---

Question 1 — Haemostasis, Coagulation Factors, Events of Coagulation, Platelets, Aspirin in Stroke [1+5+3+3]

What is Haemostasis?

Coagulation Factors

Events of Coagulation — Brief Outline

EXTRINSIC PATHWAY              INTRINSIC PATHWAY
Tissue damage                  Contact of XII with collagen
       ↓                                ↓
Tissue factor (III)            XII → XIIa
released                              ↓
       ↓                       XI → XIa
VII + TF + Ca²⁺                       ↓
       ↓                       IX → IXa
X → Xa  ←─────────────────── IXa + VIIIa + Ca²⁺ + PL
       ↓
  Xa + Va + Ca²⁺ + PL (Platelet phospholipid)
       ↓
  PROTHROMBIN ACTIVATOR
       ↓
  Prothrombin (II) → Thrombin (IIa)    [Ca²⁺ required]
       ↓
  Fibrinogen (I) → Fibrin monomer → Fibrin polymer
       ↓ (Factor XIIIa)
  Cross-linked, stable FIBRIN CLOT

Four Major Steps of Haemostasis

1. Vascular spasm - Immediate vasoconstriction due to myogenic reflex, local autacoids, and thromboxane A2 from platelets. Reduces blood flow.
2. Platelet plug formation - Platelets adhere to exposed collagen (via von Willebrand factor), become activated, and aggregate to form the primary plug.
3. Blood coagulation (clot formation) - Cascade activation of clotting factors producing fibrin, reinforcing the platelet plug.
4. Clot retraction and fibrosis - Fibrin contracts, wound edges are drawn together; eventual fibrous tissue grows into the clot.

---

Role of Platelets in Haemostasis

• Adhesion: When endothelium is injured, platelets adhere to exposed subendothelial collagen via glycoprotein receptors and von Willebrand factor (vWF).
• Activation and shape change: Platelets change from disc to spiny sphere; release contents from alpha-granules (fibrinogen, vWF, platelet factor 4) and dense granules (ADP, serotonin, calcium).
• Aggregation: Released ADP activates more platelets; thromboxane A2 (TXA2) causes aggregation and vasoconstriction. Platelets aggregate via fibrinogen bridges (GPIIb/IIIa receptors).
• Primary plug: Loose plug of platelets seals small vascular defects.
• Coagulation support: Platelet phospholipids (platelet factor 3) provide the surface for assembly of coagulation factor complexes (tenase, prothrombinase).
• Clot retraction: Thrombostenin (acto-myosin in platelets) contracts and consolidates the clot.
• Vasoconstriction: Release of thromboxane A2 and serotonin.

---

Aspirin in Prevention of Stroke

• TXA2 is a potent platelet aggregator and vasoconstrictor. By blocking its synthesis, aspirin reduces platelet aggregation.
• Most strokes (ischaemic strokes) result from thromboembolism - platelet-rich thrombi forming on atherosclerotic plaques in carotid arteries or cerebral vessels, then embolising to the brain.
• By inhibiting TXA2 synthesis irreversibly (platelets have no nucleus and cannot synthesize new COX), aspirin produces a lifelong antiplatelet effect for that platelet (~7-10 day life span).
• Low-dose aspirin (75-150 mg) is used because endothelial cells regenerate new COX and maintain prostacyclin (PGI2) production - a platelet inhibitor. High doses block both TXA2 and PGI2, negating benefit.
• Aspirin thus prevents the formation of platelet emboli that can occlude cerebral vessels causing ischaemic stroke.

---

Question 2 — Role of Neutrophils in Defence + Innate Immunity [8+4]

Role of Neutrophils in Defence

Functions:

• Neutrophils engulf bacteria, fungi, cellular debris.
• Opsonisation with IgG and C3b enhances phagocytosis via Fc and complement receptors.
• Phagosomes fuse with lysosomes to form phagolysosomes.

