ANEMIA OF ABNORMAL IRON METABOLISM

1. Iron Deficiency Anemia (IDA)

2. Anemia of Chronic Disoders (ACD)
3. SideroblasticAnemia
- Hereditary
- Idiopathic
- Secondary
4. Adrenal Abnormalities
Hypogonadims
Anemia of Pregnancy
Differentiation among anemias of Abnormal iron metabolism including:
 Microcytic Anemias
 Hypochromic Anemias
Reference ranges:
 Bone Marrow Iron – Normal
 Bone Marrow Sideroblasts – 25%-30%
 Serum Iron- 50 – 150 ug/dL(varies with age and sex)
 Sample:
- Drawn in the Morning (25% in the evening)
- 12 hour fasting period
- 12- 24 hours off from iron containing medications
 Reagent:
- 0. 05H HCl – removes iron from transferring
- Acid ferrozine – reduces Fe3+ to Fe2+
 Serum Ferritin – 30-250 ug/L (varies with age and sex)
 Reflects the body’s tissue Iron stores
 Indicator of Iron storage status
 1st Laboratory test to become Abnormal when Iron stores begin to increase
 1ug/L ferritin = 8mg tissue iron storage
 Decreased only in Iron deficiency Anemia
 Total Iron binding Capacity (TIBC) – 250- 450 ug/dL
 Transferrin concentration is measured indirectly by measuring the ability of serum transferrin to bind iron
 In contrast to Serum Ferritin: TIBC doesn’t exhibit diurnal variations
 Transferrin Saturation (Percentage) - 20%-50%
 %transferrin saturation = Serum Iron/TIBC x 100
 Normal Value: 20% to 55%
 Free Erythrocyte Protoporphyrin (FEP) - <50 ug/dLrbcs
 Normal occurrence: Red cells produce more protoporphyrin than is neeed,
 Iron is deficient; Protoporphyrin build up in RBC several times than normal
 Iron deficiency anemia and Anemia of Chronic Disease: Increased
 Thalassemia; Normal

A. Iron deficiency anemia (IDA)

 Bone Marrow Iron – Absent
 Bone Marrow Sideroblasts – 0
 Serum Iron- Decreased
 Serum Ferritin – Decreased
 Total Iron binding Capacity (TIBC) –Increased
 Transferrin Saturation (Percentage) - Decrease
 Free Erythrocyte Protoporphyrin (FEP) – Increased

B. Anemia of Chronic disorders (ACD)

 Bone Marrow Iron – Normal to Increased
 Bone Marrow Sideroblasts – 0- Few
 Serum Iron- Decreased
 Serum Ferritin – Increased
 Total Iron binding Capacity (TIBC) – Decreased
 Transferrin Saturation (Percentage) - Decreased to Normal
 Free Erythrocyte Protoporphyrin (FEP) – Increased

C. SideroblasticAnemia
 Bone Marrow Iron – Increased
 Bone Marrow Sideroblasts – Increased (Ringed cellls)
 Serum Iron- Increased
 Serum Ferritin – Inrcreased
 Total Iron binding Capacity (TIBC) –Decreased to Normal
 Transferrin Saturation (Percentage) - Increased
 Free Erythrocyte Protoporphyrin (FEP) – Mixed
 D. Lead Poisoning
 Bone Marrow Iron – Normal
 Bone Marrow Sideroblasts – Increased (may have ring cells)
 Serum Iron- Normal to Increased (adults) / Normal to decreased (child)
 Serum Ferritin – Normal
 Total Iron binding Capacity (TIBC) – Normal
 Transferrin Saturation (Percentage) - Increased
 Free Erythrocyte Protoporphyrin (FEP) – Markedly Increased

E. Thalassemias
 Bone Marrow Iron – Normal to Increased
 Bone Marrow Sideroblasts – Normal (may be ringed)
 Serum Iron- Normal to Increased
 Serum Ferritin – Normal to Increased
 Total Iron binding Capacity (TIBC) – Normal
 Transferrin Saturation (Percentage) - Normal to Increased
 Free Erythrocyte Protoporphyrin (FEP) – Normal

1. IRON DEFICIENCY ANEMIA (IDA)

- Most common anemia worldwide
- Develops:
 Nutritional deficiency
 Acute blood loss
 Peptic Ulcerations
MECHANISM OF IRON DEFICIENCY:
A. Increased physiological disorders
o Rapid growth: infants and children
o Pregnancy
o Lactation
B. Inadequate Intake
o Iron-deficient diet
o Inadequate absorption
- Celiac post- gastrict surgeries
 Achlorhydria – reduced gastric acidity that causes inadequate absorption and reduced Iron stores.
- Ingested Fe3+ must be reduced to Fe2+ to be absorbed
- This reduction process is dependent on proper gastric acidity
 Decreased Absorption surface
C. Chronic Blood Loss
o Menstrual flow
- Prevalent in women ages 18 to 44
- Largest single contributor to this kind of anemia
o Gastrointestinal bleeding – occur Men or Post- menopausal women
o Esophangealvarices - occur Men or Post- menopausal women
o Peptic Ulcer diseases - occur Men or Post- menopausal women
o Neoplastic Diseases - occur Men or Post- menopausal women
o Haemorrhoids - occur Men or Post- menopausal women
o Regular blood donation
o Chronic hemolysis (Chronic intravascular haemolytic Disease) causes excess loss in the urine (Hemoglobinuria); paroxysmal
nocturnal hemoglobinurua (CD 59)
Normally : High demand of Iron in children, Iron reserves are usually low and would be considered depleted by Adult standards.
- INFANTS are susceptible to IDA: High physiologic demand (+) Low Iron reserves
- PREGNANT WOMEN requires dietary iron of 30 – 75mg/day, to provide the needs of the fetus and her own expanded blood
volume.
On the Last half of pregnancy: Iron supplements are required
Stages of Iron Deficiency Anemia:
1. Iron replete
2. Iron deplete
3. Iron deficient erythropoiesis
4. Iron deficiency anemia

CLINICAL PRESENTATIONS:
1. Fatigue, Breathlessness, Dizziness - Gradual onset of symptoms permits compensatory mechanism to minimize the symptoms until the anemia
becomes severe.
2. PICA - persistent, compulsive eating to eat a single food or non-food items such as starch, or something crunchy
3. Murmur
4. Resess Led Syndrome
5. Plummer Vinson
6. Chelitis
PHYSICAL PRESENTATIONS:
1. Epithelial changes
 Angular stomatitis – cracks in the corner of the mouth
 Glossitis – soreness of the tongue
 Gastritis – may progress to gastric atrophy and the result of achlorhydria
 Koilonychia – flattening and spooning of the nails
2. Splenomegaly (Rare)
3. Neurological changes (None)

HEMATOLOGY:
1. Etiologic of the anemia: maybe related to Chronic Hemorrhage
2. Hemoglobin– markedly decreased in late stage – 8 g/dL or lower
3. RBC Indices:
- MCV – 75fL (59- 80fL)
- MCH – 21pg (15-26 pg)
- MCHC – 28g/dL (22-32g/dL)
4. Anemia: Microcytic - Hypochromic
5. RDW (Red Cell Distribution Width) – Increased
- Elongated Cells
- Tailed epithelial cells
- Microcytes
6. Platelets – often Increased/ slighty increased (blood is thinned)
7. White blood Cells– Normal
- Except Eosinophils (increased: Eosinophilia)
8. Reticulocytes - Normal
9. Reticulocyte Production Index – falls below 2.0
- Ineffective erythropoiesis by the bone marrow

BONE MARROW:
1. Iron stores – severely decreased
2. Marrow sideroblasts – severely decreased

CHEMISTRY:
When Iron deficiency and inflammation (concurrent) = Ferritin may be elevated (masking the Iron deficiency)*homeostatis

TREATMENT:
1. Iron supplements
 Ferrous suflate (Oral administration)
Black stool and constipation (Guaiac test)
2. Controlling the bleeding

↓Hemoglobin = ↓Ferritin, ↑ ↑↑ Protoporphyrin

Macrocytosis = ↑ central pallor
Abdominal Perinial resection – Ferritin (IL6)

2. ANEMIA OF CHRONIC DISORDERS (ACD)

- Second most common type of Anemia
- Most common type of anemia in hospitalized patients
- Most common cause of anemia in:
 Rheumatoid Arthritis
 Tuberculosis
- Associated with
A. Infectious
1. Tuberculosis
2. Pulmonary infections , Pneumonia
3. Pelvic Inflammatory diseases
4. Chronic Fundal Diseases
5. Sub-acute bacterial endocarditis
6. Osteomyelitis
7. Meningitis
B. Inflammatory – Autoimmune – Non- infectious
1. Rheumatoid Arthritis
2. Thermal injury
3. Systemic Lupus Erythematosus
4. Myocardial Infarction
C. Malignant Diseases ( >1 or 2 months duration)
1. Carcinoma
2. Hodgkin’s disease
3. Lymphosarcoma
4. Leukemia
PATHOPHYSIOLOGY:
- ACD co-exist with IDA
1. MICROORGANISM
 The Microbacterium tuberculosis bacilli harbors the Iron in the blood being transfused.
Result: Increased storagde of macrophages, microorganism will help Iron to make the disease severe!
2. ERYTHROPOIETIN
 whenever there’s accumulation of Macrophages in Kidney – IL-6 stops the release of EPO = decrease EPO
- RBC Survival (Shortened) – non-specific stimulation of macrophages = Increase activity
- Bone Marrow response (Impaired) to the RBC survival
 EPO (Decreased)
- Iron release from storage in Macrophages – decreased
= Release of activated macrophages of IL-1 (Leukocyte endogenous factor
 Increased synthesis of Apoferritin (Fe3+) within Macrophages
 Macrophages effectively traps the Iron
Results;
(in the presence of Adequate to increased storage Iron)
- Serum Iron – decreased
- Marrow sideroblasts – decreased

CLINICAL PRESENTATION/ PHYSICAL FINDINGS:

- Typical of the Primary condition causing anemia.

HEMATOLOGY:
1. Anemia: Mild to moderate
2. Anemia: Normochromic – Normochromic/ Microcytic - Hypochromic
3. Hemoglobin:>1-2 g/dL patient’s baseline
4. Reticulocyte count: Normal
5. Reticulocyte Production Index:>2.0 (reflects Hypoploriferative nature of Anemia)

BONE MARROW:
-Hepcidin: regulator of iron absorption, decrease ferritin
-Erythropoientin : Increased
1. Siderblasts: Few
2. Stored Iron:Increased andIdentifiable in Macrophages (Prussian Blue stain)
3. Serum ferritin: Increased
4. No expected Erythroid hyperplasia (compensatory mechanism of anemia)

TREATMENT:
- IRON THERAPY IS AVOIDED, the patient has adequate tissue iron stores and exogenous iron could cause Iron overload.

3. SIDEROBLASTIC ANEMIA
- “sidero” – iron constellation
- “Blast” – growing blast anemia(ringed sideroblast)
- defect in Protopophyrin synthesis
- Abnormal iron kinetics
 Results: Excess accumulation of iron, which is deposited in the mitochondria of normoblasts

Sideroblasts– iron- containing normoblasts found in normal bone marrow.

TYPES OF SIDEROBLASTS:
1. Type 1 - 4 ferritin aggregates in approximately 50% of normoblasts
2. Type 2 – atleast 6 ferritin aggregates in approximately 50% of normoblasts
3. Type 3 AKA Pathologic Ringed Sideroblast – larger granules situated in a ring or collar around the nucleus of the
normoblasts(reffered to as Iron laden Mitochiondria)
NOTE: For a diagnosis of SideroblasticAnemia, at least 15% of the normoblasts must be TYPE 3 ringed sideroblasts

A. HEREDITARY TYPE OF SIDEROBLASTIC ANEMIA

- X-linked (Males are commonly affected)
- Autosomal Recessive Pattern of Inheritance

PATHOPHYSIOLOGY:
- Delta- Aminolevulinic Acid Synthase Activity (DECREASED) – 1st enzyme in Heme synthesis
- Coenzyme: Pyridoxal -5- Phosphate (P-5-P)
 Reduced affinity for the coenzymne
 Unusual sensitivity to mitochondrial proteases
- Treatment: Pyridoxine Therapy
CLINICAL PRESENTATION/ PHYSICAL FINDINGS:
1. Early adulthood – symptoms appears
2. Iron overloading
 Balance Diagnosis:
 Hemogblobin: Decreased
 Hematocrit: Decreased
  TIBC: Decreased
 Ferritin: Increased
 Iron: Increased
3. Splenomegaly: Mild to Moderate
4. Hepatomegaly
5. Diabetes (accumulation of Iron in pancreatic cells)
6. Cardiac arrhythmias (accumulation of iron in myocardial cells)

HEMATOLOGY:
1. Anemia is sever e
2. Hemoglobin – 6. 0g/dL
3. RBC’s population – Dimorphic
- Microcytic
- Hypochromic
- Normocytic
- Normochromic
4. RBC’s morphology
- Anisocytosis
- Poikolcytosis (target cells)
BONE MARROW:
1. Eryrthroid hyperplasia with excessive Iron stored in Macrophages
2. Upto 40%Normoblasts are pathologic rigedsideroblasts
o Polychromatophilicnormoblasts
o Orthochromicnormoblasts
3. Megaloblastic changes are sometimes changes
o Pyridoxine + Folic Acid (Treatment)
CHEMISTRY:
1. Ferritin levels: high (Large amount of stored iron)
2. Serum Iron : High
3. Transferrin saturation: High
- Iron is not utilized in Hemoglobin Synthesis and so it piless up in the mitochondira.
4. Total Iron binding Capacity: Normal
5. Free Erythrocyte Protoporphyrin: Low or Normal

COMPLICATIONS:
1. Cardiac Arrhythmias
2. Liver diseases
3. Diabetes

B. IDIOPATHIC TYPPE OF SIDEROBLASTIC ANEMIA

Also referred as:
1. Refractory anemia with ringed sideroblasts (RARS)
2. Idiopathic axquiredsideroblasticanemia (IASA)

PATHOPHYSIOLOGY:
1. Aminolevulinic Acid synthase activity – decreased
2. Heme Synthase (Ferrochelatase) – decreased
3. Leukemic Transformations common in MDS (25% of individuals with IASA)

CLINICAL PRESENTATION/PHYSICAL FINDINGS:

1. Insidious onset
2. Liver or spleen mild enlargement (40% of patients)

HEMATOLOGY:
1. Anemia is moderate
2. Hemoglobin: 7-10g/dL
3. Anisocytes: Normochromic – Slightly macrocytic (some are hypochromic)
4. Poikilocytes
o Fragment Cells
o Target Cells
o Heavily stippled
o Hypochromic Cell
BONE MARROW:
1. Erythroid hyperplasia (M:E = 1:1)
2. May resemble Erythroleukemic marrow
o PAS (highly +)
o RARS (negative)
3. Ringed sideroblasts of the Basophilic normoblast stage
- 95% of thnormoblasts are Ringed sideroblasts
CHEMISTRY:
1. Transferrin saturation: Very high (>90%)
 2. Ferritin: Elevated
3. Free Erythrocyte Protoporphyrin: Increased
4. Serum Iron: Increased
5. Good prognosis:
 >30% Ringed sideroblasts
 Normal granulopoieses
 Normal megakaryopoiesis
6. Poor prognosis: it may possible progress to leukemia
 < 30% Ringed sideroblasts
 Dysplastic granulopoieses
 Dysplastic megakaryopoiesis

C. SECONDARY SIDEROBLASTIC ANEMIA

- Exposure to certain drugs
 Chloramphenicol - inhibits mitochondrial protein synthesis decreasing ALA- S and heme synthesis.
 Ethanol – reduces the activity of several enzymes in the heme synthesis pathway.
o PBG Synthase
o Uroporphyrinogen decarboxylase
o Coproporphobilinogen oxidase
o Heme synthase
 Tuberculoidal drugs
 Antineoplastic drugs
 Toxins (Lead)
- ALA- S or Heme synthesis (or both) – Interference with the activity
- Inhibition of requiring P-5- P’ by interfering with activation of Vitamin B6
 Isoniazid
 Cycloserine
 Pyrazinamide

CLINICAL PRESENTATION/ PHYSICAL FINDINGS:

- Related to the primary disease
HEMATOLOGY:
1. Anemia is moderate
2. Hemoglobin: 6-10g/dL in alcoholics
3. RBC Production: Dimorphic
4. Normochromic – Normocytic
5. Microcytic- Hyperchromic

BONE MARROW:
- 65% Ringed sideroblasts
- Alcoholics – Bone marrow shows
 Megaloblastic changes
 Vacuolization of the RBC precursors
CHEMISTRY:
1. Alcoholic patients: Nutritional defiecient
2. Iron in bone marrow : increased
3. Transferrin saturation: increased (up to 65%)
TREATMENT:
- Removal of the offending agent
 Pyridoxine + Folic Acid – aid in the reversal condition.

ADRENAL ABNORMALITIES
1. Hypoadrenalism – Addison’s Diseases – decrease in Adrenal production of Cortisol
2. Hyperadrenalism - Cushing’s Syndrome

HYPOGONADISM
- Reduction in testosterone secretion = RBC levels decreases
- Androgens – have the ability to Increased RBC Production by increasing secretion of EPO.
- Treatment: Therapeutic administration of Androgens

ANEMIA OF PREGNANCY – moderate in severity

- Hemoglobin: <10g/dL
- Most often result in deficiency; significant increase of Iron demand, less commonly Folate deficiency
- Expansion of RED CELL MASS and PLASMA VOLUME (may cause dilution effects)

PORPHYRIA
- Vampire disease, Greek word “porphyra” means purple.
- The purple-red pigment is responsible for wine-red color of porphyric urine.
- A primary abnormality of Porphyrin Biosynthesis = Excessive accumulation and Excretion of Porphyrins
- Porphobilinogen is excreted in urine (small amounts)
 -
Acute intermittent Hepatic Porphyria (elevated amounts)
-
Ehrlich’s aldehyde reagent – Detection of urine
-
Affected many members of Royal houses: Stuart, Queen of Scots, others are infected with variegate porphyria.
Classifications:
1. Clinical presentation (Acute vs. Chronic)
2. Source of Enzyme deficiency
3. Site of enzyme deficiency in the Heme Biosynthetic Pathway
TYPES OF PORPHYRIA:
A. Inherited
1. CONGENITAL ERYTHROPOIETIC PORPHYRIA (CEP)
- Also referred as Gunther’s Disease
- Rarest type of Porphyria
- Causes Cutaneous photosensitivity
- Decrease production of Uroporphyrinogen III cosynthetase = Overproduction of Uroporphyrinogen I
- Urine: Increase excretion of Uroporphyrinogen I
- Color: Pink to deep burgundy
- Exerbating Factors: Sunlight
- Clinical features: Photosensitivity, red urine and teeth, Hemolysis

2. PORPHYRIAN CUTANEA TARDA (PCT)

- Can also be acquire porphyria
- Sensitive to light and Minor trauma = skin lesions and scarring
- Autosomal Dominant trait
- Uroporphyrinogendecarxboxylase: Decreased production
- Urine: Increased amount of Uroporphyrin I
- Color: Reddish or brownish
- Exerbating Factors: Alcohol, Hepatic Injury, Iron overload
- Clinical features: Photosensitivity

3. ERYHTROPOIETIC PROTOPORPHYRIA (EP)

- Autosomal dominant Trait
- Caused: Decreased activity of Hemeferrochelatase (hemesynthetase) = FECES: Increased concentration of protoporphyrin IX
- RBC: Markedly increased in Protoporphyrin IX
- Resembles Fluorescent cytoplasm
- B CarotineBeadlets
- Choletyramine

4. ACUTE INTERMITTENT HEPATIC PORPHYRIA (AIP)

- Most common of Inherited porphyria
- Autosomal dominant trait
- Cause: Deficient in Uropophyrinogen I synthetase
- May have Neurological manifestations
- Clinical features: Abdominal pain, Psychiatric symptoms
- Sodium benzoate, Treated with chocolate intake
- Exerbating Factors: Steroid hormones

5. HEREDIATRY COPROPORPHYRIA (HCP)

- Resembles Acute Intermittent Hepatic Porphyria
- Cause: Decrease in Coproporphyrinogen oxidase = Increased amounts of coproporphyrinogen III (Urine and Feces) and
accumulation in Liver.
- Treatment:Hematin
- Exerbating Factors: Stress
- Clinical features:Photosensitivity

6. VARIEGATE PORPHYRIA (VP)

- Autosomal dominant trait
- Clinical symptoms the same with PCT (Porphyria cutaneatarda)
- Cause: Deficiency in Protoporphyrinogen oxidase
- Stimulus:
 Sulfonamides
 Barbiturates
- Treatment: Avoid exposure to sunlight
- Exerbating Factors: Stress
- Clinical features: Photosensitivity

