🦴 BONE MARROW & HAEMATOPOIESIS

📌 1. Bone Marrow and Blood Cell Formation

Bone marrow contains pluripotent stem cells that give rise to:
- Non-lymphoid stem cells → form erythrocytes (RBCs), granulocytes, monocytes, platelets.
- Lymphoid stem cells → form B cells, T cells, and Natural Killer (NK) cells.

🕒 2. Lifespan of Blood Cells

Cell Type	Lifespan
Neutrophils	6–8 hours
Platelets	~10 days
RBCs	90–120 days

Production of these cells is tightly regulated to match the rate of loss.

🧬 3. Haematopoiesis (Blood Cell Formation)

Embryonic Sites of blood cell production:
- Yolk Sac → 1st few weeks.
- Liver & Spleen → 3rd month to 2 weeks after birth.
- Bone Marrow → Starts by 4–5 months, fully active by 7–8 months.
In Adults:
- Active bone marrow remains in central skeleton: vertebrae, sternum, ribs, skull, pelvis, and ends of long bones.
- Fat replaces red marrow in long bones over time.
- In some diseases, liver and spleen can restart blood cell production (called extramedullary haematopoiesis).

🧪 4. Haematopoietic Stem Cells

Features:
- Multipotent, capable of self-renewal and differentiation.
- Identified by surface proteins like CD34.
- Used in bone marrow/stem cell transplantation.
Two main progenitor types:
- Lymphoid Stem Cells → T cells, B cells, NK cells.
- Myeloid (Trilineage) Stem Cells:
  - Granulocyte-monocyte line → neutrophils, eosinophils, basophils, monocytes.
  - Erythroid line → RBCs.
  - Megakaryocyte line → platelets.

💉 5. Growth Factors & Hormones in Haematopoiesis

These stimulate differentiation of stem cells into mature blood cells:

Erythropoietin → stimulates RBC production.
G-CSF (Granulocyte Colony Stimulating Factor) → for neutrophils.
GM-CSF (Granulocyte-Macrophage CSF) → for granulocytes and monocytes.
Thrombopoietin → for platelet formation.

Each factor works through specific receptors to promote cell growth.

Red Blood Cells (RBCs)

1. Erythropoiesis (Formation of Red Blood Cells)

Primarily occurring in the bone marrow.

Site of Erythropoiesis:

Fetus: Yolk sac (1st trimester), liver and spleen (2nd trimester)
Infants: All bones
Adults: Vertebrae, sternum, ribs, pelvis (axial skeleton)

Stages of Erythropoiesis:

Pluripotent stem cell
Myeloid progenitor cell
Erythroid burst-forming unit (BFU-E)
Erythroid colony-forming unit (CFU-E)
Proerythroblast
Basophilic erythroblast
Polychromatophilic erythroblast
Orthochromatic erythroblast (normoblast)
Reticulocyte (immature RBC)
Erythrocyte (mature RBC)

Regulation:

Controlled by erythropoietin (EPO), produced in the kidneys in response to hypoxia.
Requires adequate iron, vitamin B12, folate, and proteins.

2. Structure of Red Blood Cells

Normal Morphology:

Shape: Biconcave disc
Size: Diameter ~7.2–7.8 μm; thickness ~2.5 μm (edge), ~1 μm (center)
Volume (MCV): ~80–100 fL
Color (MCHC): 32–36%

Advantages of Biconcave Shape:

Increased surface area for gas exchange
Flexibility to pass through capillaries

3. Function of Red Blood Cells

Primary Function:

Transport of respiratory gases:
- O₂ via hemoglobin
- CO₂ via bicarbonate, carbaminohemoglobin, and dissolved form

Secondary Functions:

Acid-base buffering (via hemoglobin and bicarbonate)
Regulation of vascular tone through nitric oxide binding and release

4. Membrane Biology of RBCs

Composition:

Lipid bilayer: Phospholipids and cholesterol
Integral proteins: Band 3, glycophorins
Peripheral proteins: Spectrin, ankyrin, actin

Cytoskeletal Network:

Maintains cell shape, deformability, and structural integrity
Key components: Spectrin–actin–ankyrin complex

Defects in Membrane Proteins:

Lead to hereditary hemolytic anemias:
- Hereditary spherocytosis: Spectrin/ankyrin defect
- Elliptocytosis: Spectrin abnormality

5. Nutritional Requirements for Erythropoiesis

Essential Nutrients:

Iron: Required for hemoglobin synthesis
Vitamin B12 and Folate: DNA synthesis in erythroblasts
Amino acids: Globin chain synthesis
Vitamin C: Enhances iron absorption
Copper, Zinc: Involved in iron metabolism

Deficiency Effects:

Iron deficiency: Microcytic, hypochromic anemia
Vitamin B12/Folate deficiency: Megaloblastic anemia

6. Hemoglobin Synthesis

Structure:

Hemoglobin (Hb) = 4 globin chains + 4 heme groups
Normal adult Hb: HbA (α₂β₂), HbA2 (α₂δ₂), HbF (α₂γ₂)

Globin Synthesis:

Encoded by genes on chromosomes:
- α-chain genes: Chromosome 16
- β-chain genes: Chromosome 11

Heme Synthesis:

Begins in mitochondria → cytoplasm → ends in mitochondria
Key enzyme: δ-aminolevulinic acid synthase (ALA synthase)

Iron Incorporation:

Fe²⁺ is inserted into protoporphyrin IX to form heme
Transported by transferrin, stored as ferritin/hemosiderin

Additional Concepts

RBC Lifespan:

Senescent RBCs are phagocytosed by macrophages (mainly in spleen)

Breakdown Products:

Hemoglobin → Heme + Globin
- Globin → Amino acids
- Heme → Iron (recycled) + Bilirubin (excreted in bile)

Anaemia

Anaemia is defined as a reduction in the total circulating red cell mass, typically identified by a decrease in hemoglobin (Hb) concentration below the age- and gender-adjusted reference range. It often leads to reduced oxygen-carrying capacity of the blood.

Males: Hb < 13.5 g/dL
Females: Hb < 12.0 g/dL

2. Pathophysiologic Classification of Anaemia

Anaemia can arise from three major mechanisms:

A. Blood Loss

Acute: Trauma, surgery, gastrointestinal bleeding (e.g., peptic ulcers)
Chronic: Menorrhagia, colorectal cancer, hookworm infestation

B. Decreased Red Cell Production

Nutritional deficiencies: Iron, vitamin B12, folate
Bone marrow disorders: Aplastic anemia, myelodysplasia
Chronic disease/inflammation: Anemia of chronic disease

C. Increased Red Cell Destruction (Hemolysis)

Intrinsic (inherited): Sickle cell disease, thalassemia, G6PD deficiency
Extrinsic (acquired): Autoimmune hemolytic anemia, mechanical destruction (e.g., prosthetic heart valves)

3. Clinical Features of Anaemia

A. General Symptoms (related to hypoxia):

Fatigue
Weakness
Dizziness or lightheadedness
Shortness of breath (especially on exertion)
Headache

B. Signs on Physical Examination:

Pallor (especially of conjunctiva, palms, mucosa)
Tachycardia
Orthostatic hypotension

C. Specific Features Based on Cause:

Type	Specific Features
Iron deficiency	Koilonychia, pica, glossitis
Vitamin B12 deficiency	Neuropathy, ataxia, dementia
Hemolytic anemia	Jaundice, splenomegaly, dark urine
Aplastic anemia	Petechiae, infections (due to pancytopenia)

4. Laboratory Investigations in Anaemia

A. Basic Tests

Complete Blood Count (CBC): Hb, HCT, RBC count, MCV, MCH, MCHC, RDW
Reticulocyte Count: Reflects marrow response to anemia
Peripheral Blood Smear: RBC morphology, anisocytosis, poikilocytosis

B. Iron Studies

Serum iron
TIBC (Total Iron Binding Capacity)
Ferritin
Transferrin saturation

C. Vitamin Levels

Serum B12
Serum folate

D. Other Investigations

LDH and haptoglobin: Hemolysis markers
Bilirubin (indirect/unconjugated): Raised in hemolysis
Bone marrow biopsy: Aplastic anemia, leukemia
Stool for occult blood: GI bleeding
Hemoglobin electrophoresis: Thalassemia, sickle cell anemia

5. Morphological Classification of Anaemia (based on MCV)

Type of Anaemia	MCV	Common Causes
Microcytic	< 80 fL	Iron deficiency, thalassemia, chronic disease (rare), sideroblastic anemia
Normocytic	80–100 fL	Acute blood loss, chronic disease, aplastic anemia, early iron deficiency
Macrocytic	> 100 fL	Vitamin B12 or folate deficiency, liver disease, alcohol use, hypothyroidism, myelodysplastic syndromes

6. Reticulocyte Index (RI)

Used to assess bone marrow response:

RI < 2% → Inadequate production
RI > 2% → Increased destruction or blood loss

Classification of Anemias

A. Pathophysiologic Classification

I. Anaemia Due to Increased Blood Loss

a) Acute post-haemorrhagic anaemia
b) Chronic blood loss

II. Anaemias Due to Impaired Red Cell Production

a) Cytoplasmic Maturation Defects

Deficient haem synthesis:
- Iron deficiency anaemia
Deficient globin synthesis:
- Thalassaemic syndromes

b) Nuclear Maturation Defects

Vitamin B₁₂ and/or folic acid deficiency:
- Megaloblastic anaemia

c) Defects in Stem Cell Proliferation and Differentiation

Aplastic anaemia
Pure red cell aplasia

d) Anaemia of Chronic Disorders

e) Bone Marrow Infiltration

f) Congenital Anaemia

III. Anaemias Due to Increased Red Cell Destruction (Haemolytic Anaemias)

A) Extrinsic (extracorpuscular) red cell abnormalities
B) Intrinsic (intracorpuscular) red cell abnormalities

B. Morphologic Classification

I. Microcytic, Hypochromic Anaemia

Iron deficiency anaemia
Thalassaemic syndromes

II. Normocytic, Normochromic Anaemia

Aplastic anaemia
Pure red cell aplasia
Anaemia of chronic disorders
- Inflammation/infections
- Disseminated malignancy
- Renal disease
- Endocrine and nutritional deficiencies (e.g., hypothyroidism)
- Liver disease
Bone marrow infiltration
- Leukaemias
- Lymphomas
- Myelosclerosis
- Multiple myeloma

III. Macrocytic, Normochromic Anaemia

Megaloblastic anaemia (Vitamin B₁₂ and/or folic acid deficiency)
Congenital anaemias
- Sideroblastic anaemia

Hypochromic Anaemia

Definition

Hypochromic anaemia refers to anaemia in which red cells show decreased haemoglobin content, leading to pale (hypochromic) and small-sized (microcytic) cells. It results from defective haemoglobin synthesis.

Types

Iron Deficiency Anaemia (IDA) – Most common worldwide.
Hypochromic Anaemias other than Iron Deficiency
- Sideroblastic anaemia
- Thalassaemia
- Anaemia of chronic disorders

Iron Deficiency Anaemia (IDA)

Prevalence

Affects ~20% of women of child-bearing age
~2% in adult males
More prevalent in developing countries

Iron Metabolism

Iron Balance

Daily losses: ~1 mg in males and non-menstruating females; +0.5–1 mg in menstruating women
Sources: Dietary iron + iron recycled from senescent RBCs

Iron Absorption

Site: Duodenum & proximal jejunum
Types of Iron:
- Haem iron (from animal sources): better absorbed
- Non-haem iron: absorption aided by vitamin C, gastric HCl
Inhibitors: Antacids, milk, phytates, tannates (e.g., tea), EDTA
Mechanism:
- Non-haem iron must be reduced to ferrous (Fe²⁺) by ferric reductase
- Transported by (Divalent Metal Transporter 1) DMT1 across mucosa
- Exported to blood via ferroportin

Iron Transport

Bound to transferrin (a β-globulin made in liver)
Delivered to bone marrow for erythropoiesis
Transferrin is usually 1/3 saturated

Iron Storage

Stored in:
- Ferritin & haemosiderin (RE cells: spleen, liver, marrow)
- Functional iron: haemoglobin (65%), myoglobin (3.5%), enzymes (0.5%), transferrin-bound (0.5%)
- Storage iron: ~30%

Excretion

Daily loss: ~1 mg; up to 2 mg in menstruating women
Routes: Desquamation (gut, skin), urine, sweat, hair, nails

Pathogenesis of IDA

Iron deficiency develops when:

Supply is inadequate for haemoglobin synthesis
Initially, iron stores are used
When stores deplete → inadequate marrow supply → anaemia

Etiology of Iron Deficiency Anaemia

1. Increased Blood Loss

Uterine: Menorrhagia, miscarriages
Gastrointestinal: Ulcers, haemorrhoids, cancer, hookworm, colitis
Renal: Haematuria, haemoglobinuria
Nasal: Recurrent epistaxis
Pulmonary: Haemoptysis

2. Increased Requirements

Growth (infancy, childhood, adolescence)
Pregnancy & lactation

3. Inadequate Dietary Intake

Poverty, anorexia (esp. in pregnancy), elderly with poor dentition

4. Decreased Absorption

Gastrectomy
Coeliac disease, other malabsorption syndromes

High-Risk Groups

1. Females (Reproductive Age)

Menorrhagia, miscarriages, IUCD use
Poor diet, increased demands (pregnancy, adolescence)

2. Post-Menopausal Women

Uterine or GI tract bleeding (carcinomas)

3. Adult Males

Usually due to chronic GI bleeding
Peptic ulcer, hookworm, malignancy, aspirin use

4. Infants & Children

Rapid growth with insufficient intake
Weaning without iron-rich foods

Clinical Features

1. Anaemia Symptoms

Fatigue, weakness, exertional dyspnoea, palpitations
Pallor of skin, mucous membranes, sclerae
Pica (craving for non-food substances)

2. Epithelial Tissue Changes (with chronic IDA)

Koilonychia (spoon nails)
Atrophic glossitis (smooth tongue)

Sideroblastic Anaemias

1. Definitions

Siderocytes
– Mature red cells (erythrocytes) containing cytoplasmic iron granules.
– Stain positive with Prussian blue; when seen in Romanowsky stains, called Pappenheimer bodies.
– Normally absent in peripheral blood; appear post-splenectomy.
Sideroblasts
– Nucleated erythroid precursors (normoblasts) with cytoplasmic iron granules.
– Prussian blue–positive granules.
– Types:
  • Normal sideroblasts – Few, fine, scattered granules; ~30–50% of normoblasts in healthy marrow.
  • Abnormal sideroblasts
    – Coarse sideroblasts: Numerous scattered granules (dyserythropoiesis, hemolysis).
    – Ringed sideroblasts: Granules form a ring around the nucleus (mitochondrial iron), hallmark of sideroblastic anaemias.

2. Classification of Sideroblastic Anaemias

Hereditary (Congenital)
- X-linked defect of δ-aminolevulinic acid (ALA) synthase.
- Presents in childhood/adolescence; moderate–severe anaemia in males; female carriers asymptomatic.
Acquired
A. Primary (Idiopathic / Refractory)
- Occurs in middle-aged/elderly of both sexes.
- Considered a myelodysplastic syndrome (MDS) subtype: refractory sideroblastic anaemia.
- Marrow: erythroid hyperplasia, dysplasia, cytopenias; 10% risk of AML transformation.
B. Secondary
- Drugs & Toxins:
  • Chloramphenicol
  • Alcohol, lead
  → Usually reversible upon withdrawal.
- Hematologic Disorders:
  • Polycythemia vera, leukemias, myeloma, hemolytic anaemias.
- Miscellaneous:
  • Carcinoma, rheumatoid arthritis, SLE, other chronic inflammatory/autoimmune diseases.

3. Pathogenesis

Ringed sideroblasts form when iron-laden mitochondria accumulate around the nucleus due to impaired haem synthesis.
Hereditary form: Enzyme deficiency (ALA-synthase) → ↓ haem production.
Primary acquired form: Defect in erythroid stem cells → dysplastic maturation and defective ALA-synthase activity.
Secondary forms: Toxins or associated diseases disrupt pyridoxine (B6) metabolism or mitochondrial function.

4. Laboratory Findings

Parameter	Finding in Sideroblastic Anaemia
Hb	Moderate–severe anaemia
MCV/MCH/MCHC	↓ in hereditary; MCV ↑ or variable in acquired
Peripheral smear	Hypochromic (microcytic or dimorphic)
Marrow iron	↑ stores; ringed sideroblasts present
Erythroid precursors	Erythroid hyperplasia; dysplasia in primary acquired
Serum iron & ferritin	↑ increased; transferrin saturation ↑↑
TIBC	Normal or ↓

5. Treatment

Hereditary & Primary Acquired
- High-dose pyridoxine (vitamin B₆) – 200 mg/day for 2–3 months.
- Supportive: red cell transfusions as needed.
Secondary
- Discontinue causative agent (drug, toxin, management of underlying disease).
- Pyridoxine supplementation.

6. Differential Diagnosis of Hypochromic Anaemias

Feature	Iron Deficiency	Thalassaemia Trait	Sideroblastic Anaemia
Serum iron	↓	Normal–↑	↑
Ferritin	↓	Normal	↑
TIBC	↑	Normal	Normal–↓
Marrow iron stores	↓	Normal	↑
Ringed sideroblasts	Absent	Absent	Present

MEGALOBLASTIC ANAEMIA

Disorder of impaired DNA synthesis causing abnormal red blood cell development.