• Respiratory burst: NADPH oxidase generates superoxide (O₂⁻), which is converted to H₂O₂, hypochlorous acid (HOCl) via myeloperoxidase (MPO) - highly bactericidal.
• Nitric oxide (NO) production also contributes.

• Lysozyme: disrupts bacterial cell walls.
• Defensins: antimicrobial peptides that perforate bacterial membranes.
• Elastase, cathepsin G, proteinase 3: proteolytic enzymes.
• Lactoferrin: chelates iron, bacteriostatic.
• BPI (Bactericidal/Permeability-Increasing protein): disrupts gram-negative bacteria.

Defects in neutrophil function:

• Chronic Granulomatous Disease (CGD): NADPH oxidase deficiency - inability to kill catalase-positive organisms.
• Chediak-Higashi syndrome: Defective granule-lysosome fusion.
• LAD (Leukocyte Adhesion Deficiency): Defective integrins, inability to migrate.

---

What is Innate Immunity?

• Does NOT require prior sensitisation
• No immunological memory
• Responds within minutes to hours
• Recognizes broad patterns, not specific antigens

1. Physical/chemical barriers: Skin (keratin layer), mucous membranes, stomach acid (pH 2), lysozyme in tears/saliva, cilia in respiratory tract.
2. Cellular components:
   • Neutrophils (phagocytosis)
   • Macrophages (phagocytosis, antigen presentation, cytokine production)
   • Natural Killer (NK) cells (kill virus-infected and tumour cells)
   • Dendritic cells (link innate to adaptive immunity)
   • Mast cells and basophils (histamine release)
   • Eosinophils (anti-parasitic)
3. Humoral components:
   • Complement system (C3b opsonisation, MAC lysis, C5a chemotaxis)
   • Acute phase proteins (CRP, fibrinogen, serum amyloid A)
   • Interferons (IFN-α, IFN-β: antiviral)
   • Lysozyme, lactoferrin, defensins
4. Pattern Recognition Receptors (PRRs):
   • Toll-like receptors (TLRs) recognize PAMPs (Pathogen-Associated Molecular Patterns) like LPS, flagellin, viral RNA

---

Question 3 — Role of Lymphocytes in Immunity + AIDS [8+4]

Role of Lymphocytes in Immunity

B Lymphocytes — Humoral Immunity

• Mature in bone marrow; migrate to secondary lymphoid organs.
• On encountering antigen (with T-helper cell help), they differentiate into plasma cells.
• Plasma cells secrete immunoglobulins (antibodies: IgG, IgM, IgA, IgE, IgD).
• Some become memory B cells for long-lasting immunity.
• Antibody functions: neutralization, opsonisation, complement activation, ADCC.

T Lymphocytes — Cell-Mediated Immunity

• Recognize antigen presented on MHC class II by APCs.
• Secrete cytokines to activate B cells, cytotoxic T cells, and macrophages.
• Th1 cells: secrete IFN-γ, IL-2 → activate macrophages, cell-mediated immunity.
• Th2 cells: secrete IL-4, IL-5, IL-13 → activate B cells, allergic/parasitic responses.
• Th17 cells: IL-17 → neutrophil recruitment, mucosal defence.

• Recognize antigen on MHC class I (present on all nucleated cells).
• Kill virus-infected cells, tumour cells, transplanted cells via:
  • Perforin/granzyme pathway (apoptosis)
  • Fas-FasL interaction

• CD4+CD25+FoxP3+ cells
• Suppress excessive immune responses; maintain self-tolerance
• Prevent autoimmunity

Natural Killer (NK) Cells

• Neither T nor B cells; innate lymphoid-like cells
• Kill target cells lacking MHC class I (eg virus-infected, tumour cells)
• Use ADCC (antibody-dependent cellular cytotoxicity)

Memory Cells

---

Acquired Immunodeficiency Syndrome (AIDS)

1. HIV binds CD4 receptor + co-receptor (CCR5 or CXCR4) on CD4+ T cells, macrophages, dendritic cells.
2. Viral RNA is reverse-transcribed to DNA → integrates into host genome (provirus).
3. Progressive destruction of CD4+ T cells → immune collapse.
4. AIDS defined as: CD4 count < 200 cells/µL (normal 500-1500) OR presence of AIDS-defining illness.