B. Acquired
- Cause: Exposure to HALOGENTAED AROMATIC HYDROCARBONS
- contain one or more atoms of a halogen (chloride, fluoride, bromide, iodide) and a benzene ring
 -

MACROCYTIC ANEMIAS
The macrocytic anemias are morphological classifications of Anemia that have an
- MCV >100fL
- Megaloblastic (>130fL or >115fL) * dramatically increased
- Non- Megaloblastic (100-110 fL)
A. MEGALOBLASTIC ANEMIA:
1. Folate deficiency
2. Cobalamin deficiency
3. Antifolate drugs
4. Cancer chemotagraphy
B. NON- MEGALOBLASTIC ANEMIA:
Smear: Hypersegmented neutrophils (5-7 lobes)
1. Liver Diseases
2. Myelodysplasia
3. Reticulocytisis
4. Hypothyroidism
5. Alcoholism
6. Chronic Obstructive Pulmonary Disease (COPD)

 Vitamin B12 Deficiency

 Folayte deficieny
 Abnormalities of B12 or Folate Mechanism
 Transcobalamin deficiency
 Nitrous oxide
 Antifolate drugs
 Other defects of DNA Synthesis
1. CONGENITAL ENZYME DEFICIENCIES
 Orotic Aciduria- one or other enzyme concerned in purine or pyrimidine synthesis that can cause
Megaloblastic Anemia identical in appearance to that caused by a deficiency of B12 or folate.
2. ACQUIRED ENZYME DEFICIENCIES
 Alcohol
 Therapy with hydrocyurea - antineoplastic (anti-cancer) 
 Cytosis arabinoside /Cytarabine/ Cytosine arabinoside (ara-C)- is a chemotherapy medication used to treat
(AML), (ALL), (CML), and non-Hodgkin's lymphoma. It is given by injection into a vein, under the skin, or
into the cerebrospinal fluid.
NUTRITIONAL ASPECTS
VITAMIN B12 & FOLATE – requirement for DNA synthesis
VITAMIN B12 FOLATE
 Normal daily dietary Intake 7-30 ug 200 – 250 ug
Main foods Animal produce only Liver, green veggies, and yeast
(Meat, Milk, Eggs, Cheese)
Cooking Small effect Easily destroyed
Minimal adult daily requirement 1-2 ug 100-150 ug
Body stores 2-3 mg (sufficient for 2-4 years) 10 -12 mg (sufficient for 4 months)
Absorption Site: Ileum Site: Duodenum and jejenum
Mechanism: Intrinsic Factor Mechanism: Conversion of methyltetrahyrdofolate
Limit: 2-3 ug/day Limit: 50%-80% of dietary content
Enterohepatic circulation 5-10 ug/day 90 ug/day
Transport in Plasma Most bound to Haptocorrin; Weakly bound to Albumin
Transcobalamin essential for cell uptake
Major intracellular physiological forms Methyl- and deoxyadenosylcobalamin Reduced polyglutamate derivatives
Usual therapeutic form Hydroxycobalamin Folic(Pteroylglutamic) acid
Laboratory Diagnosis Hemoglobin: Decreased Hemoglobin: Decreased
WBC: Decreased Hematocrit: Decreased
pH: Decreased Serum Folate: Decreased
Serum B12: Decreased WBC: Decrease (no maturation of cells)
MCV: Increased RBC folate: Decrease
Serum homocystein: Increased Homocystenemia : Increased
Serum Methyl malonyl: Increase
PBS: Macrocytes, Hypersegmented
Neutrophils
BM: Megaloblast – no maturation, impaired
DNA Synthesis
Homocysteinemia: Increased

 Cells become bigger – Nucleus signals replication is not functional but RNA and Protein synthesis is still functioning.
 Ineffective erythropoiesis – phagocytize the macro cells, other cells may be included.
 Methionine – metabolism of DNA, RNA, Lipids and proteins.

The amylases will breakdown the Vitamin B12 and salivary glands produces R- protein (protects the Vitamin B12 in gastric acid)
In stomach: destroys the B12 through an acidic environment. The Hydrochloric acid converts Pepsinogen to Pepsin, which breakdown the binding of
Apoprotein and Cobalamin. It goes to Duodenum to secrete the R- protein and Intrinsic factors (which absorbs the Vitamin B12) in pancreas; R- protein
secretes protease which will help for the binding of R Protein, Cobalamin and Intrinsic Factors. Absorption happens to the ileum, Terminal ileum (breakdown
of IF and cobalamin) The Cobalamine goes to Peripheral blood.

TRANSPORT: The Transcobalamins

 HAPTOCCORIN
 Most Vitamin B12 in plasma is bound to this transport protein.
 Also referred as Transcobalamin I
 This Is a glycoprotein largely synthesized by;
 Granulocytes
 Macrophages
 Vitamin B12 level in serum bound to haptocorrin, does’nt transfer to marrow, it appears to be functionally
“dead”
 TRANSCOBALAMIN I
 Vitamin B12 is absorbed into the portal blood where it become attracted to this plasma- binding protein
 (TC) also called Transcobalamin II – which delivers Vitamin B12 to the bone marrow and other tissues.
 Deficiencies causes: Megaloblastic anemia
 Failure of Vitamin B12 to enter marrow and other cells from plasma but the
serum Vitamin B12 is normal.
ABSORPTION: 2 Mechanisms for Cobalamin
1. Passive Absorption -
- occurring equally on
 Buccal
 Duodenal
 Ileal mucosa
- It is rapid but extremely inefficient
- <1% of an oral dose is absorbed through this process.
2. Transcobalamin II – major cobalamin transport protein in plasma.
- This is synthesized by the Liver and other Tissues:
 Macrophages
 Ileum
 Endothelium
MECHANISM:
1. Dietary cobalamin is released from protein complexes by enzymes in the stomach, duodenum and jejenum; it combines rapidly with a salivary
glycoprotein that belongs to the family of cobalamin-binding proteins known as Haptocorrin (HCs)
2. In the intestine:
The Haptocorrin is digested by pancreatic trypsin and the cobalamin transferred to Intrinsic Factor.
Intrinsic Factor is a glycoprotein, synthesized and secreted by gastric-parietal cells of the mucosa in the fundus region and the body of the
stomach, and its secretion parallels that of Hydrochloric Acid.
 3. The Intrinsic Factor – Cobalamin Complex passes to the ileum, where Intrinsic Factor attaches to a specific receptor Cubilin on the microvillus
membrane of the enterocytes.
Cubilin is also present in yolk sac and renal proximal tubular epithelium.
Cubulin appears to traffic by means of Amnionless (AMN), an endocytic receptor protein that directs sublocalization and endocytosis of cubiln
with its ligand Intrinsic Factor – Cobalamin Complex.
4. The Intrinsic Factor – Cobalamin Complex enters the ileal cell where Intrinsic Factor is destroyed. After a delay of 6 hours, the cobalamin appears
in portal blood attached to transcobalamin (TC) II.
Between 0.5 and 5.0 ug of cobalamin enters the bile.

There are 3 regions of the stomach:

- 1-2 Fundus & Body – contains acid secreting gastic parietal cells and pepsinogen secreting zymogen cells
- 3 - Antrum – contains gastin-producing cells.

VITAMIN B12 and FOLIC ACID: THEIR ROLE IN DNA SYNTHESIS

Few key reactions are needed to understand Megaloblastic anemia:
1. DNA synthesis is dependent on a key structure, deoxythymidine triphosphate (dTTP)
 This structure cannot be formed when there is a defect produce deoxythymidine monophosphate (dTMP)
from deoxyuridine monophosphate, which is catalysed by the enzyme thymidylate synthetase.
2. 5, 10- Methylene-tetrahydrofolate (methylene- FH4) is required to produce dTMP.
 Cobalamin is involved in the regeneration of (methylene- FH4)
 Cobalamin deficiency = Folate deficiency
 Cobalamin deficiency – THF Starvation, or the Methylfolate Trap - MTHF accumulates in plasma while
intracellular folate concentrations fall due to failure of formation of THF, the substrate on which folate
polyglutamates are built.
3. Folate deficiency is thought to cause Megaloblastic anemia = inhibition of Thymidylate synthesis, a rate-limiting step in DNA
Synthesis in which deoxythymidine monophosphate (dTMP) is synthesized
 This reaction needs 5, 10- Methylene- THF polyglutamate as coenzyme
4. (dTMP) converted ¿ (dTTP) - Precursor of DNA Synthesis
Absence of adequate (dTTP), deoxyuridine triphosphate (dUTP) is inserted into DNA
 Results: Abnormal DNA Synthesis including DNA strand breaks.
CAUSES OF COBALAMIN DEFICIENCY
1. Malabsorption – most common
 Pernicious Anemia – loss of Intrinsic Factor production and gastric acid
 Gastrectomy – loss of Intrinsic Factor production and gastric acid
 Crohn’s Disease or Inflammatory- Bowel Disease (IBD) – loss of absorption in terminal ileum
 Resection of Terminal Ileum – loss of absorption by specific IF-B12 receptors
 Pancreatic Insufficiency – inability of absorption R-binders off B12 * trypsin
 Blind loop syndrome – bacterial overgrowth; bacteria compete for B12
 Congenital deficiency of IF - rare
 Fish tapeworm (D. latum)- competition for B12
 Zollinger- Ellison syndrome – impaired B12 absorption but no megaloblastic anemia; hypersecretion of
gastric juice which interferes the binding of B12 to IF
 Imerslund’s syndrome – inherited as autosomal recessive trait; Vitamin B12 is bound to IF but there is
deficiency of the receptor site in the terminal ileum.
2. Dietary Deficiency
 Strict vegans
 Infants breast-fed by vegetarian mothers
3. Congenital deficiency of Transcobalamin (Rare)
FOLATE (PTEROGLUTAMIC ACID)
- A yellow, crystalline, and water- soluble substance.
- Humans are unable to synthesize and required preformed folate as a vitamin.
- It is abundant in vegetables, fruit, cereals, and dairy products.
- It is heat- labile, and much is destroyed by cooking.
- Primary dietary source: Fresh uncooked fruits and vegetables.
CAUSES:
1. Inadequate Diet – most common
 Alcoholism – The formation of monoglutamate goes to the Peripheral blood which inhibits the alcohol.
 Lack of fresh fruits and vegetables
2. Malabsorption – less common
 Gluten- sensitive enteropathy (cdelical sprue)
 Tropical sprue
 Extensive small bowel resection
 Inflammatory Bowel Disease (regional enteritis)
3. Rare causes
  Hemodialysis
 Antieleptic drugs
 Anti-folate drugs
 Oral contraceptions (uncommon) – entry of monoglutamate to THT
 Exposure to nitrous oxide
 Increased requirements
o Chronic Hemolytic Anemia
o Psoriasis
o Pregnancy
EFFECTS OF VITAMIN B12 OR FOLATE DEFFICIENCY
1. Megaloblastic Anemia 7. Megaloblastic madness
2. Macrocytosis of epithelial cell surfaces 8. Cardiovascular disease
3. Sterility 9. Neural tube defects (NTD) in fetus
4. Reversible melanin skin pigmentation (Rare) - Folate or B12 deficiency in the mother predisposes
5. Osteoblast activity: decreased (Anencephaly, Spina bifida, or encephalocoele) in fetus
6. Neuropathy (for Vitamin B12 only)
PERNICIOUS ANEMIA
- Classic or Addisonian Pernicious Anemia – due to autoimmune chronic gastritis with destruction of the gastic parietal cells.
- Pernicious is not synonymous with Megaloblastic Anemia; it is a subset of megaloblastic Anemia.
- Achloryhdria – is the absence of hydrochloric acid (HCl) in the stomach,
 Important clinical representation in Pernicious anemia.
 An absence of free HCl in gastric fluid is a universal form of megaloblastic anemia.
 It results o from atrophy of the parietal cells of the stomach.
- LDH: significantly increased = INTRAMEDULLARY DESTRUCTION: increased
- Patients may have serum antibodies against gastric parietal cells;
 Anti-parietal cell antibodies
 Anti- IF antibodies
- 7 Clinical “P”s
1. Pancytopenia
2. Peripheral nephropathy
3. Posterior spinal column neuropathy
4. Pyrimidal tract signs
5. Papillary tongue atropathy
6. pH elevation (gastic fluid)
7. Psychosis (Megaloblastic psychosis)

LABORATORY ABNORMALITIES IN MEGALOBLASTIC ANEMIA:

1. Asynchrony (Age of the nuclear material – age of the cytoplasm) – this can best be appreciated by making serious comparison of
the nuclear and cytoplasmic material in megaloblastic precursol cells vs. Normoblastic precursor cells. (No DNA Synthesis, RNA
and Protein synthesis is active)
2. Hemoglobin: Decreased
3. MCV: Increased (often: >120 fL)
4. PBS: Oval macrocytes, Hypersegmented neutrophils (>5 Nuclear lobes (amongh >5% of the Neutrophils)
5. WBC: Leukopenia
6. PLT: Thrombocytopenia
7. Lactic dehydrogenase: Increased
8. Bilirubin: Increased (jaundice)
9. Haptoglobin: Decreased
10. Bone marrow:
 Megaloblasts
 Hypercellular with Erythroid hyperplasia (M:E ratio of 1:1 to 1:3) NV: 3:1
 Giant bands and Metamyelocytes

LABORATORY DIAGNOSIS:
- Schilling’s test- Standard method to diagnose Pernicious Anemia, once cobalamin deficiency is confirmed.
- (This test is already obsolete)
1. Oral: Radiolabeled cobalamin
2. Intramuscular: Large dose of unlabelled B12
o Purpose: “cold” B12 is to saturate the B12- binding sites in the serum, and thereby flush all of the orally absorbed by the
B12 in the urine, where it can be measured)
3. Urine is collected for 24 hours
o Indication: The amount of radioactivity = how much B12 is absorbed orally.
o The recovery of <6% in the urine= malabsorption of B12
o If the initial value is Abnormal, second stage is performed, which intrinsic factor is given together with the labelled B12
o An increase in the amount of B12 absorbed during the second stages = pernicious anemia

LABORATORY DIAGNOSIS of MEGALOBLASTIC ANEMIA RESULT OF ABSORPTION TESTS OF RADIOACTIVE

VITAMIN B12
TESTS COBALAMIN FOLATE DOSE OF LABEL B12 DOSE OF LABEL
DEFECIENCY DEFICIENCY GIVEN ALONE B12 GIVEN WITH
Serum Cobalamin Decreased Normal IF
Serum Folate Normal to Increased Decreased Vegan Normal Normal
Serum Methylmalonic Acid Increased Normal Pernicious anemia Low Normal
Serum Homocysteine Increased Increased or gastrectomy
(Moderate) Ileal Lesion Low Low
Erythtocyte Folate Decreased Decreased Intestinal blind- Low* Low*
loop syndrome
(*)Corrected by Antibiotic Theraphy

ANEMIA OF BONE MARROW FAILURE AND SYSTEMIC

DISORDERS
1.Primary Defect in the BM:
- Aplastic Anemia
- Pure Red Cell Aplasia (PRCA)
2. Presence of a space-occupying lesion in the BM:
- Myelophistic Anemia
3. Systemic diseases:
- Renal
- Endocrine
ANEMIA OF THE BONE MARROW

1. APLASTIC ANEMIA
- There’s a peripheral blood pancytopenia (decreased in cellular constituents: WBCs, RBCs, PLTs
- The BM is hypoplastic (underdeveloped) or aplastic.
Diagnostic criteria for Severe Aplastic Anemia:
o Bone Marrow
 Cellularity: <25% -<50% normal cellularity with <30% hematopoietic cells
 Peripheral Blood:
 Granulocytes - <0.5x109 /L
 Platelets - <20x109 /L
 Anemia - <1% reticulocytes
*Milder forms do not meet these criteria (Hypoplastic anemia to Drug toxicity)

CLINICAL CHARACTERISTIC OF APLASTIC ANEMIA

Absence of Immature myeloid cells in the Peripheral Blood
Aplastic of Hypoplastic marrow
Lack of splenomegaly

CLASSIFICATION OF APLASTIC ANEMIA

1. Primary
o Congenital Fanconi Anemia (Heterozygous recessive Disorder-diverse congential malformations)
o Acquired Idiopathic Anemia
2. Secondary
o Drugs and Chemicals
 Chloramphenicol (for bacterial infections. Ex. Use to treat conjunctivitis)
 Classic drug associated for Marrow Aplasia
 Benzene and their derivatives:
o Hydantoins
o Sulfonamides
o Gold Preparations
 Insecticides:
o Chlordane
o Chlorophentone (DDT)
o Gamma Benzene hexachloride (Lindane)
o Radiation
 Low dose irradiation = Long term
 High dose radiation with acute exposure = rapid developing Bone marrow aplasia and death
 Damages the stem cells or the Hematopoietic microenvironment
o Immune Mechanism
 Autoimmune
 Lymphocytes from patients suppresses growth of normal marrow cells in vitro
Treatment: Anti-thymocyte globulin for mediated aplasia
  Natural Killer Cells (Null Cell Lymphocytes) inhibits growth of hematopoietic cells in vitro: proposed to be
responsible for lymphocyte- mediated aplasia
 In vitro inhibition of erythropoiesis = Abnormal composition of T cell subset in the bone marrow
 Excess activation of Tac+, T3+, T11+ lymphocytes
o Infections/ Miscellanous etiologies
 Non – A, non- B hepatitis
 Military tuberculosis
 Brucellosis – ingestion of unpasteurized milk or uncooked meats of infected animals.
 Parasitic Infections
 Hypersplenism
 Paroxysmla nocturnal homoglobinuria

PATHOPHYSIOLOGY:
- Failure of the Hematopoietic stem cells growth = Hypoplastic marrow and Pancytopenia
- Deficiency in the number of bone marrow cells
- Immune suppression of stem cells
- Defect in the stem cells themselves

CLINICAL PRESENTATION:
- Fever, Pallor and Weakness
- Doesn’t generally demonstrates Splenomegaly or Lymphadenopathy
- Finding of Immature of RBC and WBC in Peripheral bloods
- Anemia
- Normocytic- Normochromic
- Neutrophils: Decreased (Increased susceptibility to Bacterila infections not on Viral infections)
- Platelets: Decreased (because of the hemmorrhage)
- No immature cells in the bone marrow
- Hypoplastic or aplastic, with increased fat and decreased hematopoietic cells.
- Bone marrow has patchy areas of cellularity.
- Hemoglobin F: elevated
- Leukocyte Alkaline Phosphatase: Increased
- Ham Acidified Serum Test – confirmatory for PNH (+)

CHEMISTRY:
- Serum Iron: elevated
- Plasma Iron Clearance: Delayed
- Iron: Adequate
- Iron uptake: Decreased – developing red cells
- Erythropoietin: Normal or Elevated

TREATMENT:
- Bone marrow transplantation (least immunogenic)
- Transfusion of needed blood products: PRBC, Platelet concentration, granulocyte concentration
- Immunosuppressive therapy
- Administration of Anti- thrombocyte (ATG) and anti- lymphocyte globulin (ALG)
- Androgens

2. CONGENITAL APLASTIC ANEMIA (FANCONI ANEMIA)

- Rare, Inherited form of AA, Inherited in an Autos ommal recessive manner
- Pancytopenic disorder with a hypoplastic to aplastic bone marrow
- Multiple Chromosomal Defect - increase the probability of Acute Myeloid Leukemia development
- May have multiple chromosomal abnormalities
 Ring chromosome
 Translocations
 Dicentric forms
 Spontaneous breaks (automatic, natural occurrence)

CLINICAL PRESENTATION:
1. Microcephaly (abnormally small brain)
2. Brown skin Pigmentation
3. Short stature
4. Malformation of the thumb
5. Internal strabismus (crossed eyes)
6. Kidney malformations
7. Genital Hyperplasia
8. Mental retardation

HEMATOLOGY:
1. Manifests 5-10 years birth
 2. Anisocytosis and Poikilocytosis - (+)
3. Hemoglobin F: markedly increase
4. Osmotic Fragility Test: Increase
5. Erythrocyte Sedimentation Rate: Increase

TREATMENT:
1. Androgens
2. Bone Marrow Transplantation
3. Death – most often secondary to haemorrhage or infection

3. PURE RED CELL APLASIA

- Inherited or acquired

CLASSIFICATION OF PURE RED CELL APLASIA

1. Acquired
 Primary
 Idiopathic
 Immune mechanism
 Ig Inhibor to RBC precursor
 Erythropoietin Inhibitor
 Secondary
 Benign tumors
 Drugs and Chemicals
 Infextions
 Hemolytic Anemia- aplastic crisis
2. Congenital - Diamond- Blackfan Anemia

ACQUIRED PURE RED CELL APLASIA

- Seen in >40 years of age
- Characterized by Severe Anemia
- Leukocyte: Normal to slighty decreased
- Platelets: Normal to slighty decreased
- Bone Marrow: Overall Normal cellularity
- Erythroid elements: Severe decreased, sometimes almost no elements seen

