect in this condition?
 (a) What is the likely diagnosis in this case?
 - Hermansky-Pudlak syndrome (HPS)

 (b) What abnormalities would you expect on platelet aggregation testing?

 - Absence of the second wave of aggregation to ADP and epinephrine
and decreased response to collagen

 (c) What is the platelet defect in this condition?

 - Absence of platelet dense granules
3/Abnormalities of glycoprotein (GP1b) are
found in which two of the following conditions?

 A. Bernard-Soulier syndrome
 B. May-Hegglin anomaly
 C. Di George (velocardiofacial) syndrome
 D. Glanzmann thrombasthenia
Correct Answer: A and C
 A. Bernard-Soulier syndrome
 B. May-Hegglin anomaly
 C. Di George (velocardiofacial) syndrome
 D. Glanzmann thrombasthenia
4/A 10 yr old is seen by the pediatrician for fever. He is noted on physical
examination to have some rhinorrhea, otitis media, and mild cervical
lymphadenopathy. His CBC is normal with exception of a platelet count of
987,000/mm3. What testing and treatment is recommended at this time?

 A. Reassurance and follow-up CBC in a few weeks

 B. Bone marrow evaluation
 C. JAK2 kinase mutation testing
 D. Alpha-fetoprotein levels
 E. Anti-platelet drug therapy
Answer: A
 This child most likely has a reactive thrombocytosis and reassurance to the
family can be provided. Bone marrow biopsy and JAK2 mutation testing are
part of the evaluation for essential (or primary) thrombocytosis, caused by an
overproduction of platelets by the bone marrow. This condition is very rare in
a child of this age with another explanation for thrombocytosis.
Thrombocytosis is associated with hepatoblastoma and is usually seen in
children younger than 10 years of age and is speculated to result from increase
thrombopoietin production from the liver. Without findings of hepatomegaly
the likelihood of this diagnosis is low and alpha-fetoprotein levels do not need
to be checked. Reactive thrombocytosis is not associated with thrombosis and
therefore anti-platelet drug therapy is not indicated.
5/A 5 year old female is referred to you because of recurrent epistaxis. The
parents are first cousins. Past medical history is significant for strabismus
surgery at 3 years of age. On physical examination you notice oculocutaneous
albinism. Platelet count and peripheral blood smear are normal. Which of the
following is most likely absent in her platelets?

 A. ADAMTS13
 B. Adenosine diphosphate
 C. Fibrinogen
 D. Platelet factor 4
 E. Thrombin
Answer:B
 Hermansky-Pudlak Syndrome, an autosomal recessive disorder, causes a
bleeding diathesis due to a lack of dense granules in the platelets which is
demonstrated on electron microscopy. The disease is associated with
occulocutaneous albinism, pulmonary fibrosis, strabismus and nystagmus.
Dense granules contain small molecules such as adenosine triphosphate,
adenosine diphosphate, serotonin and calcium. Platelet aggregation studies will
therefore show absent second wave in response to adenosine diphosphate and
epinephrine.