---

Question 4 — Haemophilia + Steps of Haemostasis + Intrinsic Pathway [2+3+7]

What is Haemophilia?

• Haemophilia A: Deficiency of Factor VIII (anti-haemophilic factor A). X-linked recessive. Most common (1:5000 males).
• Haemophilia B (Christmas disease): Deficiency of Factor IX. X-linked recessive.
• Haemophilia C: Deficiency of Factor XI. Autosomal recessive.

---

Steps of Haemostasis

1. Vascular spasm: Immediate vasoconstriction reduces blood flow. Due to myogenic reflex, neural signals, and TXA2 from platelets.
2. Platelet plug (primary haemostasis): Platelet adhesion (via vWF and collagen), activation, and aggregation form a loose platelet plug.
3. Coagulation / clot formation (secondary haemostasis): Activation of coagulation cascade producing cross-linked fibrin reinforcing the platelet plug.
4. Clot retraction: Platelets contract to pull wound edges together.
5. Fibrinolysis: Plasmin dissolves the clot once wound heals; t-PA converts plasminogen to plasmin.

---

Intrinsic Pathway of Coagulation (in detail)

STEP 1: Contact Activation (Surface Activation)
Factor XII + Collagen/Kaolin/Glass → Factor XIIa
(aided by Prekallikrein + HMWK → Kallikrein, positive feedback)

STEP 2: Factor XI Activation
XIIa + HMWK → XIa

STEP 3: Factor IX Activation
XIa + Ca²⁺ → IXa

STEP 4: Tenase Complex Assembly
IXa + VIIIa + Ca²⁺ + Platelet Phospholipid
               ↓
Factor X → Factor Xa
(Note: Factor VIIIa is activated by small amounts of thrombin)

STEP 5: Prothrombinase Complex
Xa + Va + Ca²⁺ + Platelet Phospholipid (Factor 3)
               ↓
Prothrombin (II) → Thrombin (IIa)

STEP 6: Fibrin Formation
Thrombin cleaves Fibrinogen (I) → Fibrin monomer + fibrinopeptides A & B
Fibrin monomers polymerize → Fibrin polymer (loose)
Factor XIIIa (activated by thrombin) → Cross-linked fibrin (stable clot)

• Thrombin activates Factor V, Factor VIII, Factor XIII (amplifies coagulation)
• Thrombin activates more platelets

---

Question 5 — Structure of Platelets + Granule Contents + Functions [4+5+3]

Structure of Platelets

1. Peripheral zone (glycocalyx/outer coat):
   • Glycoproteins: GP Ib (receptor for vWF, adhesion), GPIIb/IIIa (fibrinogen receptor, aggregation)
   • Phospholipid bilayer containing arachidonic acid (precursor of TXA2)
2. Sol-gel zone (cytoskeleton):
   • Actin and myosin filaments (thrombostenin) - clot retraction
   • Microtubules maintain disc shape
3. Organelle zone:
   • Alpha-granules (most abundant)
   • Dense granules (δ-granules)
   • Lambda granules (lysosomes)
   • Mitochondria
4. Membrane systems:
   • Open canalicular system (OCS): connected to surface membrane, secretory pathway
   • Dense tubular system (DTS): analogous to sarcoplasmic reticulum, stores Ca²⁺, site of TXA2 synthesis

---

Granule Contents and Their Functions

Alpha (α) Granules:

Dense (δ) Granules:

Lambda (λ) Granules (lysosomes):

• Acid hydrolases (cathepsins, acid phosphatase)
• Degrade phagocytosed material

---

Functions of Platelets

1. Haemostasis (primary): Form platelet plug at site of vessel injury
2. Coagulation support: Platelet phospholipids (PF3) provide surface for tenase and prothrombinase complexes
3. Vasoconstriction: Release TXA2 and serotonin
4. Clot retraction: Thrombostenin (acto-myosin) contracts, consolidates fibrin clot
5. Wound healing: PDGF stimulates smooth muscle and fibroblast proliferation for vessel repair
6. Vascular maintenance: Seal daily microinjuries in capillaries; maintain endothelial integrity by releasing growth factors
7. Immunity: Interact with pathogens; release antimicrobial peptides; activate complement

---

Question 6 — Erythropoiesis, Sites, Stages, Regulation, Polycythaemia [2+2+4+5+2]

What is Erythropoiesis?