CLINICAL PRESENTATION:
o Anemia develops insidiously
o Onset is very gradual, the patient’s body compensates
o Extreme pallor, only constant clinical finding.
o Splenomegaly
o Hepatomegaly
o Hepatosplenomegaly

HEMATOLOGY:
o Normocytic- Normochromic
o Reticulocyte count: Greatly decreased (or even 0%)
o Bone Marrow: Normal
o Erythroid Precursors: Extremely decrease or absent

CHEMISTRY:
o Erythropoietin: Increased
o Serum Iron: Increased
o Transferrin saturation: Increased
 Results of multiple red cell transfusion

TREATMENT:
o Red Cell transfusion
 Complications of frequent and long term RBC transfusion
 Hemochromatosis
 Hepatitis
o Treat the primary condition that causes the Aplasia
 Removal of Thymoma
o Immunosuppression for those with immunologic involvement
o Androgen

CONGENITAL PURE RED CELL APLASIA (DIAMOND- BLACKFAN ANEMIA)

- Anemia: Normocytic- normochromic
- WBC: Normal
 - PLT: Normal
- Marrow erythroblasts: Marked decrease

PATHOPHYSIOLOGY:
- The bone marriw is lack of response to EPO
- CFU- E insensitivity to EPO
- Patient lymphocytes also inhibits EPO (inhibitory)

CLINICAL PRESENTATION:
- At birth: Evident pallor, almost always evident by the age of 1 year
- Intermittent anemia & Hemosiderosis = Marked growth retardation = interferes with liver and endocrine functions
- It doesn’t demonstrate renal abnormalities
- Diagnosis is usually made during Infancy.
- Hemoglobin = 1.7g/dL – 9.4g/dL
- Expected increase NRBCs which isn’t not seen in Peripheral Blood Smear.
- Reticulocytes: <1%-<2%
- Reticulocyte Production Index: Extremely low
- Bone marrow: Cellular with markedly decreased in Red cell precursors
- Erythropoietin: Elevated
- Red Cell Survival: Normal
- Hemoglobin F: Elevated

PRINCIPAL CLINICAL AND LABORATORY FACTORS FOR DIFFERENTIATION

OF CONGENITAL DISORDERS CAUSING BONE MARROW FAILURE
FEATURES FANCONI ANEMIA DIAMOND- FANCONI ANEMIA
(Acquired) (Congenital)
Hematologic classification Aplastic Anemia Pure Red Cell Aplasia
Brown Skin Pigmentation Common Uncommon
Thumb abnormalities Common Uncommon
Renal abnormalities Common Uncommon
On-set Hematologic abnormalities 5—10 years old <1 year of age
Bone Marrow Biopsy Hypoplastic to Aplastic Cellular
Bone Marrow Aspirate Pancytopenia Markedly decrease only in Erythroid precursor
Peripheral Blood Pancytopenia RBC: Decrease WBC: Normal
Cyrogenetics Multiple Chromosomal abnormalities No associated abnormalitites

TREATMENT:
- RBC Transfusions = may complicate the patient with Hepatitis and Hemosideris
- Steroid (corticosteroid)therapy for immunologic involvement
- Splenectomy (complication: patient becomes more susceptible to infections)

MYELOPHISTIC ANEMIA
- A common finding in patiens with carcinoma (94%)
-
CHARACTERISTICS AND BIOSYNTHESIS OF HEMOGLOBIN
HEMOGLOBIN FUNCTION
- The red cells in systemic arterial blood carry 0z from the lungs to the tissues and return in venous blood with CO2 to the lungs.

HEME STRUCTURE AND BIOSYNTHESIS

 consists of a ring of carbon, hydrogen, and nitrogen atoms called protoporphyrin IX, with a central atom of divalent ferrous iron.
 Each of the four heme groups is positioned in a pocket of the polypeptide chain
 Ferrous iron in each heme molecule reversibly combines with one oxygen molecule Biosynthesis

EFFECTS OF VARIOUS FACTORS OF OXYHEMOGLOBIN DISSOCATION CURVE

- Oxyhemoglobin dissocation curve (OCD)- the relationship os described of haemoglobin, which plots the percent oxygen
saturation of haemoglobin vs. the PO2
- During oxygenation each of the 4 heme iron atoms of haemoglobin molecule can reversible bind one oxygen molecules.
Approximately 1.34 mL of O2 bound/ each gram of HgB
- Hemoglobin affinity for Oxygen relates the Partial pressure of O2
- (P50 value) - defines in terms of amount of oxygen needed to saturate 50% of haemoglobin
- Sigmoidal – indicates;
o Low haemoglobin affinity for oxygen at low oxygen tension
o High affinity for oxygen at high tension.
- Reference interval; Arterial oxygen saturation: 96%- 100%
SHIFT TO THE LEFT (Higher percentage of Oxygen saturation
 Arterial: 80 - 100mm Hg
 Venous: 30 to 50mm Hg
- Bohr Effect – influence of the PH on the release of oxygen from haemoglobin.
FACTORS LEFT RIGHT
 Occurs in the mitochondria and cytoplasm of bone marrow SHIFT SHIFT
erythrocyte precursors, beginning with the pronormoblast through the
H HB F admixture Increase N/A
circulating polychromatic (also known as polychromatophilic)
erythrocyte. O Oxygen affinity Increase Decrease
P pH Increase Decrease
 Begins in the mitochondria with the condensation of glycine and B Blood temperature Decrease Increase
succinyl coenzyme A (CoA) catalyzed by aminolevulinate synthase
to form aminolevulinic acid (ALA). In the cytoplasm, aminolevulinic E Erythrocyte 2,3 DPG Decrease Increase
acid dehydratase (also known as porphobilinogen synthase) converts C Co2, Erythrocyte 2,3 DPG Decrease Increase
ALA to porphobilinogen (PBG). PBG undergoes several
transformations in the cytoplasm from hydroxymethylbilane to coproporphyrinogen III. This pathway then continues in the mitochondria until, in
the final step of production of heme, Fe2+ combines with protoporphyrin IX in the presence of ferrochelatase (heme synthase) to make heme.

 Transferrin, a plasma protein, carries iron in the ferric (Fe3+) form to developing erythroid cells. Transferrin binds to transferrin receptors on
erythroid precursor cell membranes and the receptors and transferrin (with bound iron) are brought into the cell in an endosome. Acidication of the
endosome releases the iron from transferrin. Iron is transported out of the endosome and into themitochondria where it is reduced to the ferrous
state, and is united with protoporphyrin IX to make heme. Heme leaves the mitochondria and is joined to the globin chains in the cytoplasm.

GLOBIN STRUCTURE AND SYNTHESIS

 The four globin chains comprising each hemoglobin molecule consist of two identical pairs of unlike polypeptide chains, 141 to 146
amino acids each. Variations in amino acid sequences give rise to different types of polypeptide chains.
 Six structural genes code for six globin chains. The a- and z- globin genes are on the short arm of chromosome 16; the e-, g-, d-, and b-
globin gene cluster is on the short arm of chromosome 11
 takes place in erythroid precursors from the pronormoblast through the circulating polychromatic erythrocyte, but not in the mature
erythrocyte. Transcription of the globin genes to messenger ribonucleic acid (mRNA) occurs in the nucleus, and translation of mRNA to
the globin polypeptide chain occurs on ribosomes in the cytoplasm.

TYPES OF HEMOGLOBIN
1. Embryonic Hemoglobins
 Primitive hemoglobins formed by immature erythrocytes in the yolk sac.
 These hemoglobins include Gower I, Gower II, and Portland types.
 They are found in the human embryo and persist until approximately 12 weeks of gestation
2. Fetal Hemoglobin
 predominant hemoglobin variety in the fetus and the newborn. This hemoglobin type has two alpha and two gamma chains. The
gamma chains have 146 amino acids, as do beta chains.
 hemoglobin A replaces hemoglobin F in the circulating erythrocytes until the normal adult level of hemoglobin F
3. Glycosylated Hemoglobin (Hemoglobin A1c)
 subfraction of normal hemoglobin A is hemoglobin A1. It is formed during the maturation of
the erythrocyte.
 accurately reflects the patient’s blood glucose level over the preceding weeks and has been
recently used to monitor the control of diabetes.
  concentration of hemoglobin A1 is 3% to 6% in normal persons and 6% to 12% in both insulindependent and non–insulin-
dependent diabetics.
4. Hemoglobin A
 adult hemoglobin is predominantly of the A variety (95% to 97%), the A2 type is also found in
small quantities (2% to 3%).
 Hemoglobin A is composed of two alpha and two beta polypeptide
chains.
 Hemoglobin A2 is composed of two alpha and two delta chains. consists of four heme groups and four polypeptide chains
(organized into two alpha chains and two beta chain)
 one heme- capable to carry 1 mole of oxygen
 has 141 amino acids in each of the alpha chains and 146 amino acids in each of the beta chains
 major switch from HbF to Hb A occurs 3-6 months after birth

VARIANT FORMS THAT CAUSE DISEASE:

1. Hemoglobin H (B4) – formed by a tetramere of B chains - Thalassemia
2. Hemoglobin Barts- formed by a tetramere of y chains - Thalassemia
3. Hemoglobin S- variation in the B chain gene - Sickle cell disease
4. Hemoglobin C- variation in the B chain gene- Mild Chronic Hemolytic Anemia
5. Hemoglobin E- variation on the B chain gene - Mild Chronic Hemolytic Anemia
6. Hemoglobin AS- heterozygous form causing sickle cell trait with one adult gene and one sickle cell disease gene
7. Hemoglobin SC- heterozygous form with one sickle gene and another encoding hemoglobin C

TYPES OF HEMOGLOBIN ACCORDING TO FUNCTION:

A. Functional ARTERIAL VENOUS
1. Oxyhemoglobin- arterial blood (bright red)
2. Deoxygenated Hemoglobin- venous blood (dark red) pH 7.35-7.45 7.31-7.41
B. Non-Functional pCO2 (kPa) 4.7 - 6.0 5.5 - 6.8
1. CARBOXYHEMOGLOBIN pCO2 (mmHg) 35 -45 41 - 51
 Carbon monoxide (+) Heme Iron Bicarbonate 22-28 23-29
 1 molecule of CO (+) haemoglobin = shifts the Hemoglobin-Oxygen
(mmol/L)
dissociation curve to the left irreversible
PO2 (kPa) 10.6 - 13.3 4.0 -5.3
Result: Anoxia – increase affinity and severely impairing release of oxygen
pO2 (mmHg)  80-100 30 -40
to the tissues
 “Silent killer” – odourless and colorless gas, hypoxic sO2 (%) > 95 75
 Ligh sensitive, Typical, brilliant , cherry Red Color
 not capable to transport oxygen, hypoxia
 Chief sources:
 Gasoline motorcyles
 Illuminating gases
 Gas heaters
 Defective stoves
 Smoking of tobacco
 Carbon Monixide Affinity: 240x

2. SULFHEMOGLOBIN
 Irreversible oxidation of haemoglobin by drugs or exposure to chemicals in industrial or environmental setting.
 Drugs:
o Sulfonamides
o Phenacetin
o Nitrites
o Phenylhydrazine
 Partially denatured form of haemoglobin formed during oxidative hemolysis.
 Formed through Sulfur atom (+) pyrrole ring of heme with greenish pigment.
 During oxidation of hemoglobin: Sulfur is incorporated into heme iron rings = green hemochrome.
 Reported:
1. Patients receiving treatment with sulfonamids or aromatic amine drugs (Phenacetin & Acetanilid)
2. Patients with severe constipation (bacteremia due to Clostridium perfringens)
3. Enterogenous cyanosis

 Ineffective for oxygen transport, but can combine with carbon monoxide to form Carboxyhemoglobin.
 Cannot be converted to Normal haemoglobin, remains in cell form until it breaks down.
 Color of blood: Mauve lavender
 Oxygen Affinity: 100x lower

3. METHEMOGLOBIN
 Reversible oxidation of heme iron- ferric state = inability of haemoglobin to combine reversible to oxygen
 Cannot carry oxygen because the oxidized ferric iron can’t bind
 Hign Methemoglobin= Low Oxygen delivery to the tissues
 >30% of Methemoglobin to the blood;
Results:
 Functional anemia
  Cyanosis – bluish discoloration of skin and mucus membrane
 Hypoxia – which results dyspnea, headache, vertigo, change in mental status
 >30% of Methemoglobin to the blood; Comatose and Death
 Birth or First few months of life, Methemoglobin may occur and can be treated with Methylene blue
 Acquired form: Exposure to Exogenous oxidants
 Nitrites
 Primaquine
 Dapsone
 Bezocaine
 builds up in the circulation and if the level is above 10% individual appear cyanotic
THE ERYTHROCYTE LIFE CYCLE
HEMOLYTIC PROCESS
1. EXTRAVASCULAR HEMOLYSIS
 majority of RBCs are phagocytized and destroyed by the spleen
 heme oxygenase breaks down the hemoglobin into heme ring and the globin proteins within macrophages of the MPS
(Mononuclear Phagocytic System)
 Iron is removed, and returns to bone marrow or enters the iron storage pool
 Biliverdin is produced and transformed to bilirubin which circulates to liver bound to albumin
 The bilirubin is conjugated in the liver and excreted via bile secretions into the intestine.
 Intestinal bacteria metabolize bilirubin into stercobilinogen and stercobilin which are excreted via feces.
2. INTRAVASCULAR HEMOLYSIS
 takes place directly inside the vessel and hemoglobin is released into the plasma
 Hemoglobinemia, red tinged hemoglobin seen directly in the plasma after the
 blood sample is centrifuged is an unusual finding seen only in intravascular hemolysis

SEVERAL FATES OF THE RELEASED HEMOGLOBIN

1. Hemoglobin Complexes with Haptoglobin
2. Excess Heme complex with Hemopexin
3. Methemalbumin releases heme that binds with albumin

RELATIONSHIP OF HEMOLYSIS AND CLINICAL EVENTS

CLINICAL
PHYSICAL SYMPTOMS
EVENTS
Decrease RBC, Symptoms of anemia, pallor,
Hb, Hct fatigue, tachycardia
Increase bilirubin Jaundice
Hemoglobinemia Blood-tinged plasma
Hemoglobinuria Blood tinged urine
IRON ABSORPTION
 occurs in the proximal small intestine, predominantly the duodenum and upper jejunum
 absorption into the mucosal cells lining the GI tract
 movement across the mucosal cells into the plasma on the other side
IRON TRANSPORT
1. Transferrin
o transport iron in the plasma, B globulin
o is a plasma protein synthesized by the liver, which has a high affinity for Ferric
o each molecule of transferrin can carry up to 2 molecules of iron
IRON STORAGE
Major sites of iron storage
1. Bone marrow
2. Ferritin; Hemosderin
Storage Forms of Iron
1. Ferritin- immediate use or short term storage
2. Hemosiderin- long term storage
ANALYSIS OF HEMOGLOBIN
1. Electrophoresis
 based on the principle that hemoglobin molecules in an alkaline solution have a net
 negative charge and move toward the anode in an electrophoretic system
 can separate hemoglobins A, F, S, and C and other variant hemoglobins
 hose with a greater electropho- retic mobility than hemoglobin A at pH 8.6 are
 Classified as the fast hemoglobins.
A. Cellulose Acetate Hemoglobin Electrophoresis- pH=8.6
1. C, E, O, A2
2. S, D, G
3. F
4. A1
5. BARTS
6. I
7. H
B. Citrate Cigar Hemoglobin Electrophoresis
1. F
 2. A, E
3. ORIGIN
4. O, D, G
5. S
6. C

ERYTHROCYTE MORPHOLOGY AND INCLUSIONS

Normal mature erythrocytes (discocytes) are biconcave and disc shaped and lacks a nucleus. The number (percentage) of abnormal cells per 100 normal red
cells should be reported after counting several hundred cells.

THE VARIATIONS FROM NORMAL CAN BE CLASSIFIED AS:

1. Variation in size
2. Variation in shape
3. Alteration in color
4. Inclusions in the erythrocyte
5. Alterations in the erythrocyte distribution on a peripheral blood smear

VARIATIONS IN ERYTHROCYTE SIZE (ANISOCYTOSIS)

Red Cell Distribution Width (RDW) - numerical expression that correlates with the degree of anisocytosis
NV: 11.5% - 14.5%
1. NORMOCYTIC – normal size of the RBC; normal MCV (80-100fl)
 Acute Post Hemorrhagic Anemia
 Hemolytic Anemia
 Aplastic Anemia

2. MICROCYTIC- cell measure less that 6 um in diameter; MCV (< 80fl)

- Associated with a decrease in hemoglobin may be produced by a deficiency of iron, an impaired globulin synthesis, or a
mitochondrial abnormality affecting the synthesis of the heme unit of the hemoglobin molecule.
 Chronic disease
 Thalassemia (normal RDW)
 Iron deficiency (increased RDW) *Chronic blood loss (most common clinical finding)

3. MACROCYTIC- cells are larger than 9 um; MCV (>100fl)

- result of a defect in either nuclear maturation or stimulated erythropoiesis
-true macrocytes represent a nuclear maturation defect associated with a deficiency of either
vitamin B12 or folate
- other type of macrocytosis is caused by increased erythropoietin stimulation, which increases the
synthesis of hemoglobin in developing cells
 Chemotherapy
 Liver disease
 Alcoholism
 Megaloblastic anemia

VARIATIONS IN ERYTHROCYTE SHAPE (POIKILOCYTOSIS)

1. MEGALOCYTE/ OVAL MACROCYTE/ MACROCYTE

 Increases in this abnormality are seen in vitamin B12 and folate deficiencies and may be observed in erythrocytes that are in the
reticulocyte stage
 Asynchronous development
2. ACANTHOCYTE
 have irreversibly multiple thorny, spike- like projections which are bent at their tips that are
 irregularly distributed around the cellular membrane and may vary in size
 Unlike echinocytes, acanthocytes have few spicules
 prevalent in two very different disorders: Abetalipoproteinemia, a rare hereditary disorder, and spur cell anemia are also found in
cirrhosis of the liver with associated Hemolytic anemia, following heparin administration; in hepatic hemangioma; in neonatal
hepatitis, and postsplenectomy.
 Abetalipoproteinemia, acanthocytes represent an imbalance between erythrocyte and plasma lipids. The reason for this
imbalance is that the patient does not absorb lipids in the small intestine. This results in decreased plasma lipids, which in
turn produces a membrane defect. The loss of membrane integrity causes the cells to be more sensitive to external and
internal forces. It is characterized by pigmentary degeneration of retina (retinitis pigmentosa).
3. BLISTER CELLS
 erythrocytes containing one or more vacuoles that resemble a blister on the skin.
 has a significantly thinned area at the periphery or outer border of the cell membrane.
 The vacuoles may rupture. If rupturing does occur, distorted cells (keratocytes) and cellfragments (schistocytes) are produced.
 result from the traumatic interaction of blood vessels and circulating blood such as fibrindeposits
 hallmark of Microangiopathic hemolytic anema