 Alpha granules contain proteins such as fibrinogen, platelet factor 4, von

Willebrand factor, and thrombospondin. Thrombin is a potent platelet agonist
but is not contained in platelet granules. ADAMTS13 is a metalloprotease
responsible for cleaving ultralarge von Willebrand mutlimers and not contained
within the platelet.
. 6/A 2-year-old male was diagnosed with Bernard-Soulier syndrome at an outside
hospital and is referred to you. Remembering that this disorder is caused by a lack
of the glycoprotein complex Ib-IX, you recognize that this patient will have
recurrent episodes of bleeding secondary to
 o A. A lack of platelet activation
 o B. A lack of platelet release of ADP
 o C. A lack of clot stabilization
 o D. A lack of platelet adhesion
 o E. A lack of platelet aggregation
Correct:D. A lack of platelet adhesion
 Patients with Bernard-Soulier syndrome (BSS) lack the glycoprotein complex
Ib-IX that is essential for adhesion of platelets to the vascular endothelium
mediated by von Willebrand factor, resulting in a significant bleeding diathesis.
Platelet activation follows adhesion and is mediated by the change in platelet
shape that exposes the GPIIb/IIIa receptor, which is absent in Glanzmann
thrombasthenia. Next platelet aggregation occurs through the cross-linking of
activated platelets by fibrinogen or von Willeband factor. Propagation of the
clot occurs with the addition of procoagulants that assemble on the
aminophosphlipid rich surface of the activated platelets. Lastly, the clot is
stabilized by Factor XIIIa. Release of ADP is regulated by platelet dense
granules not by surface glycoproteins.
Acquired States of Platelet Dysfunction
During Childhood
 Liver disease
 Renal failure
 Cardiac disease
 Cyanotic congenital CHD
 Post-cardiopulmonary bypass
 Malignancy
 Drugs
Drugs and Platelet Dysfunction in
Pediatric Patients
 Aspirin
( irreversible inhibition of cyclo-
oxygenase )
 Other non-steroidal anti-inflammatory agents
 Valproic acid
 Semi-synthetic penicillin derivatives
 Psychotropic drugs
Treatment of
Qualitative Platelet Defects
 Remove or discontinue offending agent ( e.g. drug )
 Treat underlying disease ( e.g. dialysis )
 Local measures ( pressure, Gelfoam, desiccated collagen, etc )
 Tranexamic acid
 Desmopressin ( DDAVP )
 RBC transfusion if patient anemic
 Recombinant Factor VIIa
 Platelet transfusion
 Cryoprecipitate ( uremia )

Poon MC J Thromb Haemost 2004 ; 2 : 1096-1103

Disorders of Platelets

 Production and life - span

 Morphology and function
 Quantitative disorders
 Thrombocytopenia

 Thrombocytosis

 Qualitative disorders
 Platelet transfusion
Guidelines for
Platelet Transfusion in Children
 Platelet count 5-10 x 109 /L with failure of platelet production
 Platelet count <30 x 109 /L in neonate with failure of platelet
production
 Platelet count <50 x 109 /L in stable premature infant:
 With active bleeding
 Invasive procedure with failure of platelet production
 Platelet count <100 x 109 /L in sick premature infant:
 With active bleeding
 Invasive procedure in patient with DIC

Roseff SD Transfusion 2002 ; 42 : 1398-1413

Guidelines for Platelet Transfusion in Patients
with a Normal Platelet Count

 Active bleeding in association with a qualitative platelet

defect
 Unexplained, excessive bleeding in a patient undergoing
cardiopulmonary bypass
 Patient undergoing ECMO:
 with platelet count of < 100 x 109 /L

with higher platelet counts and bleeding

Roseff SD Transfusion 2002 ; 42 : 1398-1413

Platelet Refractoriness

 Suspect condition in multitransfused cases ( e.g. acute leukemia, post-

transplant ) who fail to respond to an adequate transfusion of pooled,
random donor platelets and who have no identifiable cause for 
platelet consumption e.g. fever, sepsis, DIC, splenomegaly
 Pathologic alloantibodies are generally directed against HLA antigens
 Management involves transfusion of HLA-matched single donor
( apheresis ) platelets
 Estimated Risk of Transfusion
Risk Factor Estimated Risk per Unit
Transfused*
Viral Infection
Hepatitis B 1 / 150,000
Hepatitis C 1 / 2 million
HIV 1 / 8 million

Bacterial Contamination
Red Cells 1 / 500,000
Platelets 1 / 1,000; sepsis 1 / 10,000
Immune
Acute hemolytic reaction 1 / 40,000
Delayed hemolytic reaction 1 / 7,000
Transfusion-related 1 / 5,000
acute lung injury ( TRALI )
Transfusion associated very rare
graft-versus- host-disease
* Adapted from Goodnough LT et al Critical Care Medicine 2003: 31; S678–S686
Kleinman S Transfusion Medicine Reviews 2003 ; 17 : 120-162
O’Brien SF et al Transfusion 2007 ; 47 : 316-325
patient requests information about risk of
HIV infection following a random donor
platelet transfusion. What is the correct
best estimate of the risk in North America?

1.1 in 500,000
2.1 in 2-4 million
3.1 in 10 million
2- The correct best estimate is 1 in 2-4
million.