1. Definition

Megaloblastic anaemia is a type of macrocytic anaemia caused by defective DNA synthesis, resulting in delayed nuclear maturation and large, abnormal red blood cell precursors called megaloblasts.

2. Cause

Deficiency of Vitamin B₁₂ (Cobalamin)
Deficiency of Folic Acid (Folate)
Less commonly:
- Congenital or acquired defects in vitamin B₁₂/folate metabolism
- Drugs interfering with DNA synthesis (e.g., methotrexate)

3. Pathogenesis

Impaired DNA synthesis causes asynchronous maturation:
- Nucleus lags behind the cytoplasm in development.
Leads to large nucleated precursors in bone marrow (megaloblasts) and macrocytic red cells in blood.
Results in ineffective erythropoiesis and pancytopenia in severe cases.

4. Morphological Features

Bone Marrow:
- Hypercellular with megaloblastic changes in erythroid, myeloid, and megakaryocytic lineages.
Peripheral Blood:
- Macrocytosis (↑ MCV)
- Oval macrocytes
- Hypersegmented neutrophils
- Low reticulocyte count
- Possible pancytopenia (↓ RBCs, WBCs, platelets)

5. Vitamin B₁₂ Metabolism Overview

Feature	Vitamin B₁₂ (Cobalamin)
Sources	Animal products only
Cooking	Little effect
Daily Requirement	2–4 µg
Absorption Site	Distal ileum
Absorption Mechanism	Requires Intrinsic Factor (IF)
Body Stores	2–3 mg (lasts 2–4 years)
Transport	Transcobalamin II (TC II)

6. Vitamin B₁₂ Functions

Methylation of Homocysteine → Methionine (linked to folate metabolism)
Conversion of Methylmalonyl-CoA → Succinyl-CoA
- Deficiency can cause accumulation of odd-chain fatty acids in neurons → Neurological symptoms

7. Folate Metabolism Overview

Feature	Folate (Folic Acid)
Sources	Green vegetables, liver, fruits
Cooking	Easily destroyed
Daily Requirement	100–200 µg
Absorption Site	Duodenum and jejunum
Body Stores	10–12 mg (lasts 4 months)

8. Folate Functions

Essential for DNA synthesis via:
1. Thymidylate synthase reaction
  - Converts dUMP → dTMP (thymidine)
2. Methylation of homocysteine
  - Linked with vitamin B₁₂ metabolism

9. Biochemical Basis of Megaloblastic Anaemia

Defective DNA synthesis due to lack of methyl-THF or B₁₂ leads to:
- Inhibition of dTMP synthesis
- Impaired cell division
- Megaloblastic changes in marrow and macrocytic anaemia in blood
Accumulation of methylmalonic acid and homocysteine

10. Clinical Features

Fatigue, pallor, weakness
Glossitis (beefy red tongue)
Neurological symptoms (only in B₁₂ deficiency):
- Paresthesia (tingling)
- Ataxia
- Memory loss

11. Laboratory Findings

↑ MCV (>100 fL) – Macrocytic anaemia
Hypersegmented neutrophils
↓ Reticulocyte count
↑ Serum LDH and indirect bilirubin (due to ineffective erythropoiesis)
Vitamin levels:
- ↓ Serum B₁₂ and/or folate

12. Treatment

Vitamin B₁₂ deficiency → IM cyanocobalamin or hydroxycobalamin
Folate deficiency → Oral folic acid

13. Important Clinical Tip

Folic acid supplementation can improve anaemia in both B₁₂ and folate deficiency, but it does NOT treat neurological symptoms of B₁₂ deficiency.

🧬 Sickle Cell Anaemia (HbSS)

Sickle cell anaemia is a genetic blood disorder caused by a mutation in the β-globin gene, resulting in an abnormal type of haemoglobin known as Haemoglobin S (HbS).

Molecular Basis

HbS is formed when valine replaces glutamic acid at the 6th position of the β-globin chain.
In deoxygenated states, HbS becomes insoluble, forming long polymers inside red blood cells.
These polymers distort the red blood cells into a sickle (crescent) shape.

Pathophysiology

Sickled cells are rigid and fragile, leading to:
- Blockage of small blood vessels.
- Destruction in the spleen (haemolysis).
- Reduced red cell lifespan (≈ 20 days).

🧬 Genetic Patterns

Genotype	Condition	Description
HbSS	Sickle Cell Anaemia	Homozygous for HbS; severe disease.
HbAS	Sickle Cell Trait	Heterozygous; usually asymptomatic, but crisis can occur under stress (e.g., high altitude, surgery, exercise).
HbSC, HbSD, HbSβ-thalassaemia	Sickle Cell Disease variants	Also clinically significant; grouped under “sickle cell disease”.

🩺 Clinical Features of Sickle Cell Disease

Haemolytic anaemia: Fatigue, pallor, jaundice.
Pain crises (due to vascular blockage).
Swelling of hands and feet (dactylitis in children).
Infections: Salmonella, Pneumococcus.
Delayed growth and development.
Gallstones, leg ulcers, kidney damage, visual loss.
Crises:
- Haemolytic – Increased destruction of RBCs.
- Aplastic – Often due to parvovirus B19.
- Infarctive – Organ damage from blocked vessels.

In Pregnancy

Higher risk of:
- Preterm labor, miscarriage, stillbirth.
- Anaemic and infarctive crises.
- Infections, especially post-delivery.

HbAS (Sickle Cell Trait) offers partial protection against severe falciparum malaria.

🧪 Laboratory Findings

In Sickle Cell Anaemia (HbSS):

Low haemoglobin.
Blood film shows:
- Sickle cells.
- Nucleated RBCs.
- Target cells.
- Poikilocytosis (irregular shapes).
- Macrocytes (if folate deficient).
High reticulocyte count (due to increased RBC production).
Elevated white cells and platelets (especially in crisis).
No HbA, >80% HbS, some HbF and HbA₂.

Diagnostic Tests

Sickle cell slide test (positive).
Solubility test for HbS (positive).
Haemoglobin electrophoresis:
- Confirms type and proportion of haemoglobin variants.
Urine test: May show haematuria and sickle cells.

Management

Pain relief, hydration, oxygen in crises.
Folic acid supplements.
Antibiotics and vaccines to prevent infections.
Blood transfusions during crises (especially if Hb < 6–8 g/dL).
Hydroxyurea to increase HbF and reduce sickling.
Bone marrow transplant (curative in some cases).

THALASSAEMIA

Thalassaemia is an inherited (genetic) blood disorder.
It causes reduced production of one or more globin chains.
It is a quantitative defect (less production), unlike haemoglobinopathies (which are qualitative problems in structure).

First seen in people from Mediterranean countries, hence called “Mediterranean anaemia.”

Normal Globin Genes

We have:
- 2 α-globin genes on chromosome 16 (from each parent) → total 4 α genes.
- 1 β-globin gene on chromosome 11 (from each parent) → total 2 β genes.

Types of Thalassaemia

Type	Cause	Forms
α-thalassaemia	Decreased production of α-chains	Carrier (1 gene), Trait (2 genes), HbH disease (3 genes), Hb Bart’s (4 genes)
β-thalassaemia	Decreased production of β-chains	Minor (trait), Intermedia, Major (Cooley’s anaemia)

PATHOPHYSIOLOGY

What Causes Anaemia?

α-thalassaemia: Less or no α-chains → less HbA → anaemia.
β-thalassaemia: Less β-chains → excess free α-chains → damage red cells → destruction in spleen/liver → anaemia.

α-THALASSAEMIA TYPES

1️⃣ Hb Bart’s Hydrops Foetalis (4 α-gene deletion)

Most severe form, fatal before or shortly after birth.
Forms abnormal Hb Bart’s (γ₄) with high oxygen affinity → tissue hypoxia.

Lab findings:

Severe anaemia (<6 g/dL)
Blood film: microcytosis, hypochromia, anisopoikilocytosis
Hb electrophoresis: ~90% Hb Bart’s

2️⃣ HbH Disease (3 α-gene deletion)

Forms HbH (β₄) → unstable, forms Heinz bodies.
Moderate anaemia; symptoms worsen during stress (e.g., infection).