---

Sites of Erythropoiesis

• 0-2 months: Yolk sac (primitive erythropoiesis - megaloblastic)
• 2-7 months: Liver (main site), spleen, lymph nodes (definitive erythropoiesis)
• 5 months onwards: Bone marrow gradually takes over

• All bones initially (flat + long)
• After age 5: Progressively confined to flat bones
• Adult: Red bone marrow of flat bones - sternum, ribs, vertebrae, iliac crest, skull, upper end of humerus and femur

---

Stages of RBC Development (Erythropoiesis)

Pluripotent Stem Cell (HSC)
        ↓
Common Myeloid Progenitor (CMP)
        ↓
Megakaryocyte-Erythroid Progenitor (MEP)
        ↓
Burst Forming Unit-Erythroid (BFU-E)  ← EPO promotes maturation here
        ↓
Colony Forming Unit-Erythroid (CFU-E)
        ↓
PROERYTHROBLAST (Pronormoblast)
  - Large cell, deep basophilic cytoplasm, large nucleus
        ↓
BASOPHILIC ERYTHROBLAST (Early normoblast)
  - Still basophilic (RNA-rich), nucleus smaller
        ↓
POLYCHROMATOPHILIC ERYTHROBLAST (Intermediate normoblast)
  - Haemoglobin begins to appear (acidophilic staining)
  - Mix of blue (RNA) and pink (Hb)
        ↓
ORTHOCHROMATOPHILIC ERYTHROBLAST (Late normoblast)
  - Hb fully formed, pyknotic nucleus
        ↓
RETICULOCYTE
  - Nucleus extruded; reticular network of RNA visible with supravital stain
  - Spends 1-2 days in marrow, 1 day in circulation
        ↓
MATURE ERYTHROCYTE
  - Biconcave disc, 7.2 µm, no nucleus, no organelles, HbA
  - Life span: 120 days

---

Regulation of Erythropoiesis

• A glycoprotein hormone, 90% produced by peritubular interstitial cells of kidneys (10% liver).
• Stimulus: Tissue hypoxia → stabilizes HIF-1α → increases EPO gene transcription.
• Action: Promotes BFU-E → CFU-E differentiation; prevents apoptosis of erythroid progenitors; speeds up maturation; increases reticulocyte release.

• Androgens: stimulate EPO production (explains higher Hb in males)
• Thyroid hormone, growth hormone, cortisol: promote erythropoiesis
• Estrogens: inhibit EPO
• Iron, Vitamin B12, Folate: required for DNA synthesis and haemoglobin formation
• Intrinsic factor: required for Vitamin B12 absorption
• Stem cell factor (SCF): for early progenitor proliferation

---

Polycythaemia

1. Polycythaemia Vera (Primary): JAK2 mutation (V617F, ~95%); EPO-independent proliferation of erythroid (and other) progenitors. Features: Splenomegaly, raised RBC+WBC+platelets, ruddy complexion, pruritis after bathing, hyperviscosity.
2. Secondary Polycythaemia: Appropriate (altitude, COPD, cyanotic heart disease, high-affinity Hb) or inappropriate (EPO-secreting tumours - renal cell carcinoma, hepatocellular carcinoma).
3. Relative (Spurious): Dehydration, diuretic use - plasma volume reduced, not true increase in RBC mass.