4. BURR CELLS (ECHINOCYTE/ CRENATED RBC/ SEA URCHIN CELL)

 erythrocytes having one or more spiny projections of cellular membrane; have short,
  scalloped, or spike-like projections that are regularly distributed around the cell membrane.
 Crenation can occur as the result of the physical loss of intracorpuscular water and is due to
 abnormality in the lipid content of RBC membrane
 Depletion of ATP, exposure to hypertonic solution, artifact in air drying, Anemia associated
5. ELLIPTOCYTES
 generally narrower and more elongated than ovalocytes due to bipolar aggregation of haemoglobin
 represent a membrane defect in which the membrane is radically affected and suffers a loss of integrity
 Associated clinical disorders include Hereditary elliptocytosis, Anemias associated with malignancy, Hemoglobin (Hb) C disease,
Hemolytic anemias (occasionally), Iron deficiency anemia, Pernicious anemia, Sickle cell trait, and Thalassemia.
 Pencil/ Oat cell- thinner variant
6. HELMET CELL (KERATOCYTE OF BESSIS)
 larger scooped out part of the cell
 remains after the rupturing of a blister cell and are formed as a result of the physical process of fragmentation.
7. KERATOCYTES
 erythrocytes that are partially deformed but not cut.
 the spicules, resembling two horns, result from a ruptured vacuole. Usually the cell appears like a half-moon or spindle.
 seen in conditions such as Disseminated (diffuse) intravascular coagulation (DIC)
8. KNIZOCYTE (PINCH CELL)
 triangular with 2 pallor areas
 resemble a pinched bottle.
 associated with Microangiopathic hemolytic anemias, including Hereditary spherocytosis
9. LEPTOCYTE (THIN CELLS)
 resemble target cells (codocytes) but the inner, central portion is not completely detached from the outer membrane
 thinner variant of codocyte (target cell)
 appear a very thin, flat cells with only a thin rim of hemoglobin coloring the edges
 seen in a variety of anemias including IDA, thalassemia, and other hemoglobin disorder, plus obstructive jaundice
10. PYKNOCYTES
 distorted, contracted erythrocytes that are similar to burr cells.
 are seen in acute and severe Hemolytic anemia ; glucose-6-phosphate dehydrogenase (G6PD) deficiency; and Hereditary
lipoprotein deficiency
 may be seen in small numbers during the first 2 to 3 months of life as infantile pyknocytes
11. SCHISTOCYTES (SCHIZOCYTE)
 result of the breaking apart of an erythrocyte,
 the schistocyte is about half the size of a normal erythrocyte and may have a deeper red appearance fragments of erythrocytes that
are small and irregularly shaped.
 can be seen in Hemolytic anemias related to burns and Prosthetic implants as well as Renal transplant rejections
12. SICKLE CELLS (DREPANOCYTES, MENISCOCYTES)
 resemble a crescent; at least one of the ends of the cell must be pointed
 result from the gelation of polymerized deoxygenated Hb S (Polymerization of Hb S is influenced by both lowered oxygen levels
and decreased blood pH)
 influx of sodium ions and other metabolic changes produce an extremely increased level of
 intracellular calcium ions. Alterations in the cellular contents produce cell membrane rigidity.
 The presence of sickle cells is associated with Sickle cell anemia.
13. SPHEROCYTES (BRONZE CELL)
 lost their normal biconcave shape (extremely compact, round shape)
 may appear as artefacts if a slide is examined at the thin end of a normal blood smear
 surface area of the erythrocyte to the volume of the cell contents decreases because of the
 loss of cell membrane
 they have a 14 days life span
 they are found in ABO HDN, in transfusion reactions, and in Hemolytic anemia associated with burns.
 In ABO erythoblastosis fetalis, they are a valuable diagnosis because they areabsent in erythroblastosis caused by RH compatibility.
 transfused cells (storage phenomenon (lessions of storage) appear as microscopherocytes inthe recipients blood.
 in vitro, they are formed by the action of lytic agents on red cells.
 They may also result from the fragmentation of normal erythrocytes.
 They present a paralytic cell.
 Spherocytosis is seen in an inherited disorder known as Hereditary spherocytic anemia (HSA)

14. STOMATOCYTES (MOUTH CELL)

 have a slit like opening that resembles a mouth result from increased sodium (Na+) ion and decreased potassium (K+) ion
concentrations within the cytoplasm of the erythrocyte
 increased osmotic fragility and increased autohemolysis at 37 degree Celsius
 Acute alcoholism, Alcoholic cirrhosis, Glutathione deficiency, Hereditary spherocytosis, Infectious mononucleosis, Lead poisoning,
Malignancies, Thalassemia minor, Rh null disease and transiently accompanying Hemolytic anemia
15. TARGET CELL (CODOCYTES)
 erythrocytes that resemble a shooting target (central red bull’s-eye is surrounded by a clear ring and then an outer red ring)
 the defect is related to a maldistribution of haemoglobin
  certain enzyme defects, cholesterol and phosphatidylcholine are abnormally increased within the erythrocyte and become
incorporated into the membrane lipid
 seen in the Hemoglobinopathies (Hb C disease, S-C and S-S disease, sickle cell thalassemia, and thalassemia), Hemolytic anemias,
Hepatic disease with or without jaundice, and Iron deficiency anemia as well as after a splenectomy
16. TEARDROP CELL (DACRYOCYTE)
 pear shaped cell
 diagnostic in Myelofibrosis
17. BITE CELL (DEGMACYTE)
 suggestive of Heinz bodies
 Anemia when present in significant figure

BROWN’S RED BLOOD CELL MORPHOLOGY GRADING CHART

“STAP
HS“ 1+ =
Spherocyte 1 to
1+ = aggregates
Teardrop 5/fiel
to 3 to 4 RBC
cell d
2+ = aggregates
Acanthocyt 2+ =
to 5 to 10 RBC
e 6 to Rouleaux INCLUSIONS FEULGEN SUPRAVITA WRIGH
3+ = numerous
Polychrom 10/fie STAIN L T
aggregates
atophilia ld (DNA) STAIN STAIN
with only few
Helmet 3+ = (RNA)
free RBC
cells >10/f Basophilic stippling - + +
Schistocyte ield Cabot Rings - - +
s
Howell-Jolly bodies + + +
“ Si PAP
“STOP maBA Polychromatophilia - - +
1+ =
BEB“ HO” Reticulocytes - + -
3 to
Stomatocyt Sickle Pappenheimer bodies - + +
10/fie
e cell
ld Heinz bodies - + -/+
Target cell Pappenhe
2+ =
Ovalocyte imer Grade as
11 to
Poikilocyte bodies positive only
20/fie
Burr cell Basophili
ld
Elliptocyte c
3+ =
Bizarre- stippling
>20/f
shaped Howell-
ield
RBC jolly
bodies

VARIATIONS IN ERYTHROCYTE COLOR/STAINING/ HB CONTENT (ANISOCHROMIA)

1. Anulocyte
 Thin cells that are poorly hemoglobinized and exhibit a thin peripheral ring of stained
 hemoglobin surrounding a large central clear area

2. Hypochromia

 Hb content of the cell is greatly decreased such that the central pallor is more prominent
 cells are usually microcytic when the central pallor exceeds one third of the cell’s diameter
 clinically associated with Iron deficiency anemia

3. Hyperchromia
 Hb content of the cell appears to be increased since the cell appears thicker than normal.
 The entire cell stains deep pink lacking the usual central pallor
 Spherocytes and megalocyte

4. Polychromatophilia
 used if a nonnucleated erythrocyte has a faintly blue-orange color when stained Wright stain
 lacks the full amount of hemoglobin, and the blue color is caused by diffusely distributed residual RNA in the cytoplasm.
 larger than a mature erythrocyte. If stained with a supravital stain, a poly- chromatophilic
 erythrocyte appears to have a thread-like netting within it and is called a reticulocyte
 Increased numbers of polychromatophilic erythrocytes are associated with rapid blood regeneration and increased bone
marrow activity

VARIATIONS IN ERYTHROCYTE INCLUSIONS

1. Basophilic Stippling (Punctuate basophilia)

 tiny, round, solid- staining, dark-blue granules
 Coarse basophilic stippling is sometimes referred to as punctate stippling.
  Stippling represents granules composed of ribosomes and RNA that are precipitated during the process of
 staining of a blood smear
 Associated clinically with Disturbed erythropoiesis (defective or accelerated heme synthesis), Lead poisoning, Pyrimidine-5-
nucleotidase deficiency and Severe anemias.
 mistaken as pappenheimer bodies (positive iron stains (Prussian blue) and concentrated in the periphery)

Lead poisoning – coarse basophilic and punctuate stippling

Polycythemia Vera – fine basophilic stippling
PICA (Children) – Lead poisoning
PICA (Adult) – Iron deficiency Anemia
2. Cabot rings
 are ring-shaped, figure-eight, or loop-shaped structures
 stain a red or reddish purple color and have no internal structure
 represent remnants of microtubules from the mitotic spindle
 can be seen in Lead poisoning and Pernicious anemia
3. Crystals
 Hb C crystals- appear as rodlike or angular opaque structure with dense staining crystals (bar of gold, clam shell)
 Hb H crystals- seen with a brilliant cresyl blue stain and appear as blue globules
 Hb SC crytals- Washinton monument shape
4. Heinz Bodies
 Can be seen with a stain such as crystal violet or brilliant cresyl blue represent precipitated, denatured hemoglobin and are clinically
associated with congenital haemolytic anemia, G6PD deficiency, hemolytic anemias secondary to drugs such as phenacetin, and some
hemoglobinopathies.
 -golf ball-multiple heinz bodies
5. Howell-Jolly bodies
 cells contain only one or two Howell-Jolly bodies. Although these inclusions are most frequently seen in mature erythrocytes that lack a
nucleus, they may be seen in immature, nucleated erythrocytes
 They are nuclear remnants predominantly composed of DNA (Feulgen +)abnormal accelerated erythropoiesis, because the spleen cannot
keep up with pitting these remnants from the cell.
 associated with hemolytic anemias, pernicious anemia, and particularly postsplenectomy, physiological atrophy of the spleen
6. Pappenheimer bodies
 Siderotic granules are dark-staining particles of iron in the erythrocyte that are visible with a special iron stain— Prussian blue (appear as
blue dots and represent ferric (Fe3+) ions
 aggregates of mitochondria, ribosomes, and iron particles
 associated with iron-loading anemias, hyposplenism, and hemolytic anemias
7. Malarial Parasites

RED CELL ARTIFACTS

 Factors that aid in the recognition of artifacts:
o Total hemoglobin
o Clinical history
o Repeat blood smear
o Use plastic coverslips
o Immediate examination of a small drop of fresh blood by phase microscopy

It may be caused by the following factors:

o Delay in making the smear
o Abnormal temperatire
o Drying (too slow)
o Crowding of cells in polycythemia
o Increased viscosity
o Presence of abnormal proteins
o Glass effect
o Abnormal pH
o Anionic and cationic changes of the red cell
o Pressure when making the smear
o Errors on staining
DESIGN FORMATION (AIKA- fat and oil)
 occurs when a number of RBC get bunched up and are pushed out of shape by the surrounding RBC
 caused by fat or oil on the slide, applying too much pressure on RBC
CRENATED CELLS (EDRALLYNE- sardines)
 due to shrinkage of cells in hypertonic solution or in increased pH
ROULEAUX FORMATION (SHERYL – old)
 characterized by stack of coin-like columns of RBCs
 may indicate presence of plasma globulin, which may be indicative of a disease known as multiple myeloma
 may also indicate blood sample is getting old
HEMOLYTIC ANEMIAS
Normal red cell destruction
Red cell destruction usually occurs after a mean lifespan of 120 days when the cells are removed extravascularly by the macrophages of the reticuloendothelial (RE)
system, especially in the marrow but also in the liver and spleen, as the cells have no nucleus, red cell metabolism gradually deteriorates as enzymes are degraded and not replaced
and the cells become non-viable.
The breakdown of heme from red cells liberates iron for recirculation via plasma transferrin to marrow erythroblasts, and protoporphyrin which is broken down to
bilirubin.
Bilirubin circulates to the liver where it is conjugated to glucuronides which are excreted into the gut via bile and converted to stercobilinogen and stercobilin (excreted
in feces). Stercobilinogen and stercobilin are partly reabsorbed and excreted in urine as urobilinogen and urobilin
- Globin chains are broken down to amino acids which are reutilized for general protein synthesis in the body.
- Haptoglobins are proteins present in normal plasma capable of binding hemoglobin. The hemoglobin-haptoglobin complex is removed from plasma
by the RE system.
- Intravascular hemolysis (breakdown of red cells within blood vessels) plays little or no part in normal red cell destruction.

INTRODUCTION TO HEMOLYTIC ANEMIAS

There is an increase in erythrocyte destruction initiated primarily by trapping of cells in sinuses of the spleen or liver and producing a decrease in the normal average
life span of the erythrocyte.
Most anemias have a hemolytic component, and even in the Anemias of marrow failure, the erythrocyte is somewhat defective.
- This is particularly evident in the case of Dyserythropoietic syndromes, Megaloblastic anemias, and Thalassemias.
Hemolytic disruption of the erythrocyte involves an alteration in the erythrocytic membrane

THE CAUSES OF THIS MEMBRANE ALTERATION CAN BE DIVIDED INTO:

A. Inherited hemolytic disorders (intrinsic hemolytic anemia)
B. Acquired hemolytic disorders in which a factor outside the erythrocyte acts on it (extrinsic hemolytic anemia)

COMPARISON OF INTRAVASCULAR AND EXTRAVASCULAR HEMOLYSIS

INTRAVASCULAR EXTRAVASCULAR
Site of destruction of erythrocytes Within blood cells Spleen or liver
Mechanism Activation of complement IgM or IgG Cell-mediated phagocytosis of IgM-or IgG-coated cells
Laboratory findings Hemoglobinuria & Hemosiderinuria Positive Direct Antiglobulin test
THE MAIN LABORATORY FEATURES OF INTRATRAVASCULAR HEMOLYSIS ARE AS FOLLOWS.
1. Hemoglobinemia and hemoglobinuria
2. Hemosiderinuria (iron storage protein in the spun deposit of urine)
3. Methemalbuminemia (detected spectrophotometrically by Schumm'stest).

Inherited Hemolytic Anemia

Etiology: Inherited hemolytic disorders may affect the basic membrane structure, the erythrocytic enzymes, or the hemoglobin molecules within the red cell.
Examples:
A. Structural Membrane Defects
1. Hereditary spherocytosis
2. Hereditary ellipitocytosis
3. Hereditary stomatocytosis
4. Hereditary xerocytosis Rhus disease
B. Erythrocytic Enzyme Defects
1. G6PD deficiency Pyruvate kinase deficiency
2. Glutathione reductase Hexokinase
3. Defects of the Hemoglobin Molecule
4. Hb C disorder HD S-C disorder
5. HD S-S disorder (sickle cell anemia)
6. Thalassemia
Acquired Hemolytic Anemia
Etiology: Acquired hemolytic anemias can be classified according to the agent or condition responsible for inducing the hemolysis. Amajor distinction exists between acute
hemolysis of red cells, which results from damage done directly to the cell membrane, and hemolytic anemias caused by immunologic responses.
Examples of Agents and Conditions:
A. CHEMICALS, DRUGS, VENOMS
1. Aniline
2. Nitrobenzene Copper (Wilson disease)
3. Phenacetin Lead from gasoline or paint
4. Phenol derivatives Naphthalene (found in moth balls)
5. Resorcinol and Sulfonamides

B. MICROORGANISMS
1. Bacteria: Clostridium sp., cholera, E. coli O157: HZ. typhoid fever
2. Protozoa: Leishmania, malaria (Plasmodium sp.), toxoplasmosis

C. IMMUNE MECHANISMS (ANTIBODIES)

1. Autoimmune anemia due to cold Idiopathic reactive antibodies Cold hemagglutinin disease Idiopathic or secondary
2. Lymphoproliferative disorders (CLL, malignancies, ystemic lupus erythematosus, viruses), Hemolytic disease of the fetus and newborn (HDFN)
3. Paroxysmal cold hemoglobinuria Autoimmune anemia due to warm Incompatible blood transfusion reactive antibodies Drug induced
4. Paroxysmal nocturnal hemoglobinuria
D. AGENTS
1. Severe burns

E. TRAUMATIC AND MICROANGIOPATHIC HEMOLYTIC ANEMIAS

1. Disseminated intravascular coagulation
2. Prosthetic cardiac valves
3. E. coli O157:H7
4. Thrombotic thrombocytopenic purpura (TTP)
5. Hemolytic uremic syndrome

INHERITED HEMOLYTIC ANEMIA

A. STRUCTURAL MEMBRANE DETECT
- The cell membrane allows the erythrocyte with the flexibility and resilience to undergo numerous passages through the spleen during its 120-day life
span. The ability of erythrocytes to deform and subsequently return to their original biconcave disc shape is determined by: 2.
 Flexibility of the membrane, which relies on the structural and functional integrity of the membrane skeleton
 Cytoplasmic viscosity determined primarily by haemoglobin
 Cell surface-area-to-volume ratio

PATHOPHYSIOLOGY
- Structural proteins, forming the erythrocyte skeleton, are a and B-spectrin, actin, and protein 4.1
- Red cell band 3 is the major integral membrane protein that regulates exchange and facilitates the transfer of CO, from tissues to lungs,
- Ankyrin is the major connecting protein that links the membrane skeleton to the membrane bilayer

Mutations in any of the genes coding for the major membrane proteins can;
1. Alter the amount or function of the expressed proteins
2. Compromise the integrity of the membrane
3. Contribute to abnormal erythrocyte morphology

HEREDITARY SPHEROCYTOSIS (HS)

 Results from the loss of erythrocytic membrane surface, as vesicles, due to decreasedmembrane proteins linked to spectrin-ankyrin-band 3 associations and weak
contacts between spectrin and the negatively charged llpids of the inner half of the membrane bilayer.
 Hereditary spherocytosis (HS) is the most common inherited hemolytic anemia in people of Northem European descent. Most cases (approximately three-
fourths) are inherited in an autosomal dominant fashion.
 Hereditary spherocytosis is genetically and dinically heterogeneous; the majority of cases result from mutations in the gene for ankytin.
 Hemolysis is extravascular, Occurring only in the presence of the spleen.
 The fundamental cause in most cases of HS is defective vertical attachment between the phospholipid bilayer and the cytoskeleton scaffold, thus, the cell has a
decreased surface-aron-to-volume ratio, which changes the shape of the cell from discoid to spherocyte.
 Spherocytes have reduced cellular flexibility. Spherocytic cells demonstrate an abnormal permeability to sodium ion (Nat), causing an influx of sodium at 10
times the normal rate.
 Cholelithiasis, or the presences of gallstones, is a common complication of patients with HS and occurs with greatest frequency in adolescents and young adults.

Molecular basis of hereditary spherocytosis and elliptocytosis:

 A. Hereditary Spherocytosis
1. Ankyrin deficiency or abnormalities
2. Spectrin deficiency or abnormalities
3. Band 3 abnormalities
4. Pallidin (Protein 4.2) abnormalities
B. Hereditary Elliptocytosis
1. Alpha-or Beta-spectrin mutants leading to defective spectrin dimer formation
2. Alpha-or Beta-spectrin mutants leading to defective spectrin-ankyrin associations
3. Protein 4.1 deficiency or abnormality

LABORATORY INVESTIGATIONS:
 Hemoglobin concentration can range from normal to decrease.
 Peripheral blood smearsdemonstrate the characteristic spherocytes.
 Reticulocyte count is increased (5 - 20%) - The MCV is normal or slightly decreased.
 The MCH is normal, but the MCHC is generally greater than 36%.
 The osmotic fragility test, incubated and unincubated, is the test of choice to confirm a diagnosis of HS. Red blood cells from patients suspected of
having HS are subjected to varying salt solutions ranging from isotonic saline (0.85% NaCl) to distilled water (0.0% Naa).
 The classic finding is that the osmotic fragility is increased. The abnormality may require 24-h incubation at 37°C to become obvious. Auto-
hemolysis is increased and corrected by glucose. The cells are incubated with their own plasma for 48 h with or without glucose.
 The direct antiglobulin (Coombs) test is normal, excluding an autoimmune cause of spherocytosis and haemolysis.
TREATMENT
 The principal form of treatment is splenectomy although this should not be performed unless dinically indicated because of anemia or callstones
because of the risk of postsplenectomy sensis, particularly in early childhood.
 Splenectomy should always produce a rise in the hemoglobin level to normal, even though microspherocytes formed in the rest of the RE system will
remain.
 Folic acid is given in severe cases to prevent folate deficiency.

HEREDITARY ELLIPTOCYTOSIS (HE)

 Most cases of hereditary elliptocytosis (HE) are inherited in an autosomal dominant pattern.
 This has similar dinical and laboratory features to HS except for the appearance of the blood film, but it is usually a dinically milder disorder. Hereditary
elliptocytosis is due to defective horizontal stability in the cytoskeleton.
 Most cases are caused by mutations in spectrin, resulting in impaired assembly of the spectrin tetramers. Since the spectrin tetramer assembly is defective, the
cytoskeleton is less rigid and more easily deformed. Red blood cells are squeezed into an elliptical shape as they pass through capillaries, and eventually RBCs in
HE patients become fixed in that shape.
 Cells have a nearly normal life span. Occasional patients require splenectomy
 Patients with homozygous or doubly heterozygous elliptocytosis present with a severe hemolytic anemia with microspherocytes, poikilocytes and splenomegaly
(hereditary Pyropolkilocytosis).

SOUTH-EAST ASIAN OVALOCYTOSIS

 Variant of HE common in Melanesia, Malaysia, Indonesia and the Philippines and is caused by a nine amino acid deletion at the junction of the cytoplasmic and
transmembrane domains of the band 3 protein The cells are rigid and resist invasion by malarial parasites.
 Most cases are not anemic and are asymptomatic

HEREDITARY PYROPOLIDIOCYTOSIS
 HPP is a rare autosomal recessive disorder, representing a subset of common hereditary elliptocytosis HE, seen primarily in blacks.
 It is manifested in infancy or early childhood as a severe hemolytic anemia with significant poikilocytosis.
 Bizarre red cell shapes are evident when a peripheral blood smear is examined.
 MCV values range from 55 to 74 L because of the prevalence of microspherocytes and red blood cell fragments.
 This multiformity of the poikilocytosis is unmatched in any other hemolytic disease.
Hereditary pyropoikilocytosis involves two abnormalities:
1. defective assembly of o/B spectrin tetramers
2. An absolute decrease in the amount of spectrin present.
 One consequence is decreased thermal stability of the spectrin proteins (hence the term pyro," meaning fire).
 Spectrin proteins from patients with HPP denature at approximately 45°C, whereas spectrin from normal individuals denatures at
approximately 49°C.