Lab findings:

Hb 8–9 g/dL
Blood film: microcytosis, target cells, stippling
Hb electrophoresis: 2–4% HbH

3️⃣ α-thalassaemia Trait (1 or 2 α-gene deletions)

Usually asymptomatic
Found in patients with mild microcytic anaemia not due to iron deficiency

Lab findings:

Slightly low Hb or normal
Small red cells (↓MCV, ↓MCH)
Hb electrophoresis: small amount of Hb Bart’s in newborns; normal HbA2

β-THALASSAEMIA TYPES

🅰️ Molecular Pathogenesis

Caused by mutations in β-globin gene (not deletions like α-thalassaemia)
β⁰: No β-chain production
β⁺: Reduced β-chain production

🅱️ Types

Type	Inheritance	Severity	Notes
β-thalassaemia minor (trait)	Heterozygous (one gene affected)	Mild	Often asymptomatic
β-thalassaemia intermedia	Partial defect	Moderate	May need occasional transfusions
β-thalassaemia major (Cooley’s anaemia)	Homozygous (both genes affected)	Severe	Needs regular transfusions

🔹 β-Thalassaemia Major (Cooley’s Anaemia)

Most severe form
Caused by either:
- Complete lack of β-chain (β⁰) or
- Very reduced β-chain (β⁺)
Results in more HbF (fetal Hb), not HbA
Anaemia, hepatosplenomegaly, bone deformities due to marrow expansion

Summary Table

Disorder	Genes Affected	Hb Changes	Clinical Severity
Hb Bart’s	4 α deleted	Mostly Hb Bart’s	Fatal
HbH Disease	3 α deleted	HbH (β₄)	Moderate
α-thal. Trait	1–2 α deleted	Slight Hb Bart’s in newborn	Mild/asymptomatic
β-thal. Minor	1 β mutated	Slight ↓ HbA, ↑HbA2	Mild
β-thal. Major	2 β mutated	↑HbF, ↓HbA	Severe

THROMBOPOIESIS

Thrombopoiesis is the process of platelet production in the bone marrow from megakaryocytes through cytoplasmic fragmentation.

Arises from myeloid stem cells, which also give rise to:
- Erythroid progenitors
- Granulocyte-monocyte progenitors
- Megakaryocyte progenitors → Platelets

Controlled by Thrombopoietin (TPO)

Stages of Platelet Development

Stage	Key Features
Megakaryoblast	- Earliest precursor from haematopoietic stem cell - Differentiates into promegakaryocyte
Promegakaryocyte	- Undergoes endomitosis (chromatin replicates without cell division) - Forms a polyploid cell (up to 32× diploid DNA) - Cytoplasm becomes granular
Megakaryocyte	- Large cell (30–90 μm) - 4–16 lobed nucleus with coarsely clumped chromatin - Light blue cytoplasm with red-purple granules - Cytoplasmic pseudopods fragment into platelets - One cell produces up to 4000 platelets
Platelets (Thrombocytes)	- Small (1–4 μm), discoid, non-nucleated - Contain red-purple granules - Lifespan: 7–10 days - Distribution: 70% in circulation, 30% in spleen - Remain in spleen 24–36 hours post-release

Total time for platelet formation from stem cell: ~10 days

Functions of Platelets in Haemostasis

1. Primary Haemostasis

Occurs within seconds of vascular injury → formation of platelet plug

Steps:

Platelet Adhesion
- Platelets bind to collagen in exposed subendothelium layer.
- Mediated by:
  - GpIa-IIa (integrin) → direct collagen binding
  - GpIb-IX complex via von Willebrand factor (vWF) → stabilizes adhesion
Platelet Release Reaction
- Activation → release of granule contents:
  - Dense granules: ADP, ATP, Calcium, Serotonin
  - Alpha granules: Factor V, VIII, PF4, PDGF, fibrinogen, vWF, thrombospondin
  - Lysosomal granules: hydrolytic enzymes, PAI-1
Platelet Aggregation
- Platelets cross-link via fibrinogen bridges
- Involves GpIIb-IIIa receptors on platelet surface

2. Secondary Haemostasis

Involves the plasma coagulation cascade
Results in fibrin plug formation
Takes several minutes to complete

🩸 Bleeding Disorders

Bleeding disorders are a group of conditions characterized by defective haemostasis resulting in abnormal bleeding—either spontaneously or after minor trauma/surgery.

Types of Bleeding:

Spontaneous bleeding: Small hemorrhages in skin or mucosa (e.g., petechiae, purpura, ecchymoses)
Post-traumatic/surgical bleeding: Larger bleeds (e.g., haematomas, haemarthrosis)

Classification of Causes:

Vascular Abnormalities
(e.g., fragile vessels, abnormal vasoconstriction)
Platelet Abnormalities
(quantitative or qualitative platelet defects)
Coagulation Factor Deficiencies
(intrinsic/extrinsic/common pathway factor defects)
Fibrinolytic Defects
(abnormal breakdown of fibrin clots)
Combination Disorders
(e.g., Disseminated Intravascular Coagulation – DIC)

🔬 Investigations in Bleeding Disorders

🧾 General Approach:

A. Clinical Evaluation

Detailed history (personal and family)
Site, frequency, and nature of bleeding

B. Screening Tests

To detect general haemostatic abnormalities

C. Specific Tests

To pinpoint the exact defect

🧪 Types of Investigations:

A. Vascular Haemostasis Tests:

Bleeding Time (normal: 3–8 min)
- Prolonged in:
  i. Thrombocytopenia
  ii. Platelet dysfunction
  iii. von Willebrand disease
  iv. Vascular defects (e.g., Ehlers-Danlos)
  v. Severe factor V/XI deficiency
Hess Capillary Resistance Test (Tourniquet Test)

20 petechiae in 3 cm² = Positive test
Indicates capillary fragility or thrombocytopenia

B. Platelet Function Tests:

1. Screening:

Platelet count
Bleeding time
Blood film for platelet morphology

2. Special Tests:

Adhesion tests (glass bead retention)
Aggregation tests (with ADP, collagen, ristocetin)
Granule content/release: EM or biochemical assays
Prothrombin consumption index: indirect platelet activity

C. Coagulation System Tests:

1. Screening Tests:

Coagulation Time: 4–9 minutes (limited utility)
APTT (or PTTK): Measures intrinsic + common pathway
- Prolonged in:
  i. Heparin therapy
  ii. DIC
  iii. Liver disease
  iv. Lupus anticoagulant
PT (Prothrombin Time): Measures extrinsic + common pathway

Prolonged in:
i. Oral anticoagulants
ii. Liver disease
iii. Vitamin K deficiency
iv. DIC

Fibrinogen Estimation & Thrombin Time:
- Prolonged in:
  i. Hypofibrinogenaemia
  ii. FDP elevation
  iii. Heparin presence

2. Special Tests:

Specific Factor Assays: To identify deficient clotting factor
Quantitative Assays: Immunological measurement of factors

D. Fibrinolytic System Tests:

1. Screening:

Fibrinogen levels
Fibrin degradation products (FDP)
Ethanol gelation test
Euglobulin or whole blood lysis time

2. Specific Tests:

ELISA or chromogenic assays for:

Plasminogen
Plasmin
Plasminogen activators/inhibitors
FDPs

🔄 Balance in Haemostasis:

Normal haemostasis involves:

Blood vessel integrity
Platelets
Coagulation factors
Natural inhibitors (e.g., antithrombin)
Fibrinolytic system

🛑 Disruption in any of these → bleeding diathesis

HAEMORRHAGIC DIATHESES DUE TO VASCULAR DISORDERS

Also called non-thrombocytopenic purpuras or vascular purpuras.
Characterized by mild bleeding symptoms:
- Petechiae (tiny pinpoint hemorrhages)
- Purpura (larger reddish-purple spots)
- Ecchymoses (bruises)
Bleeding is mostly confined to skin and mucous membranes.
Standard haemostasis tests (bleeding time, coagulation time, platelet count/function) are usually normal.
Bleeding results from:
- Damage to capillary endothelium
- Abnormalities in subendothelial matrix
- Abnormal vessel support or formation

A. Inherited Vascular Bleeding Disorders

Hereditary Haemorrhagic Telangiectasia (Osler-Weber-Rendu disease)
- Autosomal dominant inheritance.
- Abnormal, dilated capillaries (telangiectasias) present on skin, mucous membranes, internal organs.
- Causes recurrent nosebleeds and GI bleeding.
Inherited Connective Tissue Disorders
- Include:
  - Marfan's syndrome
  - Ehlers-Danlos syndrome
  - Pseudoxanthoma elasticum
- Defective connective tissue matrix causes fragile vessels → easy bruising.

B. Acquired Vascular Bleeding Disorders

Henoch-Schönlein Purpura (HSP)
- Immune-mediated vasculitis, mainly in children and young adults.
- Caused by IgA immune complex deposition in small vessel walls.
- Clinical features:
  - Purpuric rash (arms, legs, buttocks)
  - Haematuria
  - Abdominal pain, polyarthralgia
  - Acute nephritis
- Coagulation tests are normal.
Haemolytic-Uraemic Syndrome (HUS)
- Primarily affects infants and young children.
- Features:
  - Bleeding tendency
  - Acute renal failure
  - Hyaline thrombi in glomeruli of kidneys.
Simple Easy Bruising (Devil’s pinches)
- Common in women of reproductive age.
- Unknown cause.
Infection
- Septicaemia and severe viral infections (e.g., measles) can cause toxic endothelial damage or DIC → bleeding.
Drug Reactions
- Certain drugs can cause hypersensitivity vasculitis (also called leucocytoclastic vasculitis).
- Leads to vascular damage and bleeding.
Steroid Purpura
- Due to long-term steroid use or Cushing’s syndrome.
- Causes weakening of vascular support tissue.
Senile Purpura
- Age-related atrophy of vascular supportive tissue.
- Common on forearms and hands in elderly.
Scurvy
- Vitamin C deficiency.
- Defective collagen synthesis → bleeding in skin, muscles, GI and urinary tracts.