---

Question 7 — Physiological Basis of Blood Grouping + Hazards of Blood Transfusion [6+6]

Physiological Basis of Blood Grouping

ABO System:

• Antigens are glycoproteins/glycolipids on RBC membrane coded by ABO gene locus on chromosome 9.
• Antibodies (IgM, naturally occurring) form against absent antigens from age 3-6 months (likely from exposure to environmental antigens with similar structure).
• The H antigen is the precursor substance; A and B transferases add specific sugars.
• Group O individuals have only H antigen.

Rh System:

• Rh (Rhesus) antigens: D, C, c, E, e. Most important: D antigen.
• Rh positive (Rh+): D antigen present (~85% of population).
• Rh negative (Rh-): No D antigen.
• No naturally occurring Rh antibodies - antibodies form only after exposure (transfusion or pregnancy).
• Haemolytic Disease of Newborn (HDN/Erythroblastosis foetalis): Rh- mother sensitised by Rh+ fetal blood during first pregnancy; IgG anti-D crosses placenta in subsequent Rh+ pregnancies, causing haemolysis of fetal RBCs.
• Prevention: Rho(D) immune globulin (Anti-D) given to Rh- mothers within 72 hours of delivery or abortion.

---

Hazards of Blood Transfusion

Immunological Reactions:

1. Acute Haemolytic Transfusion Reaction (AHTR): ABO incompatibility → IgM + complement → intravascular haemolysis. Features: Fever, chills, flank pain, haemoglobinuria, renal failure, DIC. Life-threatening.
2. Delayed Haemolytic Reaction: Due to minor blood group antibodies (Kidd, Duffy, Kell); extravascular haemolysis; occurs 3-14 days post-transfusion.
3. Febrile Non-Haemolytic Reaction (FNHTR): Antibodies against donor leukocytes → cytokine release → fever, chills. Most common reaction.
4. Allergic Reactions: Urticaria, anaphylaxis (especially IgA-deficient recipients with anti-IgA antibodies).
5. Transfusion-Related Acute Lung Injury (TRALI): Donor anti-leukocyte antibodies activate recipient PMNs in lungs → non-cardiogenic pulmonary oedema.
6. Graft-vs-Host Disease (TA-GvHD): In immunocompromised recipients - donor T cells attack host tissues. Fatal if not prevented with irradiation of blood.

Infective Hazards:

1. Viral infections: HIV, Hepatitis B, Hepatitis C, CMV, HTLV-I/II
2. Bacterial contamination: Especially in platelet concentrates (Staphylococcus, Pseudomonas)
3. Parasitic: Malaria, Chagas disease

Non-Immune Hazards:

1. Transfusion-associated circulatory overload (TACO): Especially in elderly or cardiac patients; pulmonary oedema.
2. Hyperkalaemia: Stored blood releases K⁺ (especially in massive transfusion)
3. Hypocalcaemia: Citrate (anticoagulant in blood bags) chelates Ca²⁺; clinically significant only in massive rapid transfusion.
4. Hypothermia: Rapid transfusion of cold blood → cardiac arrhythmias.
5. Dilutional coagulopathy: Massive transfusion dilutes clotting factors and platelets.
6. Haemosiderosis: Iron overload in patients with multiple chronic transfusions (e.g. thalassaemia major).
7. Air embolism: Rare with modern blood bags.

---

Question 8 — Define Haemostasis + Sequence of Events + Platelets in Clotting + Lab Tests for Bleeding Disorders [2+4+3+3]

Definition

Sequence of Events (see Q1 and Q4 above for full detail)

1. Vascular spasm (immediate, seconds)
2. Primary haemostasis - platelet plug formation (minutes)
3. Secondary haemostasis - fibrin clot formation (coagulation cascade)
4. Clot retraction and wound healing
5. Fibrinolysis (plasmin dissolves clot)

Role of Platelets in Clotting (see Q1, Q5 for details)

• Provide phospholipid surface (PF3) for tenase and prothrombinase complexes
• Release calcium from dense granules (cofactor for coagulation)
• Release fibrinogen, Factor V (from alpha granules)
• Seal the primary plug until fibrin reinforces