HEREDITARY STOMATOCYTOSIS
 Can be seen in the genetic hemoglobin defect, thalassemia, and in lead poisoning, HS, and alcoholic cirrhosis.
 The cellular appearance stems from a cation abnormality, because the erythrocytes contain increased sodium (Na+) and decreased potassium (K+. Because the
intracellular osmolality is exceeded and the intracellular concentration of cations increases, water enters the cell and overhydrated erythrocytes take on the
appearance of stomatocytes or erythrocytes with a mouth-like opening. The cells are uniconcave. The MCHC is usually decreased and the MCV may be
increased. Anemia is usually mild to moderate. Bilirubin is increased and reticulocytosis is moderate. Peripheral blood smears have 10% to 50% stomatocytes.
Osmodic fragility and autohemolysis are increased. Autohemolysis is partially corrected with glucose and adenosine triphosphate (ATP). Splenectomy yields
variable responses.

HEREDITARY XEROCYTOSIS .
 Is a permeability disorder.
 In vitro, the thermal instability of spectrin suggests a defect in qualitative spectrin abnormality.
 The net loss of Intracellular K exceeds the passive Na* influx, yielding a net Na* gain. This causes the red cell to dehydrate. The MCHC increases and the red
cell appears contracted and spiculated.
 When the MCHC Increases beyond 37%, cytoplasmic viscosity increases and cellular deformability decreases. Rigid red cells are trapped in the spleen and
removed from the circulation. Peripheral blood smears demonstrate budding, fragments, microspherocytes, and bizarre red cell fragments.
 The osmotic fragility test is abnormal, especially after incubation. Autohemolysis is increased and the hemolysis is not corrected with glucose.

ERYTHROCYTE MEMBRANE DEFECTS

DISORDER INHERITANCE PROTEINS INVOLVED HEMATOLOGIC MANIFESTATIONS TREATMENT
HEREDITARY Autosomal dominant Ankyrin most common; B Spherocytes; increased osmotic fragility; majority have mild Folic acid; splenectomy for
SPHEROCYTOSIS (HS) (majority) spectrin, a spectrin, band to moderate anemia but may have exacerbations; autosomal severe cases
3 protein, others recessive cases more severe
Autosomal recessive HS
is usually due to
mutations in the a
spectrin gene
HEREDITARY Autosomal dominant a spectrin most common Eliptocytes; majority asymptomatic, without anemia; None in majority of cases
ELLIPTOCYTOSIS (HE) occasional severe anemia in Infancy
HEREDITARY Hereditary recessive A spectrin most! common Microcytosis, poikilocytosis, RBC fragments; moderate to Splenectomy
PYROPOLKILOCYTOSIS severe anemia
(HPP)
SPHEROCYTIC Autosomal dominant Unknown Moderate hemolytic anemia; elliptocytes and spherocytes on Splenectomy for severe
ELIPTOCYTOSIS smear; Increased osmotic fragilit cases
SOUTHEAST ASIAN Autosomal dominant Band 3 protein No hemolysis or anemia; increased resistance to malarial None
OVALOCYTOSIS infection

RHnull DISEASE (RH DEFICIENCY SYNDROME)

 is a rare hereditary disorder causing mild, at disease, compensated chronic hemolytic anemia.
 This disorder is associated with stomatocytosis, spherocytosis, and the deletion of all RhHr determinants induding the Landsteiner-Weiner (LW) antigen from the
red blood cells.
 Rhe cells are abnormally permeable to K and partly cornpensate for this leakiness and the resultant cation deficiency by reinforcing the number and activity of
Na-KATPase pumps.
 The abnormal red cell morphology may be related to the increased K* leak rate flooint The resulting anemia is mild but variable.
 Typically, the hemoglobin concentration is between 11 and 13 g/dL and reticulocytes are: moderately increased.
 Many of the red blood cells are spheroidal or stomatocytic.
 Hemoglobin F levels are often elevated.

ACANTHOCYTOSIS
 Dense contracted or spheroidal red blood cells with multiple thorny projections or spicules.
 Acanthocytes are prevalent in two very different constitutional disorders:
 abetalipoproteinemia and spur cell anernia.
 Abctatipoproteinemia is a rare derangement of lipid metabolism resulting from a genetic inability to synthesize apolipoprotein B (apos), the protein that coats
chylomicrons. Moderate anemia may develop in young children, but adults suffer from only mild anemia. MCV, MCH, MCHC, and osmotic fragility are normal.

SPUR CELL HEMOLYTIC ANEMIA

 This form of acanthocyte-associated hemolytic anemia is seen in patients with established alcoholic cirrhosis.
 Hemolysis usually becomes severe and may necessitate maintenance transfusions.
 In most patients, the hemoglobin concentrations level off at 5 to 7 g/dL.
 Reticulocyte counts fluctuate between 10% and 20%, and S.Cr-labeled red cell half-life survival may be as short as 5 to 6 days. .
 Other causes of acanthocytosis can include neonatal hepatitis, infantile pyknocytosis, theMalcod blood group, and severe malnutrition (e.g., anorexia nervosa).
 86%

NEUROACANTHOCYTOSIS (NA)
 is a heterogeneous group of neurodegenerative disordersassociated with acanthocytosis in peripheral blood.
 Clinically, NA is characterized by acombination of neurobehavioral changes.
 The cause of the disorder is unknown, but wo dlab recent evidence is increasing that the erythrocytic membrane is defective, with major KITA integral
membrane protein band 3 being reported in a few cases.

ERYTHROCYTIC ENZYME DEFECTS

o Erythrocytic enzyme defects are inherited.
o The anemias in this group are caused by deficiencies of;
1. Glucose-6-phosphate dehydrogenase (G6PD)
2. Pyruvate kinase (PK)
3. Methemoglobin reductase

GLUCOSE-6-PHOSPHATE DEHYDROGENASE (G6PD DEFICIENCY)

 The gene for G6PD is located on the X chromosome, so G6PD deficiency is inherited as an X-linked trait. As for all X-linked traits, men develop the disease, whereas
women are usually asymptomatic carriers. The female heterozygotes have an advantage of resistance to Falciparum malaria.
 Glucose 6-phosphate dehydrogenase (G6PD) is a housekeeping enzyme critical in the redox metabolism of all aerobic cells and the most common sarebic erythrocyte
enzyme defidency.
 Glucose-6-Phosphate Dehydrogenase is the first enzyme in the hexose monophosphate shunt, which is required to generate the reduced fom of nicotinamide adenine
dinucleotide phosphate (NADPH).
 NADPH is required for the regeneration of alutathione by the enzyme glutathione reductase.

G6PD Glucose6-phosphate + NADP —6-phosphogluconate +NADPH

 An excess intermediate product oxidized glutathione, accumulates in the red cell because of the absence of NADPH and forms Insoluble complexes with
haemoglobinthat result in Heinz best formation. This damages the cell, resulting in hemolysis.
 The aggregates of oxidized hemoglobin are plucked out of the cell by the spleen,resulting in characteristicblte or blister cells. - The level of G6PD is highest in
reticulocytes and declines with increasing age of thered cell.
 In normal cells, the half-life of the enzyme is approximately 62 days
 CLASSIFICATION OF G6PD VARIANTS BASED ON THE LEVEL OF ENZYME ACTIVITY AND THE SEVERITY OF HEMOLYSIS
AND ANEMIA:
Class I variants Severe enzyme deficiency (<10% of normal) and chronic hemolysis
Class 2 variants Severe enzyme deficiency, but intermittent instead of chronic hemolysis
Caucasians (G6PD Mediterranean)
Class 3 variants Mild to moderate enzyme deficiency (10-60% of normal), with intermittent hemolysis usually precipitated by infection or
oxidative drugs or chemicals
(African variant)
Class 4 variants Normal enzyme activity and no anemia or hemolysis
Class 5 variants Increased enzyme activity, without anemia or hemolysis
CLINICAL FEATURES: THE MAIN SYNDROMES THAT OCCUR ARE AS FOLLOWS.
1. Acute hemolytic anemia in response to oxidant stress, e.g. drugs, fava beans or Infections. The acute hemolytic anemia is caused by rapidly developing
intravascular hemolysis with hemoglobinuria.
2. Neonatal jaundice
3. Rarely, a congenital non-spherocytic hemolytic anemia.

MANIFESTATIONS:
1. Episodes of hemolysis are indicated by sudden onset of jaundice, pallor, dark urine, andabdominal or back pain.
2. The hemoglobin level typically drops about 3 to 4 g/dL.
3. The blood smear may show "bits" and "blister colls polychromasia, schistocytes, and microspherocytes.
4. Serum bilirubin and lactic dehydrogenase levels are increased;
5. haptoglobin is decreased.
6. A reticulocyte response becomes evident at about 5 days and is maximal at 10 days. Since neticulocytes have probective G6PD levels, hemolysis stops, and the
hemoglobin returns to normal after about 2 to 4 weeks even if the oxidative drug is continued Quantitatively decreased level of G6PD; a positive autohemolysis
test result, and The presence of Heinz bodies on peripheral blood smears prepared for Heinz body screening.
7. Heinz bodies are not visible on routinely stained, Wright stain preparations.

AGENTS THAT MAY CAUSE HEMOLYTIC ANEMIA IN GLUCOSE-6-PHOSPHATE DEHYDROGENASE (G6PD) DEFICIENCY
 Infections and other acute illness (e.g, Diabetic ketoacidosis)
 Fava beans (possibly other vegetables): Uncooked fava beans are a notorious cause of hemolysis in patients with G6PD Mediterranean (faviam).
 Drugs
UNSAFE FOR CLASS I, II AND III PROBABLY SAFE FOR CLASS II AND III
VARIANTS VARIANTS
1. Acetanilid 1. Acetaminophen
2. Furazolidine 2. Pyrimethamine
3. Nalidixic Acid 3. Aspirin
4. Naphthalene 4. Isoniazid
5. Nitrofurantoin 5. Ascorbic Acid
6. Phenazopyridine 6. Phenacetin
7. Phenylhydrazine 7. Chloramphenicol
8. Primaquine 8. Phenytoin
9. Sulfa antibiotics 9. Chloroquine
10. Thiazolsulfone 10. Qunidine
11. Toluldine blue 11. Diphenhydramine
12. Trinitrotoluene 12. Quinine
13. Doxorubicin 13. Vitamin K
PYRUVATE KINASE DEFIDENCY
 Is the second most common RBC enzymopathy and the most common enzyme deficiency in the Embden-Meyerhof (glycolytic pathway.
 However, it is far less common than G6PD deficiency.
 It occurs most commonly in people of Northern European and Mediterranean descent.
 It is inherited in an autosomal recessive manner.

PATHOPHYSIOLOGY
 The human PK-LR gene. codes for red cell PK.
 PK is essential in the Embden-Meyerhof pathway of anaerobic glycolysis.
 Mature erythrocytes lack mitochondria and are exclusively dependent on anaerobic glycolysis for the generation of ATP.
 Erythrocytes with PK deficiency generate less adenosine triphosphate (ATP) and NADH from glucose.
 There is deceased Nat K+-ATPase activity, with consequent cellular dehydration and result in cell shrinkage, distortion of the shape of the cell, and increased
membrane rigidity.
 The exact mechanism of hemolysis is unknown, but it is thought that there are abnormalities in membrane function. 2,3-Diphosphoglycerate (DPG) accumulates
in RBCs; since Increased 2,3-DPG facilitates 02 unloading,
 These changes subsequently lead to premature destruction of erythrocytes in the spleen and liver as well as hemolytic anemia.

CLINICAL MANIFESTATIONS
 The red calls become rigid as a result of reduced adenosine triphosphate formation.
 Patients with this disorder have elevated 2,3-DPG because of the abnormal enzyme block.
 The severity of the anemla varies widely (hemoglobin 4-10 g/dL) and causes relatively mild symptoms because of a shit to the right in the oxygen (02)
dissociation curve caused by a rise in Intracellular 2,3-diphosphoglycerate (2,3-DPG).
 Jaundice is usual and gallstones frequent. Frontal bossing may be present.
 The blood film shows polkilocytosis and distorted "price" cells, particularly post splenectomy
 Laboratory tests show that autohemolysis Is Increased but, in contrast to HS, it is not comected by alucoee; direct enzyme assay is needed to make the diagnosis.
 Splenectomy may alleviate the anemia but does not cure it and is indicated in those patients who need frequent transfusions. Neonatal hyperbilirubinemia is
common and may require exchange transfusion.
 Peripheral blood smears of patients with PK deficiency usually appear as normochromic, normocytic erythrocytes with varying degrees of polychromatophilia
(reticulocytosis)

METHEMOGLOBIN REDUCTASE DEFIDENCY

  Hemoglobin that is oxidized from the ferrous to the ferric valence state is called methemoglobin.
 Approximately 1% of circulating hemoglobin in normal individuals is methemoglobin.
 Hereditary deficiency of the enzyme NADH-methemoglobin reductase results in Increased levels of methemoglobin.
 A deficiency in the enzyme, also called NADH diaphorase, can result from inheritance of an autosomal recessive tralt or in conjunction with haemoglobin M
disease, or as a result of exposure to toxic substances or various drugs.
 The predominant clinical manifestation of methemoglobin reductase deficiencyis cyanosis because the methemoglobin cannot carry oxygen to the tissues.

OTHER RED BLOOD CALL ENZYME DEFICIENCIES;

GLUCOSEPHOSPHATE ISOMERERE DEFIDENCY: causes an abnormality in anaerobic glycolysis and is the third most common red blood cell enzyme deficiency
ENZYME INHERITANCE HEMATOLOGIC MANIFESTATIONS OTHER MANIFESTATIONS
Glucose phosphoisomerase Autosomal recessive Neonatal hyperbilirubinemia;
Hemolytic anemia of
variable severity
Gluthathione synthetase Autosomal recessive Hemolytic anemia Metabolic acidosis
Hexokinase Autosomal recessive Hemolytic anemia
Phosphofructokinase Autosomal recessive Hemolytic anemia Glycogen storage recessive
Aldolase Autosomal recessive Hemolytic anemia Glycogen storage recessive
Phosphoglycerokinase X-linked Hemolytic anemia Mental retardation; Myoglobinuria
Triosephosphate myoglobinuria Autosomal recessive Hemolytic anemia Progressive neurologic abnormalities
Pyrimidine 5 nudeotidase Autosomal recessive Hemolytic anemla with recessive prominent basophilic stippling
AOQUIRED HEMOLYTIC ANEMIAS
 The acquired hemolytic anemias (with one exception) Involve abnormalities that arecoctrinsic to the erythrocyte.
 The Paroxysmal Nocturnal Hemoglobinuria (PNH), which is an acquired genetic lesion consumption to the acquired = extrinsic rule is resulting in Increased
susceptibility of red blood cells (RBC) to hemolysis by the complement cascade.
 The clues that with an acquired hemolytic anemia are an increased reticulocyte count (or reticulocyte production index), increased bilirubin and lactic
dehydrogenase,and decreased haptoglobin

1. IMMUNE HEMOLYTIC ANEMIAS

The easiest way to approach the immune hemolytic anemias is to divide them according to the mechanism of hemolysis and the type of mediating antibody
 DIRECT COMPLEMENT-MEDIATED (INTRAVASCULAR) HEMOLYSIS VERSUS
PHAGOCYTOSIS BY MACROPHAGES OF THE RETICULOENDOTHELIAL SYSTEM (EXTRAVASCULAR HEMOLYSIS)

MECHANISMS OF IMMUNE HEMOLYSIS

DIRECT COMPLEMENT MEDIATED PHAGOCYTOSIS by MACROPHAGES of RES
Intravascular Extravascular
IgG or IgM antibody that fixes complement Mediated by IgG or complement on surface
Complement cascade completed through Predominantly occurs in spleen and/or liver
membrane attack complex (MAC; C5b-9)

 WARM-REACTIVE ANTIBODIES, USUALLY IMMUNOGLOBULIN G (IGG), VERSUS

COLD REACTIVE AND BODLES, USUALLY IGM

IgG VERSUS IgM ANTIBODIES

IgG ANTIBODIES (WARM) IgM ANTIBODIES (COLD)
(Usually warm reactive React at cooler temperatures; initiate complement sequence
May or may not fix complement Antibodies dissociate from RBC at warmer core temperatures; complement
components remain on RBC surface
Hemolysis usually actravascular; predominantly in spleen; liver is Hemolysis usually extravascuslar; predominantly in liver; spleen is usually a minor
usually a minor participant participant
Intravascular hemolysis is rare Intravascular hemolysis may occur
Foy receptors on splenic macrophages recognize IgG on RBC Complement receptors on Kupffer's cells recognize inactivated complement on RBC
surface surface (iC3b))
Presence of complement components Synergistic with IgG; more High-titer antibody may activate enough complement to complete complement
avid phagocytosis cascade, Initiate Intravascular hemolysis; Example: antibodies to A or B blood group
antigens

IMMUNE HAEMOLYTIC ANEMLA CAN BE FURTHER DIVIDED INTO:

AUTOLAMUNE HEMOLYTIC ANEMIAS
 Associated with warm-type antibodies
 Associated with cold-type antibodies
 Associated with both warm- and cold-type antibodies

ISOINMUNE HEMOLYTIC ANEMIAS

 Hemolytic Disease of the fetus and newborn (HDFN)
 Rh Incompatibility
 ABO Incompatbility

DRUG-INDUCED HEMOLYTIC ANEMIA

 Adsorption of immune complexes to red cell membrane
  Adsorption of drug to red cell membrane
 Induction of autoantbody to drugs
 Non-immunological adsorption of immunoglobulin to red blood cell membrane

MECHANISMS OF IMMUNE HEMOLYSIS

1. DIRECT COMPLEMENT MEDIATED (INTRAVASCULAR)
 Caused by IOM or IgG antibodies that fbx complement. The complement cascade must proceed to the terminal membrane attack complex (MAC; C5b - 9)
 Direct complement-mediated hemolysis is less common than hemolysis due to phagocytosis by macrophages of the reticuloendothelial system.
2. MACROPHAGES OF THE RETICULOENDOTHELIAL SYSTEM (EXTRAVASCULAR)
 Macrophages of the reticuloendothellal system have receptors for the Fc component of IgG (Fc receptors) and for complement components.
 The presence of IgG and complement on RBC surfaces results in complete or partial phagocytosis of the erythrocyte.
 Phagocytosis occurs primarily in the spleen and/or liver by splenic macrophages or Kupffer cells in the liver.

COLD-REACTIVE IMMUNE HEMOLYTIC ANEMIA

 Cold-reactive Immune hemolytic anemia is generally mediated by IgM antibodies that react minimally at approximately 4 to 18°C.
 IgM antibodies may cause intravascular hemolysis if the antibody is present in high titer; more often hemolysis is extravascular, predominantly in the liver.
 IgM antibodies are large and can bridge the distance between two RBCs; thus, IgM antibodies by themselves are able to agglutinate RBCs. (In contrast,
IgG antibodies are smaller and alone are not able to agglutinate RBCS.)
 There are many causes of cold-reactive immune hemolytic anemia. Most of these fall into the category of cold agglutinin disease. Cold agglutinin disease,
in turn, is divided into primary (Idiopathic) and secondary forms.
 Cold-reactive immune hemolytic anemia is less common than the warm-reacting type, accounting for approximately 10 to 20% of immune hemolytic
anemias.

CAUSES OF COLD-REACTIVE IMMUNE HEMOLYTIC ANEMIA

1. COLD AGGLUTININ DISEASE
a. Primary (Idiopathic)
b. Secondary
 Infections
 Autoimmune disorders
 Lymphoproliferative disorders
2. PAROXYSMAL COLD HEMOGLOBINURLA (PCH)
 Do not confuse cold agglutinins with a cryoglobulins. Cold agglutinins are antibodies that cause agglutination of erythrocytes at cold
temperatures.
 Cryoglobulins are antibodies that aggregate at cold temperatures (no erythrocytes involved).
A. PRIMARY COLD AGGLUTININ DISEASE
 Occurs with no obvious precipitating cause. It usually occurs in older individuals (peak age about 70 years) and is more common in women than men.
 The course is usually chronic, lasting for months or years. The anemia associated with idiopathic cold agglutinin disease is usually modest; the main symptoms
are related to agglutination of erythrocytes on exposure to cold rather than the anemia.
 Agglutination occurs in the fingers, toes, nose, and ears and causes cyanosis of those areas (acrocyanosis).
 Some patients have a lymphoproliferative disorder such as chronic lymphocytic leukemia (Cu), Waldenström's macroglobulinemia, or non-Hodgkin's lymphoma.
 The antibody is monoclonal, usually IgM with kappa light chain (IgM).
 Primary cold agglutinin disease thus represents a monoclonal gammopathy.