HAEMORRHAGIC DIATHESES DUE TO PLATELET DISORDERS

Bleeding due to platelet disorders can result from:

A. Thrombocytopenia (Low Platelet Count)

Defined as <150,000/μL.
Clinically significant bleeding when <20,000/μL.
Bleeding features:
- Purpura, mucosal haemorrhages, prolonged bleeding after trauma.

Causes of Thrombocytopenia

Impaired production (e.g., bone marrow failure)
Increased destruction (e.g., autoimmune or drug-induced)
Splenic sequestration (platelets trapped in enlarged spleen)
Dilutional loss (massive transfusion)

Important Causes

1. Drug-Induced Thrombocytopenia

Drugs form drug-antibody complexes damaging platelets.
Common drugs:
- Chemotherapeutics (alkylating agents, anthracyclines, antimetabolites)
- Antibiotics (sulfonamides, rifampicin, penicillins)
- Cardiovascular drugs (digitoxin, thiazides)
- Others: diclofenac, acyclovir, heparin, ethanol
Clinical features:
- Acute purpura
- Very low platelet count (often <10,000/μL)
- Bone marrow: normal or increased megakaryocytes
Treatment:
- Discontinue offending drug
- Avoid re-exposure
- In severe cases: steroids, plasmapheresis, platelet transfusion

2. Heparin-Induced Thrombocytopenia (HIT)

Unique from other drug-induced causes.
Features:
- Moderate thrombocytopenia (not usually <20,000/μL)
- Thrombosis, not bleeding, is common
Mechanism:
- Autoantibodies against PF-4-heparin complex
- Activates endothelial cells → clot formation
Occurs 5–10 days after starting heparin.
Diagnosis: Based on 4 Ts:
1. Thrombocytopenia
2. Thrombosis
3. Timing
4. No other causes

✅ Immune Thrombocytopenic Purpura (ITP)

An acquired immune disorder characterized by immune-mediated destruction of platelets with normal or increased megakaryocytes in the bone marrow.

Pathogenesis:

🔹 Acute ITP:

Common in children, post-viral illness (e.g., hepatitis C, HIV, CMV).
Mechanism: Immune complexes or cross-reacting antibodies destroy platelets.
Self-limited, usually resolves in weeks to 6 months.

🔹 Chronic ITP:

Seen in adults, more in women (20–40 yrs).
Often idiopathic but can be associated with SLE, HIV/AIDS, thyroiditis.
Mechanism:
- IgG autoantibodies against platelet glycoproteins (Gp IIb/IIIa, Gp Ib/IX).
- Platelet destruction occurs primarily in the spleen via the reticuloendothelial system (RES).

Clinical Features:

Petechiae, easy bruising, mucosal bleeding (gums, nose, menorrhagia, GI, urinary).
No lymphadenopathy; splenomegaly may occur in chronic ITP.
Rare: Intracranial hemorrhage.

🧪 Laboratory Findings:

Low platelet count (10,000–50,000/μl).
Peripheral blood smear: Large platelets.
Bone marrow: Increased megakaryocytes with abnormal morphology.
Anti-platelet antibodies detectable (IgG).
Reduced platelet lifespan (<1 hr vs. normal 7–10 days).

💊 Treatment:

Acute ITP: Spontaneous recovery in 90% cases.
Chronic ITP:
- Corticosteroids (1st line) – suppress antibody production and macrophage activity.
- Immunosuppressants: Vincristine, azathioprine, cyclophosphamide.
- Splenectomy: Removes site of destruction and antibody production.
- Platelet transfusion: For emergency bleeding only.

✅ Thrombotic Thrombocytopenic Purpura (TTP) & Hemolytic-Uremic Syndrome (HUS)

Both are thrombotic microangiopathies (TMAs).
Characterized by:
1. Microangiopathic hemolytic anemia
2. Thrombocytopenia
3. Widespread microvascular thrombosis (platelet-fibrin thrombi)

🔬 Pathogenesis:

Unlike DIC, coagulation cascade is not the primary driver.
Initiated by endothelial injury → release of vWF → platelet-rich microthrombi.
Triggers include: Pregnancy, cancer, HIV, chemotherapy, mitomycin C.

⚠️ Clinical Features:

TTP: Classic pentad
1. Fever
2. Thrombocytopenia
3. MAHA (schistocytes, negative Coombs)
4. Neurologic symptoms (confusion, stroke-like episodes)
5. Renal failure

🧪 Laboratory Findings:

Thrombocytopenia
MAHA: Negative Coombs test; schistocytes on smear
Leukocytosis, ± leukaemoid reaction
Bone marrow: Normal or ↑ megakaryocytes, ± myeloid hyperplasia
Biopsy: Shows platelet-fibrin thrombi in small vessels without vasculitis

B. THROMBOCYTOSIS

Thrombocytosis is defined as a platelet count > 400,000/μl.

Types:

Primary (Essential) Thrombocytosis / Thrombocythaemia:
- A type of myeloproliferative neoplasm.
- Associated with clonal proliferation of megakaryocytes.
Secondary (Reactive) Thrombocytosis:
- Occurs in response to another condition:

Massive haemorrhage
Iron deficiency anaemia
Severe sepsis
Marked inflammation
Disseminated malignancies
Haemolysis
Post-splenectomy

Clinical Significance:

May cause bleeding or thrombotic complications.
The exact mechanism of how elevated platelet count leads to these outcomes is not well understood.

Management:

Reactive thrombocytosis usually does not require specific treatment.
Management focuses on treating the underlying cause.

C. DISORDERS OF PLATELET FUNCTIONS

Overview:

Suspected in patients with:
- Skin and mucosal bleeding
- Prolonged bleeding time
- Normal platelet count
Can be hereditary or acquired.

I. HEREDITARY DISORDERS OF PLATELET FUNCTION

Classified based on primary functional defect:

1. Defective Platelet Adhesion:

Platelets fail to stick to damaged endothelium.
Examples:
- Bernard-Soulier Syndrome:
  - Autosomal recessive
  - Deficiency of Gp Ib-IX complex (vWF receptor)
  - Results in defective adhesion to subendothelium.
- von Willebrand Disease:
  - Deficiency or dysfunction of vWF
  - Affects both platelet adhesion and factor VIII levels

2. Defective Platelet Aggregation:

Platelets fail to stick to each other.
Example:
- Glanzmann’s Thrombasthenia:
  - Deficiency of Gp IIb-IIIa complex (fibrinogen receptor)
  - Impaired aggregation with ADP or collagen

3. Disorders of Platelet Release Reaction:

Initial aggregation is normal, but release of granule contents (ADP, 5-HT, prostaglandins) is defective.
Result from intracellular storage pool deficiencies or signaling defects.

II. ACQUIRED DISORDERS OF PLATELET FUNCTION

1. Aspirin Therapy:

Inhibits cyclooxygenase (COX) enzyme (converts arachidonic acid into prostaglandin H₂ (PGH₂))
Reduces thromboxane A₂ and prostaglandin synthesis
Leads to:
- Impaired platelet aggregation and release
- Prolonged bleeding time
Clinically used to prevent thromboembolic events in patients at risk for myocardial infarction or stroke

2. Other Acquired Causes:

Various systemic conditions interfere with platelet function:
- Uraemia
- Liver disease
- Multiple myeloma
- Waldenström’s macroglobulinaemia
- Myeloproliferative disorders

COAGULATION DISORDERS

Overview

Coagulation disorders involve abnormalities in plasma coagulation factors, either inherited or acquired.
They are less common than platelet or vascular bleeding disorders.
Bleeding pattern: Unlike petechiae or purpura (typical of platelet disorders), coagulation disorders show:
- Large ecchymoses
- Deep muscle hematomas
- Hemarthroses (bleeding into joints)
- Bleeding in body cavities (e.g., CNS, GIT, urinary tract)

Diagnosis:

Screening Tests

Whole blood coagulation time
Bleeding time
Activated partial thromboplastin time (APTT/PTTK)
Prothrombin time (PT)
Coagulation factor assays

Types of Coagulation Disorders

A. Hereditary Coagulation Disorders

Usually due to single factor deficiency, most commonly:

Haemophilia A (Classic Haemophilia) – Factor VIII deficiency
Haemophilia B (Christmas Disease) – Factor IX deficiency
von Willebrand Disease (vWD) – Defective or deficient vWF

1. Haemophilia A (Classic Haemophilia)

Inheritance: X-linked recessive → affects males; females are usually carriers.
Pathogenesis:
- 90%: Low factor VIII level
- 10%: Normal levels but reduced activity
- Factor VIII synthesized in liver, complexes with vWF in plasma
- <5% activity → symptomatic disease
Clinical Features:
- Delayed bleeding post-injury
- Recurrent hemarthroses, muscle hematomas, hematuria
- Rare: intracranial/oropharyngeal bleeds
Lab Findings:
- ↑ APTT
- Normal PT
- ↓ Factor VIII activity
- Normal bleeding time & platelet count
Treatment:
- Factor VIII concentrates
- Cryoprecipitates
- Risk: transmission of HIV, hepatitis from blood products

2. Haemophilia B (Christmas Disease)

Deficiency of: Factor IX
Inheritance and Clinical features: Same as Haemophilia A
Diagnosis:
- ↓ Factor IX activity (essential to differentiate from Hemophilia A)
Treatment:
- Factor IX enriched plasma or fresh frozen plasma
- Risk: hepatitis, DIC, thrombosis

3. von Willebrand’s Disease (vWD)

Most common hereditary coagulation disorder
Inheritance: Autosomal dominant (affects both sexes)
Defective Protein: von Willebrand factor (vWF)
- Encoded on chromosome 12
- Synthesized in endothelial cells, megakaryocytes, platelets
- Facilitates platelet adhesion
Types:
- Type I (Most common): Mild ↓ vWF
- Type II: Normal vWF quantity, ↓ function
- Type III: No vWF, severe disease
Clinical Features:
- Mucosal bleeding (e.g., gums, nose), menorrhagia
- Post-surgical bleeding
Lab Findings:
- ↑ Bleeding time
- Normal platelet count
- ↓ Factor VIII activity
- ↓ vWF levels
- Defective platelet aggregation with ristocetin
Treatment:
- Cryoprecipitates or Factor VIII concentrates

B. Acquired Coagulation Disorders

Usually involve multiple factor deficiencies

1. Vitamin K Deficiency

Vitamin K-dependent factors: II, VII, IX, X, Protein C, Protein S
Causes:
- Neonates: immature liver, sterile gut, low milk content
- Adults: poor diet, malabsorption, liver disease
Lab Findings:
- ↑ PT and APTT
Treatment:
- Parenteral vitamin K

2. Coagulation Disorders in Liver Disease

Liver synthesizes most coagulation factors and inhibitors (e.g., Protein C, S, AT III)
Bleeding in liver disease due to:
- Portal hypertension (→ splenomegaly, thrombocytopenia)
- Decreased synthesis of factors and inhibitors
- Vitamin K malabsorption
- Systemic fibrinolysis or DIC
Lab findings:
- ↑ PT, APTT
- Mild thrombocytopenia
- ↓ vitamin K-dependent factors
- Normal fibrinogen

Summary Table: Lab Findings in Major Coagulation Disorders

Disorder	BT	PT	APTT	Factor Assay	Platelet Count
Haemophilia A	N	N	↑	↓ Factor VIII	N
Haemophilia B	N	N	↑	↓ Factor IX	N
von Willebrand Disease	↑	N	↑	↓ vWF & Factor VIII	N
Vitamin K Deficiency	N	↑	↑	↓ II, VII, IX, X	N
Liver Disease	N	↑	↑	Multiple ↓	↓ (mild)

WHITE BLOOD CELLS (LEUCOCYTES): NORMAL AND REACTIVE

Types of White Blood Cells

White blood cells (WBCs) are divided into two main categories:

Granulocytes (contain granules in cytoplasm)
- Neutrophils
- Eosinophils
- Basophils
Agranulocytes (Non-granular leucocytes)
- Lymphocytes: T cells, B cells, and Natural Killer (NK) cells
- Monocytes

🧬 GRANULOPOIESIS (Formation of Granulocytes)

Site of Production

All granulocytes are made in the bone marrow.
They are part of the myeloid series.

Maturation Stages

Myeloblast – Earliest precursor, no granules, large nucleus with 2–5 nucleoli
Promyelocyte – Larger than myeloblast, contains primary (azurophilic) granules
Myelocyte – Appearance of secondary (specific) granules, nucleus off-center
Metamyelocyte – Nucleus becomes indented like a kidney or horseshoe
Band Cell – Immature granulocyte with band-shaped nucleus
Segmented Granulocyte – Mature form: neutrophil, eosinophil, basophil

Regulation

Controlled by G-CSF and GM-CSF (colony-stimulating factors)

🧪 MONOCYTE-MACROPHAGE SERIES

Stages

Monoblast – Similar to myeloblast, may show phagocytosis
Promonocyte – Larger cell with indented nucleus, fine granules
Monocyte – Enters blood, then tissues to become a macrophage

Function

Phagocytosis (ingestion of microbes/debris)
Secrete enzymes and cytokines (e.g., TNF-α, IL-1)

🛡️ LYMPHOPOIESIS (Formation of Lymphocytes)

Sites

Primary lymphoid organs: Bone marrow (B cells), Thymus (T cells)
Secondary lymphoid tissues: Lymph nodes, spleen

Types of Lymphocytes

T Cells – Cell-mediated immunity
B Cells – Humoral immunity (make antibodies)
Natural Killer (NK) Cells – Part of innate immunity

Maturation Stages

Lymphoblast – Large cell, fine chromatin, 1–2 nucleoli, basophilic cytoplasm
Prolymphocyte – Slightly condensed chromatin, small nucleolus
Lymphocyte – Mature form (small round cell with dense nucleus)

🔬 MATURE WHITE BLOOD CELLS

Type	Normal % in Blood	Key Functions
Neutrophils	50–70%	Phagocytosis, inflammation, bacterial kill
Lymphocytes	20–40%	Immunity (T, B, NK cells)
Monocytes	2–10%	Phagocytosis, become tissue macrophages
Eosinophils	1–6%	Allergic reactions, parasitic defense
Basophils	<1%	Histamine release, allergic responses

🧫 Markers of Myeloid Cells

Stage	Markers
Myeloid lineage	CD33, CD13, CD15
From myelocyte onward	CD11b, CD14
Band + mature neutrophils	CD10, CD16
Monocytes	CD14 (LPS receptor)

Lymphocytes

Morphology

Size: Small (9–12 μm) and Large (12–16 μm).
Nucleus: Round or slightly indented with coarsely clumped chromatin.
Cytoplasm: Scanty, basophilic.
Plasma Cells:
- Derived from B lymphocytes.
- Eccentric nucleus with cartwheel pattern chromatin.
- Deeply basophilic cytoplasm with a pale perinuclear halo.
- Not found in normal peripheral blood; seen in Multiple Myeloma.
Reactive Lymphocytes (Turk Cells):
- Found in viral infections.
- Have basophilic cytoplasm, resembling plasma cells.

Types and Functions

T lymphocytes (Thymus-dependent)
- Mature in thymus.
- Involved in cell-mediated immunity (CMI).
- CD markers:
  - CD3+: General T cells.
  - CD4+: Helper/delayed hypersensitivity.
  - CD8+: Cytotoxic/killer cells.
B lymphocytes (Bone marrow-dependent)
- Give rise to plasma cells.
- Mediate humoral immunity (HI).
NK Cells (Natural Killer Cells)
- Morphologically similar to lymphocytes.
- Lack B/T cell markers.
- Part of innate immunity – kill virally infected cells and tumors.

Pathologic Variations

Lymphocytosis (> 4,000/μL):
- Acute infections: Pertussis, hepatitis.
- Chronic infections: Tuberculosis, syphilis.
- Hematologic disorders: Leukemias, lymphoma.
- Relative lymphocytosis: Viral infections, thyrotoxicosis, neutropenia.
Lymphopenia (< 1,500/μL):
- Acute infections.
- Bone marrow failure.
- Immunosuppressive therapy.
- Irradiation.

Monocytes

Morphology

Size: Largest WBC (12–20 μm).
Nucleus: Large, central, oval/notched/horseshoe-shaped.
Chromatin: Fine, reticulated.
Cytoplasm: Pale blue, contains fine granules and vacuoles.

Functions

Phagocytosis of antigens/microorganisms.
Antigen presentation to lymphocytes.
Inflammation mediation via prostaglandin release, acute phase response.

Tissue macrophages (RE system) derive from monocytes.

Pathologic Variations

Monocytosis (> 800/μL):
- Infections: Tuberculosis, syphilis, malaria, typhus.
- Convalescence: After acute infections.
- Haematopoietic disorders: Leukemias, lymphomas.
- Malignancies: Breast, ovary, stomach cancer.
- Collagen vascular diseases.