Lab Tests for Investigation of Bleeding Disorders

---

Question 9 — 60-Year-Old Post-Gastrectomy with Anaemia and Neurological Symptoms [10+5]

i) Mechanisms of Clinical Features

Mechanism of Anaemia:

• The stomach secretes Intrinsic Factor (IF) from parietal cells of the gastric body/fundus.
• IF is essential for absorption of dietary Vitamin B12 in the terminal ileum.
• After gastrectomy (especially total gastrectomy), parietal cells are removed → no intrinsic factor → Vitamin B12 malabsorption.
• B12 stores last 3-5 years, so symptoms appear months to years post-surgery.

• Vitamin B12 is required for DNA synthesis (as a cofactor for thymidylate synthesis via methyl-malonyl pathway).
• Deficiency → impaired nuclear maturation while cytoplasm continues to grow → megaloblastic erythropoiesis.
• Megaloblasts undergo premature intramedullary destruction (ineffective erythropoiesis).
• Peripheral blood shows macrocytic anaemia (MCV > 100 fL), hypersegmented neutrophils, macroovalocytes.

• Iron deficiency anaemia: Loss of gastric acid reduces non-haem iron absorption; early post-gastrectomy.
• Folate deficiency: Achlorhydria impairs folate absorption.
• Combined deficiency: Mixed picture possible.

Mechanism of Neurological Symptoms (Subacute Combined Degeneration of Spinal Cord, SACD):

• Vitamin B12 is required for synthesis of methionine from homocysteine (via methionine synthase).
• Methionine → S-Adenosyl Methionine (SAM) → essential for myelin synthesis.
• B12 deficiency → failure of myelin synthesis → demyelination of posterior and lateral columns of spinal cord.

• Posterior column degeneration: loss of vibration sense and proprioception, sensory ataxia, positive Romberg sign
• Lateral corticospinal tract degeneration: upper motor neuron signs - weakness, hyperreflexia, extensor plantar responses
• Peripheral neuropathy: glove-and-stocking sensory loss
• Cognitive impairment: "megaloblastic madness" - depression, memory loss, psychosis
• Optic atrophy (rare)

• Folate deficiency causes megaloblastic anaemia but NOT neurological involvement (because folate does not participate in the methylmalonyl-CoA → succinyl-CoA pathway critical for myelin).

---

ii) Management

• FBC (macrocytic anaemia, hypersegmented PMNs), peripheral blood film
• Serum B12 level (low)
• Serum folate, red cell folate
• Serum iron, TIBC, ferritin
• Methylmalonic acid (MMA) elevated - specific for B12 deficiency
• Homocysteine elevated (in both B12 and folate deficiency)
• Schilling test (historically) - now rarely used
• Upper GI endoscopy / surgical history review

• Since IF is absent post-gastrectomy, oral B12 is NOT reliably absorbed (except in pharmacological doses via passive diffusion).
• Intramuscular (IM) Hydroxocobalamin or Cyanocobalamin:
  • Loading: 1000 µg IM daily for 1 week, then weekly for 4 weeks
  • Maintenance: 1000 µg IM every 3 months for life
• Alternatively, high-dose oral B12 (1000-2000 µg/day) can work via passive diffusion (1% absorbed without IF) - useful if IM contraindicated.
• Neurological recovery occurs if treated early; established irreversible damage may not recover.
• Do NOT give folate alone in suspected B12 deficiency - can precipitate/worsen SACD.

• Oral ferrous sulphate if iron-deficient; monitor ferritin.

• Ensure adequate B12-rich foods (meat, dairy, eggs) though absorption remains limited without IF.