B. SECONDARY COLD AGGLUTININ DISEASE

 Cold agglutinin disease (CAD) is most often related to infections, primarily Mycoplasma pneumonine or Epstein-Barr virus (EBV) (infectious mononucleosis).
 It can occasionally occur with other viral infections (adenovirus, cytomegalovirus, rubella, mumps, HIV), bacterial infections (Legionella, Escherichia coli,
Listeria), malaria, syphilis, and others.
 The patients are usually young and otherwise healthy. The onset is abrupt, usually as the infection is resolving.
 The patients present with pallor, jaundice, and the other signs of acute hemolytic anemia.
 Massive acute intravascular hemolysis and acute renal failure may occur but are fortunately rare.
 The antibodies are polyclonal and usually directed against the "I/i blood group".
 The antibody is usually IgM and binds to red cells best at 4°C. IgM antibodies are highly efficient at fixing complement and both intravascular and extravascular
hemolysis can occur. Complement alone is usually detected on the red cells, the antibody having eluted off the cells in warmer parts of the circulation.
Interestingly, in nearly all these cold AIHA syndrome the antibody is directed against the "I" antigen on the red cell surface. In infectious mononucleosis it is
anti-i.

LABORATORY TESTING:
1. THE DIRECT ANTIGLOBULIN TEST (DAT OR DIRECT COOMBS' TEST)
 Tests for antibody or complement on the patient's RBCs.
 The direct Antiglobulin test uses the patient's cells and adds a reagent serum.
 It is performed by adding antibodies directed against human IgG, complement components, or both (the antiglobulin or Coombs' reagent) to the patient's
RBCs and seeing if the cells agglutinate.
 If there is IgG or complement on the surface of the RBCs, then the added antibodies will cause the cells to agglutinate (positive DAT). If there is no IgG or
complement on the RBC surface, the cells will not agglutinate.
 In cold agglutinin disease, there is complement on the patient's RBCS but no immunoglobulin.
2. THE ANTBODY SCREEN (INDIRECT ANTIGLOBULIN TEST OR INDIRECT COOMB’S TEST)
 Tests for unexpected anti-erythrocyte and bodies in the patient's serum. The patient’s serum is added to panels of reagent red cells.
 After the cells and serum have incubated, the cells are washed and an antiglobulin reagent is added. If there are unexpected antibodies in the patient's
serum, the reagent cells will agglutinate; otherwise, the reagent cells do not agglutinate. Unexpected antibodies are those that should not be present in a
normal person; for example, antibodies against the A or B blood group antigens would be expected in a person who lacks those antigens.
 Antibodies against other RBC agents would be unexpected.
 In cold agglutinin disease, there is usually an unexpected antibody in the patient's serum that reacts best at cold temperatures.
PAROXYSMAL COLD HEMOGLOBINURIA
 It is caused by a peculiar biphasic IgG antibody that reacts and fixes complement at cold temperatures. After rewarming, the complement cascade goes to
completion with formation of the membrane attack complex and intravascular hemolysis due to complement lysis.
  The antibody has been designated the Donath-Landsteiner antibody and is usually directed against the p blood group antigen. Syphilis used to be the most
common cause, but now most cases occur in children, and are related to viral infection (measles, measles vaccine, mumps, adenovirus, EBV,coplasma
pneumonia. Occasional cases are related to systemic lupus erythematosus (SLE).
 Paroxysmal cold hemoglobinuria is a relatively common cause of acute hemolytic anemia In children
Warning: Do not confuse paroxysmal cold hemoglobinuria (PCH) with paroxysmal noctural hemoglobinuria (PNH).

THREE CLINICAL FORMS OF PAROXYSMAL COLD HEMOGLOBINURIA:

1. An acute form that follows an Infection
2. A chronic form associated with tertiary or congenital syphilis
3. A chronic idiopathic form.

CLINICAL MANIFESTATIONS:
 Patients experience intermittent episodes of pain in the back, legs, or abdomen; fever; nausea; vomiting; and headache following exposure to cold. The plasma may be
red during the acute episode. The urine will be dark red or black, Clearing over a few hours.
 The anemla may be severe.
 The DAT is positive for complement but not for IgG The diagnosis is confirmed by the Donath-Landsteiner test.
 The patient's serum is incubated in ice water with group, P+ erythrocytes and fresh normal serum. The mbcture is warmed to 37°C, and if the cells hemolyze on
rewarming, the test is positive.

WARM-REACTIVE IMMUNE HEMOLYTIC ANEMIA

 More common than the cold reactive varlant (at least 70% of Immune hemolytic anemias).
 It can be primary (Idiopathic), secondary to a wide varlety of different conditions, or drug related.
 The Idiopathic variety is more common in women and the older population.

PATHOPHYSIOLOGY
 The antibody is usually a “penaqgglutiinn," meaning that the patient's antibody will react with virtually all cells.
 In most cases, the antibody appears to be recognizing some antigen in the Rh blood group system, although it is usually impossible to define a specific antigenic reactivity.
 The antibody is almost always an IgG) Occasionally, an IgA or IgM antibody will be seen along with the IgG or, exceptionally, alone. When the cells are coated with IgG
and complement (C3d, the degraded fragment of (3) or complement alone, red cell destruction occurs more generally in the RE system.
 Red cell destruction is primarily by phagocytosis by splenic macrophages In many cases, the phagocytosis is partial rather than complete;

CLINICAL FEATURES
- The disease may occur at any age, in both sex and presents as a hemolytic anemia of varying severity.
- The spleen is often enlarged. The disease tends to remit and relapse.
- It may occur alone or in association with other diseases, or arise in some patients as a result of methyldopa therapy.
- When associated with idiopathic thrombocytopenic purpura (ITP), which is a similarcondition affecting platelets, it is known as Emmans' syndrome.

CAUSES OF WARM-REACTIVE IMMUNE HEMOLYTIC ANEMIA

1. Primary (Idiopathic)
2. Secondary
a. Infections
b. Autoimmune disorders: SLE, Rheumatoid arthritis
c. Lymphoproliferative disorders: CLL, non-Hodgkin's lymphomas, myeloma
d. Hodgkin's lymphoma
e. Thymoma
f. Ovarian dermoid cyst or teratoma
g. Carcinomas
h. Hypogammaglobulinemia
I. AIDS
3. Drug related

LABORATORY FINDINGS
 Spherocytosis prominent in the peripheral blood. The DAT is positive as a result of IgG,
 IgG and complement or IgA on the cells. The autoantibody shows specificity within the Rhesus system.
 The antibodies both on the cell surface and free in serum are best detected at 37°C.

DRUG-RELATED IMMUNE HEMOLYTIC ANEMIA

 Drugs and medications are common causes of immune hemolytic anemia (~10–20% of cases).
 The hemolysis is almost always of the warm-reactive type.
 It is common for patients on a variety of medications to develop a positive DAT;
 However, actual hemolysis is rare.

THREE MECHANISMS OF DRUG-RELATED IMMUNE HEMOLYTIC:

1. DRUG ADSORPTION (PENICILIN) TYPE
- The drug binds tightly to the RBC surface, and an antidrug antibody reacts with the drug that is bound to the red cell.
- The DATs should for IgG, with or without complement.
- Hemolysis is usually extravascular in the spleen; rarely, there may be intravascular hemolysis if the antibody fixes complement.
- The hemolysis is usually subacute, not severe.

2. NEOANTIGEN (FORMERLY CALLED IMMUNE COMPLEX) TYPE

- There is a complex of drug and antidrug antibody, which binds to an antigen on the red cell.
- The antibody can be elther IgM or IgG and is often complement foxing. The antibody often has relatively low avidity; it binds to the complex on the cell
membrane, foxes complement, and then dissociates from the cell surface.
- The DAT is therefore positive for component but usually not for immunoglobulins.
- Hemolysis is usually intrarmeanlex, often sudden and severe, and maybe associated with acute renal failure. The reaction may occur with low doses of the
medication.
- Medications that can cause this type of reaction include cephalosporin antibiotics, quinine, quinidine, and sobophen.

3. AUTOIMMUNE (METHYLDOPA) TYPE

- Methyldopa (Aldomet) is capable of inducing an autoimmune reaction.
- The antibody is directed against a red cell antigen, not against the drug itself.
 - The characteristics are similar to those of idiopathic warm-reactive immune hemolytic anemia.
- The DAT is positive for IgG with or without complement and may be positive even in the absence of the drug.
- The antibody screen may be positive. A similar reaction can be seen with levodopa and procainamid

2. NON-IMMUNOLOGIC ACQUIRED HEMOLYTIC ANEMIAS

A. MECHANICAL TRAUMA
1. MALFUNCTIONING MECHANICAL HEART VALVE
- Erythrocytes are crushed by the valve leaflets as they close, resulting in fragmentation and a chronic intravascular hemolysis.
- Hemoglobin is filtered by glomeruli and phagocytized by renal tubular epithelial cells.
- The iron is converted to hemosiderin and eventually lost as the tubular epithelial cells are shed into the urine; this may result in iron deficiency.
- The diagnosis can be made by noting a heart murmur from the malfunctioning valve, schistocytes on blood smear, and a positive urine hemosiderin test.
- The treatment is iron supplementation and replacement of the malfunctioning valve if necessary.
2. MARCH HEMOGLOBINURIA
- Can occur during long marches or marathons, resulting from erythrocytes being crushed in the capillaries of the soles of the feet as they pound against the
surface.
- This can be prevented by wearing thicker socks and softer-soled shoes
3. MLCROANGIOPATHIC HEMOLYTIC ANEMLAS
- (Sometimes called thrombotic maroangiopathles): thrombotic thrombocytopenic purpura (TTP), hemolytic-uremic syndrome (HUS),
preeclampsia/eclampsia, malignant hypertension, disseminated intravascular coagulation (DIC)
- Fibrin strands form within the capillaries, slicing erythrocytes into fragments as they pass through.

B. ACANTHOCYTOSIS
1. HEREDITARY ABETALIPOPROTEINEMIA.
2. END-STAGE LLVER DISEASE
3. SEVERE STARVATION, ANOREXIA NERVOSA

C. SEVERE HYPOPHOSPHATEMIA
1. INTRAVENOUS HYPERALIMENTATION LACKING PHOSPHOROUS SUPPLEMENTATION
2. SEVERE STARVATION
3. ALCOHOLISM
4. PROLONGED THERAPY WITH PHOSPHATE-BINDING ANTACIDS

D. WILSON'S DISEASE; COPPER POISONING

- Due to a mutation in the gene for ceruloplasmin, a copper transport protein.
- Patients accumulate excessive amounts of copper in their tissues, particularly the liver.
- Some patients with Wilson's disease develop an abrupt onset of hemolysis due to sudden release of copper from the liver.
- Excess Inorganic copper damages RBC membranes, disrupts cellular metabolism, and accelerates the oxidation of hemoglobin, all of which result in decreased
cell survival.
- Hemolytic episodes tend to occur relatively early in the course of Wilson's disease, often in the patient's twenties, and may be the initial symptomatic
manifestation of the disease. Hemolytic episodes are usually transient and self-limited but may be recurrent and severe.

E. OXIDATIVE DRUGS OR CHEMICALS

- Oxidative drugs and chemicals may cause hemolysis in people with apparently normal erythrocytes, as well as in patients with G6PD and other enzyme
deficiencies.
- Examples of drugs that have been implicated include sulfonamides, phenazopyridine (Pyridium), nitrofurantoin (Furadantin), phenacetin, and cisplatin.
- Chemicals that have been implicated in hemolysis include chlorates, nitrates, naphthalene (mothballs), methylene blue and others.
- Treatment is to stop the drug or exposure and provide support as necessary.
F. SEVERE BURNS
G. VENOMS
1. BROWN RECLUSE SPIDER
2. SNAKES (COBRAS)

H. INFECTIONS
1. DIRECT INFECTION OF ERYTHROCYTES: MALARIA, BABESIASIS, BARTONELLOSIS, TRYPANOSOMIASIS
2. CLOSTRIDIUM PERFRINGENS SEPTICEMIA
3. OTHER: GRAM-POSITIVE AND GRAM-NEGATIVE SEPTICEMIA, LEPTOSPIROSIS, BORRELIA, OTHERS

I. PAROXYSMAL NOCTURNAL HEMOGLOBINURIA (PNH)

- PNH is a rare, acquired, clonal disorder of marrow stem cells in which there is deficient synthesis of the glycosylphosphatidylinooltel (GPI) anchor, a structure
that attaches several surface proteins to the cell membrane.
- It results from mutations in the X chromosome gene coding for phosphatidylinositol glycan procain A (PIG-A) which is essential for the formation of the GPL
anchor.
- The lack of surface molecules decay-activating factor (DAF, CD55) and membrane Inhibitor of reactive hyals (MIRL, CD59) render red cells sensitive to lysis
by complement and the result is chronic intravascular hemolysis.
- Chronic intravascular hemolysis of PNH is mediated by the alternative pathway of complement.
- PNH expresses itself with hemolytic anemla, bone marrow failure, and thrombosis. Intravascular hemolysis in this disorder is characterized by Intermittent
(paroxysmal) sleep associated (nocturnal) blood in the urine (hemoglobinuria).
- Hemosiderinuria is a constant feature and can give rise to iron deficiency which mayexacerbate the anemia.
- CD55 and CD 59 are also present on white cells and platelets.

DIAGNOSIS
- The CBC shows anemia, which can vary from mild to severe.
- The anemia is usually mildly macrocytic but can be microcytic and hypochromic If iron deficiency has developed.
- Leukopenia and/or thrombocytopenia are also common. Lactic dehydrogenase and bilirubin may be elevated during exacerbations of hemolysis.
- A test for urine hemosiderin will be positive.
- The bone marrow examination in PNH generally shows erythroid hyperplasia; bone marrow cellularity is usually increased but can also be normal or decreased.
- The traditional diagnostic tests are the added serum (Ham's) best and the sucrose hemolysis text.
- Both tests depend on activating the complement system and demonstrating increased sensitivity of the cells to complement-mediated lysis.
- The sucrose hemolysis test is more sensitive, but the Ham's test is more specific.
- Demonstration of a decreased expression of GPI-anchored proteins on RBCs or leukocytes by flow cytometry has been shown to be more sensitive than either of
these tests.
ALLOIMMUNE HEMOLYTIC DISONSE OF THE NEWBORN
DEFINITION OF TERMS:
 ISOIMMUNIZATION/ALLOIMMUNIZATION
- the development of specific antibodies as a result of antigenic stimulation using material derived from the red blood cells of another individual of the
same species.
- An immune response generated in an individual or strain of one species by an alloantigen from a different individual or strain of the same species.

 ISOANTIGEN - an antigen existing in alternative (allelic) forms, thus inducing an immune response when one form is transferred to members who lack it; typical
Isoantigens are the blood group antigens; also called alloantioen.

 AUTOANTIGEN - Immunology, Any self-antigen

Example:
1. Glomerular basement membrane
2. Mitochondria,
3. Muscle,
4. Parietal cells,
5. Thyroglobulin
6. others, which may evoke production of autoantibodies

 SENSITIZATION - administration of an antigen to induce a primary immune response; exposure to allergen that results in the development of hypersensitivity.

ALLOIMMUNE HEMOLYTIC DISEASE OF THE FETUS AND NEWBORN (HDFN)

- Caused by the action of transplacentally transmitted maternal Immunoglobulin (Ig) G antibodies on paternally inherited antigens present on fetal red cells but absent on
the maternal red cells.
- Matemal IgG antibodies bind to fetal red cells, causing hemolysis.
- As a consequence of the hemolytic process, anemia, extramedullary hematopoiesis, and neonatal hyperbilirubinemla sometimes result in fetal loss or neonatal death or
disability.
- The discovery of the rhesus (Rh) factor by Landsteiner and Weiner in 1940 led to further elucidation of the condition by Levine and colleagues, who established that
erythroblastosis fetalis was caused by Immunization of an Rh-negative mother by the red blood cells from an Rh-positive fetus.
- Antibodies produced by the sensitized mother crossed the placenta in the next pregnancy and coated the fetal Rh-positive cells, leading to hemolysis and thus to
anemia, hydrops, and severe neonatal jaundice secondary to hemolysis.

RANDOM HEMATOLOGY BOARD EXAMINATION RECALLS

1. The size of the blood drop used for smear preparation: (2-3mm)
2. The distance of the blood drop for the edge of the label: (0.25 inches)
3. Right shift (decreased affinity to O2) is associated with: (increased body temperature, 2,3-DPG, CO2 and decreased blood pH)
4. Left shift (increased affinity to O2) is associated with: (decreased body temperature, 2,3- DPG, CO2 and increased blood pH)
5. Microcytic RBCs are associated with: (Chronic Disease, Iron Deficiency Anemia and others)
6. Macrocytic RBCs are associated with: (Vitamin B12 Deficiency, Folic Acid Deficiency and others)
7. PK (prekallikrein) is detected through: (Activated Partial Thromboplastin Time (APTT))
8. What is the effect of kaolin contaminated with thromboplastin in PTT: (shortened PTT)
9. Size of the unfilled portion of capillary tube in microhematocrit: (10-15 mm.)
 10. Length of capillary tube: (75mm)
11. Length of plug in capillary tube: (4-6mm)
12. What is the cell that is seen with nuclei with demarcating membrane: (Promegakaryocyte)
13. Bone marrow aspiration is performed in: (sternum, tibia and iliac crest)
14. Rouleaux formation is seen in: (Multiple Myeloma, Macroglobulinemia, Hyperparaproteinemia)
15. What is seen in 2nd Trimester of pregnancy?: (Neutrophillia)
16. What Factor group is consumed during coagulation?: (Thrombin group)
17. Degree of Hypochromia measured as 1/3 is termed as: (Normal)
18. RBC with reference to size: (Microcytosis, Anisocytosis, Macrocytosis)
19. Not used in actual RBC description: (Hyperchromia)
20. Stem Cell to blast 5 days, Lifespan with tissue phase 9-10 days. Given the characteristics, what is the cell?: (Granulocytes)
21. Stem Cell to blast 5 days, 8-11 days lifespan. Given the characteristics, what is the cell?: (Thrombocytes)
22. Length of needle usually used in routine phlebotomy: (1.0-1.5 inches)
23. Most preferred site of puncture[vein]: (median cubital)
24. Angle of needle for extraction is?: (15-⁰)
25. Gauge number usually used for phlebotomy: (19,20,21)
26. Gauge in tuberculin syringe: (25)
27. Gauge of needle used in bleeding of donors in blood collection centers: (16)
28. Size of the drop of blood used in smear preparation: (2-3 mm); *2-3cm is the measure ofsmear for AFB staining [Microbio.]
29. Length of needle usually used in routine phlebotomy: (1.0-1.5 inches)
30. Most preferred site of puncture[vein]: (median cubital)
31. Angle of needle for extraction is?: (15-⁰)
32. Gauge number usually used for phlebotomy: (19,20,21)
33. Gauge in tuberculin syringe: (25)
34. Gauge of needle used in bleeding of donors in blood collection centers: (16)
35. Size of the drop of blood used in smear preparation: (2-3 mm); *2-3cm is the measure of smear for AFB staining [Microbio.]
36. Diurnal variation in blood cells is observed in: (Neutrophils; decreased in AM, increased in PM)
37. MCV is computed from: (RBC count and Hematocrit)
38. STUDY PLATELET ESTIMATES!!
39. ESR in Wintrobe tube is read using: (Left graduation, Top is zero)
40. Disposable ESR plastic tubes is called: (Dispette)
41. Leukemia without maturation is also known as: (M2)
42. Measure of erthropoiesis: (Reticulocyte count)
43. Effect of increased Hemoglobin in ESR: (increased ESR)
44. In DIC the D-dimer test would yield (+) result after how many hours?: (4 hours)
45. Presence of blood clot will have what effect on RBC count using automated counters?: (decreased)
46. Primary (azurophillic) granules appear in what stage?: (Promyelocyte)
47. In what stage can you identify a specific WBC?: (Myelocyte)
48. Thalassemia is a disease associated with: (quantitative defect in Hemoglobin)
49. Hemoglobinopathy is a general term for the ___________ defect in Hemoglobin.: (Qualitative)
50. What type of hematocrit method is performed using Wintrobe tube?: (macro Hct)
51. Horn like/ Helmet like cells: (keratocyte)
52. Sickle RBCs are also called as: (drepanocyte)
53. . Over-anticoagulated blood has this effect on ESR and Hematocrit: (both values are decreased)
54. Identify the cell. Large cell with nuclei, without budding thrombocyte, with small reddish blue granules: (Megakaryocyte)
55. Leukocytosis happens in non-pathologic conditions such as: (Smoking, Stress, Emotionalchanges, after eating)
56. Mode of action of Heparin as anticoagulant: (anti-thrombin)
57. Cells also called as immunocytes: (B-cells)
58. Lavander top tube in phlebotomy contains: (EDTA)
59. Cells present in acute inflammation: (Neutrophils)
60. Cells producing antibodies: (B-cells, Plasma Cells)
61. Cells responding to tissue invading parasitic infection and allergy: (eosinophils)