Eosinophils

Morphology

Size: 12–15 μm (similar to neutrophils).
Nucleus: Bilobed.
Granules: Coarse, deep red, contain:
- Major basic protein.
- Peroxidase (stains intensely).
- IL-3, IL-5, adhesion molecules.
- Charcot-Leyden crystals (in asthmatic lungs).

Functions

Mediate allergic reactions.
Respond to parasites and antigen-antibody complexes.

Pathologic Variations

Eosinophilia (> 400/μL):
- Allergic disorders: Asthma, urticaria
- Parasitic infections.
- Skin diseases.
- Haematologic diseases: CML, Hodgkin's.
- Malignancy, irradiation, autoimmune diseases.
Eosinopenia (< 40/μL):
- Due to adrenal steroids or ACTH.

Basophils

Morphology

Appearance: Like other granulocytes but with:
- Coarse, dark blue basophilic granules.
- Granules may obscure the nucleus.
Contents: Heparin, histamine, serotonin (5-HT).
Degranulation leads to histamine release (allergic reactions).

Pathologic Variations

Basophilia (> 100/μL):
- Rare; seen in:
  - CML
  - Polycythaemia vera
  - Ulcerative colitis
  - Post-splenectomy

Leukaemoid Reactions

Definition

Marked reactive leucocytosis mimicking leukemia.
Lacks features of leukemia: splenomegaly, lymphadenopathy, haemorrhages.
Divided into myeloid and lymphoid types.

Myeloid Leukaemoid Reaction

Causes

Infections: Pneumonia, TB, sepsis, endocarditis.
Toxins: burns, mercury.
Malignancies: Multiple myeloma, metastasis.
Severe hemorrhage/hemolysis.

Laboratory Features

WBC count raised (not > 100,000/μL).
Immature granulocytes (myelocytes, metamyelocytes) < 15%.
<5% blasts.
Toxic granulation, in neutrophils.
NAP/LAP score high (unlike in CML).
Philadelphia chromosome negative.
Bone marrow: Myeloid hyperplasia

Lymphoid Leukaemoid Reaction

Causes

Viral infections: CMV, measles.
Rarely due to malignancies.

Laboratory Findings

WBC not > 100,000/μL.
Mature lymphocytes predominate, may mimic CLL.

MYELODYSPLASTIC SYNDROMES (MDS)

A clonal hematopoietic stem cell disorder characterized by:
- Dysmyelopoiesis (abnormal development of marrow elements).
- Cytopenias in peripheral blood.
- Hypercellular marrow with ineffective hematopoiesis.
Also called:
- Preleukemic syndromes

Classification

1. FAB Classification (1983)

Based on blast count and morphology:

Refractory Anaemia (RA)
Refractory Anaemia with Ringed Sideroblasts (RARS)
Refractory Anaemia with Excess Blasts (RAEB)
Chronic Myelomonocytic Leukaemia (CMML) features of both myelodysplastic syndrome (MDS) and myeloproliferative neoplasm (MPN)
RAEB in Transformation (RAEB-t) A preleukaemic condition with 20–30% blasts in the bone marrow and/or ≥5% blasts in the peripheral blood.

2. WHO Classification (2002)

Refined criteria, especially regarding blast % and genetic features:

Refractory Anaemia (RA): <5% blasts, no blasts in blood.
RARS: Similar to RA but with >15% ringed sideroblasts in marrow.
Refractory Cytopenia with Multilineage Dysplasia (RCMD): Cytopenias in ≥2 lineages, <5% blasts, monocytosis.
RCMD with Ringed Sideroblasts (RCMD-RS): RCMD + >15% ringed sideroblasts.
RAEB-1: 5–9% marrow blasts, <5% blasts in blood.
RAEB-2: 10–19% marrow blasts.
MDS Unclassified (MDS-U): Dysplasia without blasts, <5% marrow blasts.
MDS with isolated del(5q): <5% blasts, normal/increased megakaryocytes, 5q deletion.

Pathophysiology

Etiology

Primary (idiopathic) MDS
Secondary MDS:
- Post-radiation or chemotherapy
- After aplastic anemia treatment

Cytogenetic Abnormalities

Found in ~50% of MDS cases
Common: Trisomy, translocations, deletions
Aneuploidy associated with leukemic transformation

Molecular Abnormalities

p53 mutations
Increased apoptosis in marrow
Mitochondrial gene mutations → sideroblastic anemia & disordered iron metabolism

Clinical Features

Common in elderly males (>60 years)
Therapy-related MDS may occur at any age, ~10 years post-treatment

Symptoms

Often asymptomatic, found on routine CBC
May present with:
- Fatigue, pallor, weakness (from anaemia)
- Fever
- Weight loss
- Splenomegaly (20% of cases)

Laboratory Findings

Peripheral Blood

Cytopenias: Bi- or pancytopenia
Anaemia: Usually macrocytic or dimorphic
TLC: Usually normal; CMML may have ↑TLC
Neutrophils: Hypogranular, hyposegmented
Blasts: Present in peripheral smear; correlate with marrow blasts
Platelets: Thrombocytopenia; large

Bone Marrow

Cellularity: Normal, hypercellular, or hypocellular
Erythroid lineage: Dyserythropoiesis (abnormal nuclei, megaloblasts, ring sideroblasts)
Myeloid lineage: Hyposegmented and hypogranular precursors; increased blasts in some types
Megakaryocytes: Abnormal nuclei; often reduced in number

Treatment & Prognosis

Difficult to treat
Stem cell transplantation: Only curative option
Response to chemotherapy is generally poor

Prognosis

Varies with type:
- RA, RARS, 5q- syndrome: Long survival (years)
- RAEB-1, RAEB-2: Poor prognosis (survival in months)
Patients often succumb to:

Infections
Progression to AML

Leukaemias

Leukaemias are cancers of the blood-forming tissues in the body, such as the bone marrow and lymphatic system. They lead to the overproduction of abnormal white blood cells (called leukaemic cells).

Key characteristics:

Neoplastic: These are new, uncontrolled growths (tumors).
Clonal disorder: The abnormal cells come from a single transformed cell and multiply rapidly.
Malignant transformation: A normal stem cell (immature cell that can become any blood cell) turns cancerous.
The cancerous cells grow uncontrollably and suppress the production of normal cells like red cells (→ anemia), white cells (→ increased infections), and platelets (→ bleeding tendency).
These leukaemic cells are immature and dysfunctional, meaning they can’t perform their normal immune functions.

Causes of Leukaemia (Aetiology / Transformation Triggers)

Leukaemia often arises due to genetic damage to haematopoietic (blood-forming) cells. This damage may be caused by:

Ionizing Radiation
- Example: Radiation from atomic bombs, radiotherapy.
Chemicals
- Especially benzene
Viruses
- Certain viruses (e.g. HTLV-1)
Cytotoxic (anticancer) drugs
- Drugs used for treating cancer (e.g. alkylating agents)
Genetic & Hereditary Factors
- Oncogene activation: Certain genes (oncogenes) become abnormally active and promote cancer.
- Chromosomal abnormalities: Like translocations (gene swapping between chromosomes), deletions, or extra chromosomes (trisomies).
- These abnormalities may also influence the prognosis (outcome) of the disease.

Classification of Leukaemias

Leukaemias are classified in two ways:

1. Based on speed of progression:

Acute: Rapid progression, affects immature (blast) cells.
Chronic: Slower progression, affects more mature cells.

2. Based on cell of origin:

Myeloid: From bone marrow cells that normally make red cells, platelets, and some white cells.
Lymphoid: From cells that would normally develop into lymphocytes (B or T cells).

➤ Main types:

Acute Myeloid Leukaemia (AML)
Acute Lymphoblastic Leukaemia (ALL)
Chronic Myeloid Leukaemia (CML)
Chronic Lymphocytic Leukaemia (CLL)

Acute Leukaemias (AML & ALL)

What happens?

The bone marrow produces immature "blast" cells uncontrollably:
- AML: Myeloblasts or monoblasts.
- ALL: Lymphoblasts.
These blasts crowd out normal cells, leading to:
- Anemia → fatigue, pallor
- Neutropenia → infections
- Thrombocytopenia → bleeding, bruising
Blasts may also spill into the peripheral blood, a hallmark feature of leukaemia.

Symptoms & Signs

Common to both:

Fever (due to infection or disease activity)
Fatigue (due to anemia)
Bleeding/bruising (due to low platelets)
Bone pain (due to marrow expansion)

AML-specific:

Gum hypertrophy (infiltration of leukemic cells (especially monoblasts) into the gingival tissue)
Skin lesions
Chloromas: Green-colored tumors of leukaemic cells (common in the face).
Bleeding problems, especially in acute promyelocytic leukaemia (a subtype of AML).