---

Question 10 — Erythrocyte Membrane Structure + Shape Maintenance + Osmotic Fragility + Hereditary Spherocytosis [5+4+4+2]

Schematic Diagram of Erythrocyte Membrane

OUTSIDE
─────────────────────────────────────────────────────────────
Glycocalyx (glycolipids, glycoproteins - ABO, Rh antigens)

[LIPID BILAYER]
Outer leaflet: Phosphatidylcholine, Sphingomyelin
Inner leaflet: Phosphatidylethanolamine, Phosphatidylserine

TRANSMEMBRANE PROTEINS:
- Band 3 (anion exchanger): Cl⁻/HCO₃⁻ exchange; anchors cytoskeleton
- Glycophorin A, B, C, D: sialylated glycoproteins; negative surface charge (prevents RBC-RBC adhesion); blood group antigens

CYTOSKELETON (just beneath lipid bilayer):
- Spectrin (α+β heterodimers) - forms horizontal meshwork
  anchored via:
  • Ankyrin (connects spectrin to Band 3)
  • Protein 4.1 (connects spectrin-actin junction to glycophorin C)
  • Actin protofilaments (short F-actin; junction complexes)
  • Dematin (protein 4.9)
  • Adducin (caps actin)
─────────────────────────────────────────────────────────────
INSIDE

---

How the Biconcave Shape is Maintained

1. Spectrin cytoskeleton: The horizontal spectrin meshwork resists shear forces. When deformed in capillaries, spectrin unfolds and refolds elastically - the RBC returns to biconcave shape.
2. Lipid bilayer-cytoskeleton coupling: The vertical connections (spectrin-ankyrin-Band 3; protein 4.1-glycophorin C) prevent the lipid bilayer from detaching from the skeleton. Detachment → loss of membrane → sphere formation.
3. Surface area-to-volume ratio: RBCs have excess membrane (SA:V ratio higher than a sphere of same volume). This excess allows biconcave shape and remarkable deformability.
4. Membrane lipid composition: The asymmetric distribution of phospholipids (phosphatidylserine inward) and cholesterol content regulate membrane curvature.
5. ATP-dependent processes: Maintenance of cation gradients (Na⁺/K⁺ ATPase) maintains cell volume; ATP-dependent lipid flipping maintains lipid asymmetry.

---

Osmotic Fragility

• Water enters the cell by osmosis → cell swells
• Normal biconcave RBCs can expand their volume (due to excess membrane SA) before the critical haemolytic volume is reached
• Spherocytes have less excess membrane → they lyse at higher saline concentrations (increased fragility)
• Hypotonically-stressed flat cells (target cells in thalassaemia) resist lysis (decreased fragility)

• RBCs placed in decreasing concentrations of NaCl (from 0.85% isotonic → 0.0% distilled water)
• Normal RBCs: begin haemolysing at 0.45-0.5% NaCl; complete at 0.30-0.35%
• Spherocytes: begin haemolysing at higher concentrations (0.6-0.7%) → increased fragility
• Target cells: complete haemolysis only at very low concentrations → decreased fragility

---

Hereditary Spherocytosis

• Mutations in spectrin (α or β), ankyrin, Band 3, or protein 4.2 genes.
• Most common: Ankyrin (ANK1) mutation - disrupts spectrin-Band 3 vertical link.
• Weakened vertical interaction → membrane lipids detach from skeleton → membrane vesiculation → loss of surface area.
• Reduced SA:V ratio → cell becomes spherical.
• Spherocytes are rigid (cannot deform) → trapped and destroyed in splenic cords (extravascular haemolysis).

• Anaemia (mild to severe)
• Jaundice (unconjugated hyperbilirubinaemia)
• Splenomegaly
• Pigment gallstones (bilirubin stones)
• Aplastic crisis (Parvovirus B19 infects erythroid precursors → sudden drop in Hb)
• Increased osmotic fragility

• Spherocytes on peripheral smear (no central pallor)
• Increased MCHC (>36 g/dL) - most discriminating feature
• Negative Coombs (DAT) test (unlike autoimmune haemolysis)
• Increased osmotic fragility

• Folic acid supplementation (to meet increased erythroid demand)
• Splenectomy (definitive treatment in moderate-severe disease): eliminates site of haemolysis; prevents aplastic crises. Vaccinate pre-splenectomy (pneumococcal, meningococcal, Hib).
• Blood transfusion in aplastic crisis

---