Effects of Different Factors in Smear

Preparation:
Thin Smear Thick Smear
Factors
Preparation Preparation
Pressure ↑ ↓
Angle ↓ ↑
Size of Blood
↓ ↑
Drop
Speed ↓ ↑

Effects of Different Factors in Smear Preparation:

Westergren Wintrobe
300 mm 115mm (11.5cm)
Graduation lines= 0-200 0-100
Used for ESR and
Used for ESR ONLY
macrohematocrit
Characteristics of DIC (Disseminated Intravascular
Coagulation)
Secondary to infection or sepsis
N. meningitidis (meningococci) –Waterhouse Friedrichsen
Syndrome
D-dimer positive within 4hrs after DIC onset
Decrease fibrinogen within 4-24 hours after onset of DIC
Decrease platelet count after 48 hours after DIC onset

Association of RBC shape variants

RBC shape variant Disease Association
Acanthocyte Mc Leod phenotype
Spur cells Abetalipoproteinemia
Dacryocyte Splenomegaly (Fibrosis of splenic process)
Stomatocytes Rh null cells
Codocytes (Target cells/ leptocyte) Thalassemia

Poikilocytosis Grading in Smears

Poikilocyte per Effects of different conditions in RBC count,
Grading Hematocrit and Hemoglobin
OIO Field
0-2 Within normal limits Age ↑RBC, Hct, Hb
3-10 1+
Gender ↑ in males
10-20 2+
20-50 3+ Smoking ↑
>50 4+

NCCLS Approved Order of Draw for Evacuated Tubes

Order Tube Color-Test Additive
1 Yellow- Microbio. culture SPS
2 Light Blue- Coagulation study 3.2% Sodium Cirate
Non-additive or Silica
3 Red-Routine Chemistry
particles
4 Green-Blood Gas Analysis Heparin
Lavander-Routine
5 EDTA
Hematology
6 Gray-Glucose Test Sodium fluoride

Classification of Acute Leukemia

M0 Acute Undifferentiated Leukemia
Acute Myeloblastic Leukemia without
M1
Maturation
M2 Acute Myleblastic Leukemia with Maturation
Acute Promyelocytic Leukemia (assoc. with
M3
DIC)
M4 Acute Myelomonocytic Leukemia (Naegeli)
M5 Acute Monocytic Leukemia (Schillings)
M6 Acute Erythroleukemia (Di-Guglielmo)
M7 Acute Megakaryocytic Leukemia
HEMOLYTIC ANEMIAS
HEMATOPOIETIC AND LYMPHOID NEOPLASMS
MAJOR CHARACTERISTICS:
1. Aggressiveness - Acute vs. Chronic (Survival and maturation)
 Acute
o Lymphoid – Acute Lymphoblastic Leukemia
According to cell lineages:
 T and B Lymphocytes
o Myeloid – Acute Myeloid Leukemia (acute non-lymphoblastic leukemia)
According to cell lineages:
 Myeloblast
 Promyelocyte
 Monoblast
 Normoblast
 Megakaryoblast
 Chronic
o Lymphoid- Chronic Lymphocytic Leukemia
According to cell lineages:
 T and B Lymphocytes
 Plasma Cells
o Myeloid- Chronic Myeloproliferative Disorders
According to cell lineages:
 Myelocytes
 Metamyelocyte
 Stabs
 Neutrophils, Eosinophils, Basophils, Monocytes
 Red blood cells
  Platelets
2. Lineage- Lymphoid vs. Myeloid
o Myeloid - CFU–L and CFU-GEMM
o Lymphoid – Neoplasms derived from CFU- L
3. Predominant site of involvement
o Blood/Bone Marrow - Leukemia
o Tissue- Lymphomas (Lymphocytes)
o Granulocytic sarcoma (Myeloid Cells)
LYMPHOMAS, HODGKIN’S DISEASE, and MYELOMAS
Primary Tumors of lymph nodes are:
1. Non- Hodgkin’s Lymphomas- derived form lymphocytes either T or B cells.
Older classifications Systems:
 Rappaport
 Working Formulation
 Updated Kiel
Newest Classification Systems:
 Revised European American Lymphoid (REAL)
 World Health Organization (WHO)
2. Hodgkin’s Lymphoma – presence of a characteristic cell called a Reed- Sternberg cells (a malignancy of lymphocytes)
Older classifications Systems:
 Rye
Newest Classification Systems:
 World Health Organization (WHO)
Tumors of Plasma Cells:
1. Myeloma – used for plasma cells. Tumors in the bone marrow.
2. Plasmacytoma – usually used for plasma cells. Tumor outside the bone marrow.
CHRONIC MYELOPLORIFERATIVE DISORDERS
1. Chronic myelogenous leukemia- Chronic granulocytic leukemia (Granulocytes)
2. Polycythemia vera - Polycythemia rubra (Erythrocytes)
3. Primary thrombocythemia – Essential Thromocythemia (Megakaryocytes)
4. Idiopathic myelofibrosis – Gynogenic myeloid metaplasia (Megakaryocytes that drives fibroblasts to make reticulin fibers.
MYELODYSPLASTIC SYNDROMES – also referred as “Preleukemia” or “Smoldering Acute Leukemia”
1. Impaired mutation and differentiation – Clonal disorders of Hematopoietic stem cells = Ineffective Hematopoiesis.
2. Decreased number of cells having abnormal appearance (dysplastic) and possibly function.
3. Evolves into Acute Leukemia
Sub-classification:
A. Refractory Anemia
1. With ringed sideroblasts (RARS) COMPARISON OF CHARACTERISTICS OF ACUTE AND CHRONIC
2. Without ringed sideroblasts (RA) LEUKEMIA
B. Refractory Cytopenia with Multilineage Dysplasia (RCMD) CHARACTERISTIC ACUTE LEUKEMIA CHRONIC LEUKEMIA
C. Refractory Anemia with Excess Blasts (RAEB) ONSET Abrupt Gradual
MYELODYSPLASTIC- MYELOPLORIFERATIVE DISORDERS MORBIDITY Months Years
The diseases in this category are: AGE All Adult/ Older -60 years old
1. Atypical Chronic Myelogenous Leukemia (aCML) WBC Depends - variable Increase WBC
2. Juvenile Myelomonocytic Leukemia (JMML) PREDOMINANT Blast, Immature cells Matured
3. Chronic Myelomonocytic Leukemia (CMML) CELLS
LEUKEMIA THROMBOCYTOPEN Present Variable
 Develops subsequent to the malignant transformation IA
 Mutation and the altered expression of the specific genes NEUTROPENIA Present Variable
 Capable of proliferation and self-renewal ORGANOMEGALY Mild Marked
 Translocations are the most consistent and specific chromosomal
abnormalities that are found in human leukemia cells
 Bennett (Scotland) Virchow (Germany) – assigned the term “weisses blut” (White blood)
Factors related to the occurrence of the Leukemia:
1. Genetic and immunological factors (translocations) 4. Genetic abnormalities and associations
2. Occupational and environmental exposure 5. Viral agents
3. Chemical and Drug exposure 6. Secondary causes
ACUTE LEUKEMIA
1. ACUTE MYELOGENOUS LEUKEMIA
 Most common leukemia in the first months of life, increased also in the middle and later years.
 >30% (FAB) – of all nucleated cells in BM are myeloblast.
 >20% (WHO) – of all nucleated cells are myeloblast.
Clinical Findings in Acute Leukemia:
PATHOGENESIS:
A. Bone Marrow Infiltration
1. Neutropenia – fever, malaise, lethargic, paleness
2. Anemia
3. Thrombocytopenia – Ecchymosis, hematoma, bleeding, petechiae, haemorrhage
B. Medullary Infiltrations
1. Marrow (Arthalgia)
C. Extramedullary infiltration
1. Liver, Spleen, Lymph nodes, Thymus (Organomegaly) – mild
2. Central nervous system – vomiting, headaches
3. Gums, mouth – gingival bleeding
4. Skin – granulocytic sarcoma (lesion)

WHO CLASSIFICATION OF ACUTE LEUKEMIA

1. Acute myeloid leukemia with recurrent genetic abnormalities
2. Acute myeloid leukemia with multi-lineage dysplasia
3. Acute myeloid leukemia and myelodysplastic syndromes (therapy related)
4. Acute myeloid leukemia (not otherwise categorized)
 Acute myeloid leukemia (minimally differentiated)
 Acute myeloid leukemia (without maturation)
 Acute Myeloid leukemia (with maturation)
 Acute Myelomonocytic Leukemia
 Acute monoblastic and monocytic Leukemia
 Acute erythroid leukemia
  Acute megakaryoblastic leukemia
 Acute basophilic leukemia
 Acute panmyelosis with myelofibrosis
 Myeloid sarcoma
5. Acute leukemia of ambiguous lineage
 Undifferentiated Acute Leukemia
 Mixed phenotype acute leukemia with t(9;22)
 Mixed phenotype acute leukemia with (v;11q23)
 Mixed phenotype acute leukemia (B lymphocytes myeloid cells)
 Mixed phenotype acute leukemia (T-myeloid)

FRENCH, AMERICAN, BRITISH (FAB) – based on morphologiocal characteristic of Wright-stained cells in

Peripheral blood and bone marrow with supplementary cytochemical stains.
DESIGNATIO DESCRIPTIVE NAME
N
M0 Acute Myeloblastic Leukemia
M1 Acute Myeloblastic Leukemia without maturation
M2 Acute Myeloblastic leukemia with maturation
M3 Acute Promyelocytic Leukemia (Hypergranular)
M3b Acute Promyelocytic Leukemia (Mircogranular)
M4 Acute Myelomonocytic Leukemia aka Naegli-Type Monocytic Leukemia
M4Eo Acute Myelomonocytic Leukemia with Eosinophilia
M5a Acute Monoblastic Leukemia (poorly differentiated)
M5b Acute Monoblastic Leukemia (with differentiated)
M6 Erythroleukemia Diguglielmo
M7 Acute Megakaryoblastic Leukemia
L1 Acute Lymphoblastic Leukemia (Children)
L2 Acute Lymphoblastic Leukemia (in older children and adult)
L3 Acute Lymphoblastic Leukemia (Burkitt’s Lymphoma)

ACUTE MYELOID LEUKEMIA WITH RECURRENT GENETIC ABNORMALITIES

T(8;21) (q22;q22) Acute Myeloid Leukemia
inv(16) (p13q22) Acute Myeloid Leukemia with abnormal bone marrow Eosinophils
t(16:16) (p13;q22)
T(15:17) (q22;q22) Acute Promyelocytic Leukemia
C11q23 Acute Myeloid Leukemia
8;14 Acute Lymphoblastic Leukemia (Burkitt’s Lymphoma)
8:21 Acute Myeloblastic leukemia with maturation
t(9;22) Mixed phenotype acute leukemia
(v;11q23) Mixed phenotype acute leukemia

ACUTE MYELOGENOUS LEUKEMIA CYTOCHEMICAL STAINING

CYTOCHEMICAL STAIN CELLS
Peroxidase Myeloid (Myeloblast, Promyelocyte, Neutrophils)
Sudan B
Naphthol ASD Chloroacetate Myeloid (Myeloblast, Promyelocyte, Neutrophils)
A-Naphytl Acetate Monocytic (Myeloblast, Promyelocyte, Neutrophils)
A-Naphtyl Butyrtate
PAS Erythroblast, Megakaryoblast
Acid Phospahtase

2. ACUTE LYMPHOBLASTIC LEUKEMIA

 Most common of all leukemia
 Predominant in Children (Ages 2-10 years old)
 Patient may have:
 Fatigue
 Bleeding
 Febrile with Lymphadenopathy
 Splenomegaly
 Hepatomegaly
 CNS involvement and social infiltration
 Mutation of lymphoid precursor cells have their orgin in the marrow and thymus, at a particular stage of maturation
 Accumulate in intramedullary and extramnedullary sites
 Anemia, Thrombocytopenia, Neutropenia, Over-population of Lymphocytes in tissues as well.
 WBC Count: >100x109/L

FAB MORPHOLOGICAL CLASSIFICATION OF ACUTE LYMPHOBLASTIC LEUKEMIA

1. L1 - ACUTE LYMPHOBLASTIC LEUKEMIA (CHILDREN)

 Cell size: Small, Regular
 Nuclear Chromatin: Fine or condensed
 Nuclear Shape: Regular, cleft or indentation possible
 Nucleoli: Indistinct
 Cytoplam: Scant
2. L2 - ACUTE LYMPHOBLASTIC LEUKEMIA (IN OLDER CHILDREN AND ADULT)
 Cell size: Large, mixed sizes
  Nuclear Chromatin: Fine or condensed
 Nuclear Shape: Irregular, cleft or indentation more common
 Nucleoli: 1 or 2 prominent
 Cytoplam: Variable, often moderately abundant
3. L3 - ACUTE LYMPHOBLASTIC LEUKEMIA (BURKITT’S LYMPHOMA)
 Cell size: Large
 Nuclear Chromatin: Fine
 Nuclear Shape: Regular, round or oval
 Nucleoli:1 or 2 prominent
 Cytoplam :Deeply basophilic, vacuolated

WHO CLASSIFICATION OF ACUTE LYMPHOBLASTIC LEUKEMIIA

1. PRECURSOR B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA (ALL)

 Congenetic subgroups (with oncogenes involved);
o t(9;22)(q34;q11); BCR/ABL – Philadelphia chromosome
o t(v;11q23); MLL rearranged (MLL=myeloid-lymphoid leukemia gene)
o t(1;19)(q23;p13);E2a/PBXI
o t(12;21)(p12;q22); TEL/AMLI
 Characteristically express B cell- associated antigens such as CD19 and CD20
 Most cases express CD10 (cALLA – Common Acute Lymphoblastic Leukopenia Antigen):
- CD34 (human progenitor cell antigen)- is frequently expressed
 Cell surface immunoglobulin expression is absent, but there may be MU (heavy chain) present in the cytoplasm.
 Terminal deoxynucleotidyl transferase (TDT), a nuclear marker is often present.

2. PRECURSOR T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA

 Characteristically expresses T-cell antigen such as CD2. CD5 and CD7.
 CD1a may be present- CD4 and CD8 (the helper and T suppressor subset antigens are characteristically either absent or both expressed.
 CD3 may be present in cytoplasm but absent from the cell surface.

3. MATURE B-CELL (BURKITT-CELL LEUKEMIA)

 Characteristically by the presence of cell surface immunoglobulin, with the light chain restriction
(Either kappa or lambda light chain, but not a normal mixture of both) and B-cell antigens such as CD 19 and CD 20
 CD10 (cALLA) may be present or absent

DIFFERENTIATION BETWEEN AML & ALL

ACUTE MYELOID LEUKEMIA ACUTE LYMPHOID LEUKEMIA
AGE Adult Children
BLOOD Myeloblast, Promyelocyte Lymphoblast
MORPHOLOGY (+) rods – peroxidase (-)
CYTOCHEMIST (+) Sudan Black B (-) Sudan Black B
RY
LINEAGE (-) TDT (+) TDT

LYMPHOPROLIFERATIVE DISORDERS AND PLASMA CELL DISORDERS

Lymphoid malignancies:
- Chronic Lymphocytic Leukemia/ Small Lymphocytic lymphoma (SLL)
- Hairy Cell Leukemia
- Sezary syndrome (Leukemic phase of mycosis fugoides)
- Prolymphocytic Leukemia
- Hodgkin’s and Non- Hodgkin’s Leukemia
Plasma malignancies: - is a complication of multiple myeloma, seen late in the progression of the disease as Plasma cells overtake the normal BM Elements
- Multiple Myeloma (MM)
- Waldenstrom’s Macroglobulinemia

CHRONIC LYMPHOCYTIC LEUKEMIA/ SMALL LYMPHOCYTIC LYMPHOMA (SLL)

o Proliferation of B lymphocytes
o It is the common chronic leukemia with a prediction for men over women
o It is often discovered by accident, as a result of other complaints
o The WBC counts are exaggerated with many over 100,000x109/L
o The M:E ratio is 10 or 20:1
o May experience fatigue, weight loss, paleness
o Erythroid hyperplasia
o Direct antiglobulin test (DAT) (+)
o Chromosomal abnormalities: mostly found often by Fluorescence in situ hybridization (FISH)- deletions of 13q
o Has a highly characteristic immunophenotype: expression of B-cell markers (CD19,CD20, CD23, CD5)
Features:
 Predominantly older age group
 Lymphadenopathy and splenomegaly RAI STAGING FOR CHRONIC LYMPHOCYTIC LEUKEMIA
 Lymphocytosis (>5,000/L) STAGIN MODIFIED RAI STAGING
 Predisposition to Infection G
 Autoimmune phenomena, particulary autoimmune haemolytic anemia 0 Bone marrow and blood Low Risk Stage 0
 Transformation to large cell lymphoma (Ritcher’s syndrome) lymphocytosis
 Immunophenotype 1 Lymphocytosis with enlarge
nodes
 CD5 expression
2 Lymphocytosis with enlarged Intemediate Risk Stage 1
 Immunoglobulin light chain and CD20 spleen or liver or both &2
 Expression of CD23; absence of EMC-7 3 Lymphocytosis with anemia
Molecular Genetics:
4 Lymphocytosis with High Risk Stages 3
 Variable region Genes thrombocytopenia &4
 Zeta-chain-associated Protein 70
 Thymidine Kinase
 CD38
 MicroRNA: use for the expression of proto-oncogenes, tumour suppressor
 expression profile can distinguish normal B cells from malignant B cells
 It is associated with prognostic factors and disease progression
 Abnormal – responsible for self- sufficiency
Treatment:
 Irradiation for enlarged spleen and lymph nodes
 Fludarabine – induces apoptosis
 Ankylating agents
 Monoclonal antibodies
 Allogenic stem cells transplants
HAIRY CELL LEUKEMIA (LEUKEMIC RETICULOENDOTHELIOSIS)
- Fragile appearing mononuclear cell with hair-like or ruffled projections of the cytoplasm
- Nuclear material in these cells is round or dumbbell shaped with a spongy appearance of the chromatin.
- Approximately 50% of cells in PBS.
- Most patients are older, in their 5th decade with more males than females being affected.
- Abdominal discomfort is a frequent presenting symptom; more than 80% of patients show massive spleens that misplace the stomach.
- Monocytes and neutrophils are decrease.
- Bleeding, Infections and anemia
- Neutrophils and monocytes are greatly reduced.
- Bone marrow aspirated are usually unsuccessful and lead to a dry tap (fibrotic stage)
- CD Markers: CD22, CD11, CD25, and CD103
- Cytochemical stain: TRAP (tartrate-resistant acid phosphatase stain)
- Isoenxyme 5 is especially abundant
- Therapeutic splenectomy will provide improvement in the cytopenia and hypersplenism as well as providing an improvement in physical symptoms such as abdominal fullness and
satiety.
- Interferon alpha and 2 chlorodeoxyadenosine (2-CdA) have offered positive remissions.
-
SEZARY SYNDROME (LEUKEMIC PHASE OF MYCOSIS FUGOIDES)
- T cell lymphomas may present with a cutaneous manifestation in some patients = Mycosis fungoides (Red itchy areas, Red man syndrome)
- This disease may progress, the spleen, bone marrow, and lymph nodes become involved, which is the leukemic phase of T-cell lymphoma.
- Sezary cells approximately 8-20 um, with a convoluted, cerebriform, ovoid nucleus. This can be mistaken for monocytes. Concentrated cytoplasm (Thicker and compact)
- Pathognomonic for cutaneous T-cell lymphoma and individuals who progress to this phase have decreased survival rates.
- Sezary cells: CD2, CD3. CD4, and CD5 markers