ALL-specific:

CNS involvement (headache, vomiting, nerve palsies – in later stages).
Lymphadenopathy and hepatosplenomegaly (enlarged liver and spleen).

FAB Classification of Acute Myeloblastic Leukaemias (AML)

FAB Type	% of Cases	Morphology (Cell Features)	Cytochemical Reactions
M0: Minimally Differentiated AML	~2%	Blasts show no clear myeloid features under microscope; identified by myeloid markers only.	Myeloperoxidase: Negative
M1: AML without Maturation	~20%	Myeloblasts dominate; very few granules or Auer rods (fused primary (azurophilic) granules).	Myeloperoxidase: Positive (+)
M2: AML with Maturation	~30%	Myeloblasts and promyelocytes present; Auer rods may be seen.	Myeloperoxidase: Strongly Positive (+++)
M3: Acute Promyelocytic Leukaemia (APL)	~5%	Abundant abnormal promyelocytes; multiple Auer rods common.	Myeloperoxidase: Strongly Positive (+++)
M4: Acute Myelomonocytic Leukaemia (Naegeli Type)	~30%	Both myeloid and monocytic cells mature; myeloid cells resemble M2 type.	Myeloperoxidase: Moderately Positive (++)Non-specific esterase: Positive (+)
M5: Acute Monocytic Leukaemia (Schilling Type)	~10%	Two subtypes: M5a: Poorly differentiated monoblasts M5b: Differentiated promonocytes & monocytes	Non-specific esterase: Strongly Positive (++)
M6: Acute Erythroleukaemia (Di Guglielmo’s Syndrome)	<5%	Predominantly erythroblasts (>50% of cells); myeloblasts also increased.	Erythroblasts: PAS Positive Myeloblasts: Myeloperoxidase Positive
M7: Acute Megakaryocytic Leukaemia	<5%	Blasts are variable in shape; show platelet lineage by reacting with antiplatelet antibodies.	Platelet peroxidase: Positive

FAB Classification of Acute Lymphoblastic Leukaemia (ALL)

FAB Type	Typical Age Group	Morphology (Appearance of Lymphoblasts)	Cytochemistry
L1: Childhood ALL(Common in B-ALL and T-ALL)	More common in children	Uniform (homogeneous) small lymphoblastsScant cytoplasmRound, regular nuclei with small/inconspicuous nucleoli	PAS: Variable (±)Acid Phosphatase: Variable (±)
L2: Adult ALL(Mostly T-ALL)	More frequent in adults	Large and varied (heterogeneous) lymphoblastsVariable cytoplasmIrregular or cleft nuclei with large nucleoli	PAS: Variable (±)Acid Phosphatase: Variable (±)
L3: Burkitt-type ALL(B-cell ALL subtype)	Uncommon	Large, uniform (homogeneous) lymphoblastsRound nuclei with prominent nucleoliAbundant cytoplasmic vacuoles (often seen in Burkitt lymphoma)	PAS: Negative (–)Acid Phosphatase: Negative (–)

Epidemiology

ALL: Common in children, especially boys.
AML: More frequent in adults.
In Africa, both types occur equally in children under 15.

If not treated:

Fatal within weeks to months due to severe infections or bleeding, as normal marrow function is lost.

Chronic Lymphocytic Leukaemia (CLL)

Cancer of mature B-lymphocytes (sometimes T-cells).
Cells look mature but don’t function properly.

Common in older adults, especially males >60.
In Africa, seen in younger individuals, sometimes associated with chronic malaria and enlarged spleen.

Symptoms

Often no symptoms early on; discovered on routine blood test.
Later:
- Weight loss
- Swollen lymph nodes
- Enlarged spleen/liver
- Recurrent infections due to poor immunity
- Anaemia: From autoimmune destruction or enlarged spleen
- Bleeding/bruising: From low platelets (marrow failure)

Stage	Description	Clinical Findings	Risk Category
0	Lymphocytosis only	Absolute lymphocytosis in blood and bone marrow (>5 × 10⁹/L)	Low Risk
I	Lymphocytosis + lymphadenopathy	Enlarged lymph nodes (cervical, axillary, inguinal)	Intermediate Risk
II	Lymphocytosis + splenomegaly and/or hepatomegaly	Palpable spleen and/or liver	Intermediate Risk
III	Lymphocytosis + anemia (Hb <11 g/dL)	Fatigue, pallor, other signs of anemia	High Risk
IV	Lymphocytosis + thrombocytopenia (platelets <100 × 10⁹/L)	Easy bruising, bleeding, petechiae	High Risk

Outcome

Generally slow-growing.
Rarely changes into acute leukaemia.
Death usually from infection, marrow failure, or organ damage.

Chronic Myeloid Leukaemia (CML)

Cancer of myeloid cells (cells that make red cells, granulocytes, platelets).
Strongly associated with the Philadelphia chromosome (Ph+):
- A t(9;22) translocation creates the BCR-ABL gene, which makes the cells divide uncontrollably.

Common in young to middle-aged adults.
In developing countries, also found in children.

Stage	Clinical Features	Hematologic Findings	Bone Marrow / Cytogenetic Findings	Comments
1. Chronic Phase	- Often asymptomatic or mild symptoms- Fatigue, weight loss- Splenomegaly	- WBC count elevated- Basophilia and eosinophilia- <10% blasts in blood or marrow	- Normal to hypercellular marrow- Philadelphia chromosome (t(9;22)) present in >95% cases	- Most patients diagnosed at this stage- Excellent response to tyrosine kinase inhibitors (e.g., imatinib)
2. Accelerated Phase	- Increasing symptoms: fatigue, bone pain, fever- Progressive splenomegaly	- 10–19% blasts in blood or marrow- Platelets >1,000,000 or <100,000/µL- Basophils ≥20%	- Increasing cytogenetic abnormalities- Reduced response to therapy	- Transition phase between chronic and blast phase
3. Blast Crisis	- Severe symptoms: anemia, bleeding, infections- May resemble acute leukemia	- ≥20% blasts in blood or marrow- Possible presence of extramedullary blast proliferation (e.g., in skin, CNS)	- May show additional mutations or chromosomal abnormalities- Blasts may be myeloid or lymphoid	- Poor prognosis- Requires aggressive treatment (chemotherapy, stem cell transplant)

Blood Picture

Very high white cell count (can be >300 × 10⁹/L).
Presence of immature and mature myeloid cells in blood.

Haematopoietic System

🦴 BONE MARROW & HAEMATOPOIESIS

📌 1. Bone Marrow and Blood Cell Formation

🕒 2. Lifespan of Blood Cells

🧬 3. Haematopoiesis (Blood Cell Formation)

🧪 4. Haematopoietic Stem Cells

💉 5. Growth Factors & Hormones in Haematopoiesis

Red Blood Cells (RBCs)

1. Erythropoiesis (Formation of Red Blood Cells)

Site of Erythropoiesis:

Stages of Erythropoiesis:

Regulation:

2. Structure of Red Blood Cells

Normal Morphology:

Advantages of Biconcave Shape:

3. Function of Red Blood Cells

Primary Function:

Secondary Functions:

4. Membrane Biology of RBCs

Composition:

Cytoskeletal Network:

Defects in Membrane Proteins:

5. Nutritional Requirements for Erythropoiesis

Essential Nutrients:

Deficiency Effects:

6. Hemoglobin Synthesis

Structure:

Globin Synthesis:

Heme Synthesis:

Iron Incorporation:

Additional Concepts

RBC Lifespan:

Breakdown Products:

Anaemia

2. Pathophysiologic Classification of Anaemia

A. Blood Loss

B. Decreased Red Cell Production

C. Increased Red Cell Destruction (Hemolysis)

3. Clinical Features of Anaemia

A. General Symptoms (related to hypoxia):

B. Signs on Physical Examination:

C. Specific Features Based on Cause:

4. Laboratory Investigations in Anaemia

A. Basic Tests

B. Iron Studies

C. Vitamin Levels

D. Other Investigations

5. Morphological Classification of Anaemia (based on MCV)

6. Reticulocyte Index (RI)

Classification of Anemias

A. Pathophysiologic Classification

B. Morphologic Classification

Hypochromic Anaemia

Definition

Types

Iron Deficiency Anaemia (IDA)

Prevalence

Iron Metabolism

Iron Balance

Iron Absorption

Iron Transport

Iron Storage

Excretion

Pathogenesis of IDA

Etiology of Iron Deficiency Anaemia

1. Increased Blood Loss

2. Increased Requirements

3. Inadequate Dietary Intake

4. Decreased Absorption

High-Risk Groups

1. Females (Reproductive Age)

2. Post-Menopausal Women

3. Adult Males

4. Infants & Children

Clinical Features

1. Anaemia Symptoms

2. Epithelial Tissue Changes (with chronic IDA)

Sideroblastic Anaemias

1. Definitions

2. Classification of Sideroblastic Anaemias