PROLYMPHOCYTIC LEUKEMIA
- Rare disorder shows a majority of circulating prolymphocytes.
- Phenotyping by flow cytometry usually shows expression of B-cell markers, absence of CD5, and relatively bright surface immunoglobulin with light chain restriction.
- Individuals with PLL will show strong CD20 markers and Sig activity and will also be positive for CD19 and CD20.
Striking Appearance of Plasma Cells in certain Pathologic States:
1. Flame Cells – Red staining cytoplasm
2. Russell bodies /Grape, berry, morula – large staining globules & numerous globular bodies
3. Red staining, crystalline, rod-shaped bodies present in the cytoplasm
Immunoglobulin and Range of Activity:
1. IgG- Secondary immune response, MATURATION SERIES
precipitating antibodies, hemolysins, virus PLASMABLAST PROPLASMACYTE PLASMACYTE (PLASMA
neutralizing antibodies. (Primary reactor) CELLS)
2. IgA – Secretory antibody, protects airways Size 18-25 um in diameter 15-25 in diameter 8-20 um in diameter
and gastrointentinal tract Cell shape may be oval
3. IgM- Primary immune response. Cytoplasm Basophilic cytoplasm Intensely basophilic: usually Moderately abundant, but less than in
4. IgD- Lymphocyte activator and suppressor Abundant bluer in the blast stage, Non- the previous stage, Deeply basophilic
5. IgE – Antibody found in respiratory and Granular granular Large, well-defined hof next to
More abundant than in the blast nucleus
gastrointestinal tract/ parasitic infections. stage Non-granular usually
MULTIPLE MYELOMA A lighter staining area in the
- Occurs in older age among men more than cytoplasm
among women Next to the nucleus is often
- Several environmental and occupational visible.
factors are thought to contribute to the clonal This is termed a hof or
perinuclear halo.
proliferation of plasma cells
Nucleus Nuclear chromatin is more Round or oval Chomatin is condensed and coarse
- The classic bone lesions associated with the
clumped than in the reticular Eccentrically located Exhibits ”cartwheel” like pattern
multiple myeloma are sharp “punched out” lymphocyte Chromatin is coarser and Round or oval in shape
osteolytic lesions (skull, pelvis, ribs, femur) Exccentric (the nucleus is off densely stained Eccentric
center, located at one side of the Ocassional nucleoli may be seen No nucleoli are visible
cell
Perinuclear halo may be present
Round or oval in shape
Multiple nucleoli that may or
may not be visible
 - Aberrations in Chromosome 13 have been particularly well studies and include monosomy; deletions or translocations of the chromosome.
Characterized by a triad of abnormalities:
1. Accumulation of plasma cell in the bone marrow
2. Bone Lesions – “post- punched out”
3. Production of a monoclonal immunoglobulin (Ig) or (Ig fragments)
Pathophysiology:
3 distinct pathways:
1. Acceleration of plasma cells in the Bone Marrow
o Plasma cells accelerate in multiple myeloma
o Some may even develop colorless inclusions called Russel bodies or other crystalline inclusions.
o Flame cells may also be visualized in IgA myelomas and appear as plasma cells with a striking deep cytoplasm.
2. Activation of bone resorption factors or osteoclast
o With increased activity, bone loss is inevitable and this pathology usually brings forward the single most frequent complaint from MM patient- bone pain (compressed
back bone and sternum)
o Serum calcium is also greatly increased.
o Bone resorption – release of calcium.
3. Production of an abnormal monoclonal protein
o Usually IgG on serum immunoelectrophoresis, this is seen as an M spike.
o May lead to complications from hyperviscocity in the plasma such as blurred vision or headache.
o Increase plasma production.
Symptopms:
1. Fatigue – Anemia
2. Excessive thirst and urination – excess calcium which needs to secrete
3. Nausea – excess calcium
4. Bone back pain and ribs- compression of bone deposits of calcium
5. Bone fractures – loss of calcium
6. Unexpected infections – immunity (compromised)
7. Weakness and numbness in the legs – loss of nerve numbness due to bone compression.
Screening and diagnosis:
1. CBC, possibly a bone marrow, urinalysis
- Pancytopenia: N/N anemia; increased ESR, calcium, urine protein and uric acid
- The peripheral smear may also show a blue coloration on macroscopic examination due to excess proteins.
- May resemble Rouleaux formation (afribrinogenemia and globulinemia)
2. Protein panel: - abnormal serum electrophoresis
- Serum beta- macroglobulin in the early stage of MM, this protein is at a low levels
- Elevated levels >6 ug/mL are seen later in the disease and usually indicate higher tumor burden and poor prognosis.
- Result of excess of kappa and lambda light chains. When urine is heated to 56 celcius, BJP precipitates out and will re-disolve at higher temperature. As the urine is cooled,
precipitates will once again appear and will dissolve upon cooling.
Prognosis and Treatment: Chemotherapy, Radiation, Glucocorticoids, Interferon- alpha
WALDENSTROM’S MACROGLOBULINEMIA
- Dr. Jan Waldenstroms (IgM)
- Presence of pleomorphic B-lineage cells at different stages of maturation, such as small lymphocytes, lymphoplasmacytoid cells (abundant basophilic cytoplasm but lymphocyte-
like nuclei), and plasma cells- thus, the name plasmacytoid lymphocytes.
- Express the CD40 ligand (CD154), which is a potent inducer of B-cell expansion.
- Characterized by Lymphoplasmoploriferative disorder with infiltration of the bone marrow and a monoclonal immunoglobulin M (IgM) protein.
- Hyperviscocity syndrome – is a complication experience by these patients (Overload of IgM – macromolecules)
- Smear may show Rouleaux and Plasmacytoid lymphocytes.
- As a subset of the abnormal IgM protein, cryoglobulins may form in some patients, which leads to Raynaud’s phenomenon and bleeding.
- Chemotherapy is available for these patients, and plasmapheresis may be used as a means to reduce the IgM concentration.
- Treatment for many patients consist of:
o Plasmapheresis
o Chemotherapy
o Immunotherapy with monoclonal antibodies
o Stem cell transplantations
o Interferon

LYMPHOMA
- Overploriferation of abnormal cells of the lymphoid tissue (lymphocytes, histiocytes, and reticulum cells)
- The spilling of these cells into the PB results in a leukemic phase of the disease.
- Monoclonal Gammopathy of undetermined (MGU) – active signs
- Etiology and clinical features:
1. Epstein Barr Virus (EBV)
2. Diagnosed between 15-35 years old, also found in over 50 population.
- General physical findings: a swollen painless lymph node is felt
- Classification is based on lymph node structure, cell predominance and cell differentiation and on the histological presence of Reed-Sternberg (RS) cells in Hodgkin’s Lymphoma.
HODGKIN’S DISEASE VERSUS NON-HODGKIN’S LYMPHOMAS
HODGKIN’S DISEASE NON-HODGKIN’S LYMPHOMAS
Orderly contiguous spread Noncontagious, widely disseminated spread
Predominant centreal and axial lymoh node involvement: rare peripheral Frequent involvement of both central and axial and peripheral lymph nodes
node involvement Frequent involvement of mesenteric nodes and Waldeyer’s ring
Mesentric nodes and Waldeyer’s ring involved seldom or late Extranodal presentation (Common)
Extranodal presentation (Rare) No-Reed-sternberg cells
Presence of Reed-Sternberg (Large binucleated cell) each nucleus has a
large nucleolus

HODGKIN’S LYMPHOMA – distinct clinical

 - Lymphoma in which RS cells are found in the disease tissue.
- The Epstein-Barr virus (EBV) genome has been detected but its role in the pathogenesis is unclear.
- Diagnosis is made based upon the cellular features seen in lymph node biopsy, which may feature a Reed- Sternber cell, a largemultinucleated cell resembling a “owl’s eye”
- Characterized by a persistent defect in the cellular immunity with abnormalities in the T lymphocytes, IL- 2 production, and increased sensitivity to suppressor monocytes and
normal T suppressor cells.
- Aneuploidy or deviation from the diploid number of chromosomes, resulting from the gain or loss of chromosomes or form polyploids is a characteristic feature of Hodgkin’s
disease.
- Hyperploidy is observed in the majority of Hodgkin disease tumors that have an abnormal karyotype. A gain of chromosome 1,2.5,12 and 21 is recurring numerical abnormality;
structural rearrangements involving chromosome 1 are frequently observed.
RYE Classification:
a. Lymphocyte predominance (LP)
 Uncommon variety of Hodgkin’s disease
 Small, normal appearing lymphs, benign histiocytes, rareReed-sternberg cell
b. Mixed cellularity
 Mixture of lymphs, histiocytes, plasma cells, Reed-sternber cells/ variants of R-S Cells
RYE Classification determined by lymph node biopsy:
c. Lymphocyte depletion
 Sparse lymphs, predominace of R-S cells variants
 Many past cases of LD Hodgkin’s may have been misdiagnosed and were really non-Hodgkin’s lymphoma
d. Nodular sclerosing
 Schlerosis forms bands of collagen that subdivided the tissue into distinct nodules
 Classic R-S cells
 Variant R-S cells called Lacunar cells.
CLASSIC HODGKIN’S LYMPHOMA
1. NODULAR SCLEROSIS – Collagen bands extend from the node capsule to encircle nodules of abnormal tissue. A characteristic lacunar cell variant of the Reed-Sternberg cell is often
found. The cellular infiltrate maybe of the lymphocyte-predominant, mixed cellularity or lymphocyte- depleted type; eosinophilia frequent
2. MIXED CELLULARITY – The Reed-sternberg cells are numerous and lymphocyte numbers are intermediate.
3. LYMOHOCYTE DEPLETED – There is either a reticular pattern with dominance of Reed-Sternberg cells and sparse numbers of lymphocytes or a diffuse fibrosis pattern where the
lymph node is replaced by disordered connective tissue containing few lymphocytes. Reed-Sternberg cells may also be frequent in this latter subtype.
4. LYMPHOCYTE RICH -Scanty Reed-Sternberg cells; multiple small lymphocytes with few eosinophils and plasma cells, nodular and diffuse types.
5. NODULAR LYMPHOCYTE- PREDOMINANT – Reed- Sternberg cells are absent, abnormal polymorphic B cells.

NON- HODGKIN’S LYMPHOMA

- 3x more common than Hodgkin’s lymphoma and maybe present as painless cervical lymph node involvement
- The lymph nodes may be enlarged, and the disease may spread to the gastrointestinal and respiratory systems, skin, liver, and spleen.
- The cause is unkown
- Predisposing factors seem to be chemicals, ionizing radiation and certain viruses.
- Reed-Sternberg cells are not present.
- The diagnostic scheme is divided into low grade, intermediate grade or high grade on the different histgological tyoes of lymphocytic cells.
- Radioation and Chemotherapy may be successful in obtaining remission, but relapses for non-Hodgkin’s lymphoma are frequent.
- Primary cutaneous T-cell lymphoma
o Infiltraion of the skin
o Included in this category are: Mycoses fungoides and Sezary syndrome

DIFFUSE LARGE B- LYMPHOMA

- The most frequent type of NHL, which accounts for approximately 40% of new cases of lymphoma.
- >1/2 of the patients with diffuse large B-cell lymphoma are older than 6 0 years of age.
- Three different types of diffuse large B-cell lymphomas can be defined based on gene expression subgroups:
 Germinal center, B-cell like lymphoma that express high levels of genes characteristics of germinal center, B-cell like lymal germinal center B gene.
 Activated B-cell lymphoma, which expressed gene characteristic of mitogenically activated blood B cells.
 New subgroups, type 3 diffuse large B-cell lymphoma, which has a heterogenous gene expression, that suggest it includes more than one subtype of lymphoma
Staging of Non- Hodgkin’s Lymphoma:
STAGING OF NON-HODGKIN’S LYMPHOMAS –
Systemic symptoms associated with NHL
The staging system for NHL – ANN ARBOR STAGING SYSTEM
- Fever >38% without infection or other cause Localized-stage disease (1-2) is better than advance-stage disease (3-4)
- Severe drenching night sweats STAG FEATURES
- Loss of <10% of body weight within the 6 months prior to diagnose, E
not explained by dieting 1 Single lymph node or site; may be extranodal (designated stage IE)
Presence of absence of symptoms is designated with a suffix (A or B) 2 More than one lymph node or site on the same sideof the diaphragm (either above or
- Systemic symptoms absent below, but not both)
- Systemic symptoms present 3 Disease on both sides of the diaphragm
MANTLE CELL LYMPHOMA 4 Involvement of liver and or bone marrow
- Approximately 5 to 10%of lymphoma cases in the United states are MCL, occurring predominantly in older individuals, with a significant male predominance.
- The majority cases are advance stage at diagnosis, with a high incidence of bone marrow involvement.
- Characterized by generally small cells, with moderate nuclear irregularity (rounder than the cleaved cells of follicular lymphomas, more irregular than the round nuclei of small
lymphocytic lymphoma)
- There is a strong association with a translocation between chromosome 11 and 14 (t(11;14))
- Considered by some people to be the worst of all lymphoma types.
- Mantle cell lymphoma has a characteristic immunophenotype, Like B-cell CLL/SLL, it coexpresses B-cell markers and CD5, usually considered a T-cell marker.
BURKITT’S LYMPHOMA
- Working formulation: Diffuse Small Non-cleaved Cell
- It is very high grade NHL. It is very common in parts of Africa (endemic Burkitt’s) but also occurs less commonly in the United State (sporadic Burkitt’s)
- Burkitt- cell leukemia (designated ALL-L3) in FAC classification of acute leukemia) is the leukemic equivalent of Burkitt’s lymphoma.
- Association with EBV and high prevalence in areas of endemic malaria.
 - There is a universal association between Burkitt’s lymphoma and translocations of the c-MYCO protooncogene to one of the immunoglobulin genes (heavy or light chain)
- >80% have t(8;14); c-MYC on chromosome 8 is translocated to the immunoglobulin heavy chain gene (IgH) on chromosome 14. The remainder involve c-MYC and either the
kappa light chain gene on chromosome 2 or the lambda light chain gene on 22.
- The classic presentation is a huge rapidly growing jaw mass in a young child.
- Formerly had a dismal prognosis. Now, with very intensive chemotherapy.
- The prognosis in children has improved significantly, however, the prognosis in adults remains poor.
- Chemotherapy may result in a tumor lysis syndrome in patients with a high tumor bulk.
- Hydration, alkalanization of the urine and allopurinol are usually used to prevent acute renal failure.
- The histological pattern is described as a “starry-sky” appearance

MYELODYSPLASTIC SYNDROMES
- Group of clonal disorders of multipotential hematopoietic stem cells.
- Characterized by increase BM failure with quantitative and qualitative abnormalities of all 3 myeloid cell lineages = Stem cell lesion (Neoplasm)
- Dysplasia - abnormal development of tissue.
- Hallmark: ineffective hemopoiesis - Cytopenia (Normal – increased cellularity)
- Tendency: May progress to AML (Acute Myeloid Leukemia), death commonly occur before it develops.
- Chemotherapy and Radiotherapy(given for malignancies)
- Other names: Preleukemia Dysmyelopoietic syndrome, Oligoblastic leukemia, and Refractory anemia.
2 types of MDS:
1. De novo (new cases unrelated to any other treatments)
 Secondary cases related to prior to therapy, usually alkylating therapy or radiation
 Risks:
o Exposure to Benzene
o Radiation
o Petrochemical employees
o Cigarette smokers
o Patients with Fanconi’s Anemia
2. Patients undergoing Immunosuppressive therapy – 2-5 years transformation after the agents have been administered.
Laboratory Findings:
 Pancytopenia: Marcocytic/ Dimorphic- Hypochromic
 Normoblasts may be present
 Reticulocytes: Low/Decreased
 Granulocytes: Reduced/ Frequently shows lack of granulations
 Pelger abnormality: Single/ Bilobe nucleus is present
 BM: Apperance of Ring sideroblasts = iron deposition in mitochondria of erythroblast
 Cells are difficult to identify because the granulocyte precursors are defective in primary and secondary granules.
 Megakaryocytes are abnormal with micronuclear, small binuclear or polynuclear forms.
Cytogenetic abnormalities:
 More frequent in secondary MDS
 Most common constitue partial/total loss of chromosome5 or 7 (Trisomy 8)
 5q Syndrome - Elder females, the loss of DIFFERENTIATION OF THE CLASSIFICATION OF THE MYELODYSPLASTIC SYNDROME
chromosomes bands q13 to q33 with macrocytic SUBTYPE FAB (FRENCH-AMERICAN- WHO (WORLD HEALTH
anemia, normal and raised platelet counts and BRITISH) ORGANIZATION)
micromegakaryocytes. Investigate group devised a working Revised the work and presented their
 20% - RAS oncogene (N-RAS) mutation classification for the MDS in 1981 based classification of the MDSs based on the
 15% - FMS oncogene on a study of 50 cases additional knowledge gained from molecular,
immunological and cytogenetic studies
Dysplastic changes:
1. Dysterythropoiesis – Red Cells Refractory Anemia (RA) Dysplastic changes in blood and BM Disorder of Red cells
 Nuclear budding <1% myeloblasts in blood Anemia resistant to treatment
 Ringed sideroblasts <<5% myeloblast in marrow <1% myeloblast in PB
 Internuclear bridging <5% myeloblast in BM
Hyperplasia with Megablastoid features;
 Dimorphism multinuclearity
 Megaloblastic asynchrony Refractory anemia with >15% nucleated erythroid precursors are 15% Ringed sideroblasts
 Multinuclearity Ringed sideroblast pathologic ringed sideroblast BM: Erythroid hyperplasia
2. Dysgranulopoiesis – White Cells (RARS) <1% myeloblast in blood <5% Myeloblast
 Abnormal staining throughout the <5% myeloblasts in the marrow Liver/Spleen: Iron overloading
cytoplasm Refractory Anemia with 1-5% myeloblasts in blood BM failure: 2 or more cell lineages are affected
 Hyposegmentation excess blast (RAEB) 5-20% myeloblasts in the marrow 50% multiple chromosomal abnormalities
Markedly erythroid hyper plasia with nuclear &
 Hypersegmentation
cytoplasmic dysplastic changes
 Nuclear with little segmentation <10% blast in PB
 Missing primary granulations Refractory Anemia with 20-30% myeloblasts in marrow or Auer- (TAN combo)
 Granules that are poorly stained excess blast in rod present BM: Abnormalities (ALL) myeloid cell lines
3. Dysthrombopoiesis – Platelets transformation (RAEB-t) with 5% to 19% blast
 Mircomegakaryocytes Increase blast cells in blood and BM= Diagnosis
 Abdnormal granules for RAEB – Poor prognosis.
 Giant platelets Chronic Myelomonocytic >1000 monocytes/L in blood >1000 monocytes/L in blood
Leukemia (CMML) Monocytosis in marrow Monocytosis in marrow
Factors indicating progression to Leukemia in MDS:
Myelodysplastic syndrome PLT and WBC = decreased
1. Disease stable, if there is little increase in marrow blast unclassifibable Neutropenia and Thrombocytopenia are
count and the original karyotype is unchanged. common
2. Progressive rise in the blast count usually indicates Deleted 5q Seen in female patients
transition to acute leukemia Deletion of long arm chromosome 5
3. Sudden change in karyotype that may progress into acute PLT: normal to decreased
leukemia. Marked anemia with macrocytes
<5% blast in PB – associated with long survival
4. Abnormal karyotype develops without subsequent increase
times – good prognosis
in blast; acute leukemia may or may not develop.
CHRONIC MYELOMONOCYTIC LEUKEMIA (CMML)
- Clonal hematologic malignancy that is characterized by feautures of both MPN and MDS. This form of leukemia is much less frequent than the acute variety.
- PB: persistent Monocytosis (>1000 monocytes/L)
- PB/BM: >20% myeloblast, nonblast, promonocytes
- No Ph Chromosome or BCD-ABL 1 fusion gene.
- No evidence of PDGFRA/PDGFRB (Platelet Derived Growth Factor receptor – alpha or beta)
- Diagnosis: Clonal cytogenic/ molecular genetic abnormality
- Monocytosis (Atleast 3 months)

Other classifications:
1. ACUTE CHRONIC MYELOID LEUKEMIA (BCR-ABL1-)
- Features of MPN & MDS
- Characterized by leucocytosis (Neutrophils)
- Multilineage dysplasia is common
2. JUVENILE MYEMONOCYTIC LEUKEMIA
- Is a disorder of childhood
- Characterized by the proliferation of G&M lineages
- <20% blasts and promonocytes in PB%BM aspirates
- Erythroid and megakaryocytes abnormality: Present
- BCR-ABL1 mutation: Absent
- RAS-MARK Pathway: Present

3. MYELODYSPLASTIC/MYELOPROLIFERATIVE NEOPLASM (UNCLASSIFIABLE)

- Meets the definition of MDS/MPN but doesn’t meet the criteria of CMML

SUMMARY POINTS
 MDS - RAS& Cytopenias (one or more cell lineages)
 BM/PBS: Dysplastic changes of WBC, RBC, PLTs
 MDS blas counts: <20%
 Weaksness, Infwection, Easily bruising
 WHO – six classification of MDs
 Dysthrombopoeitic changes:
 Micromegakaryocytes
 No abnroaml granulation
 Giant platelets

 Dysgranulopoietic changes:
 Abnormal granulation of mature cells
 Hypersegmentation,
 complete lack of granulation
 Dyserythropoietic changes:
 Multinuclear red cell precursor
 Bizarre Nuclear Changes
 Nuclear bridging
 Macrocytes
 Dimorphism