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PY2.1-13 | Haematology — Part 1

Blood Composition — Plasma and Formed Elements (PY2.1)

Blood is a specialised connective tissue — yes, it's a tissue, not just a fluid. It consists of two main components:

Figure: Blood Composition — Plasma and Formed Elements (PY2.1)

1. Plasma (55% of blood volume) — the liquid matrix
Plasma is 90% water and 10% dissolved substances:
• Plasma proteins (7–9 g/dL):
- Albumin (60% of plasma proteins) — maintains oncotic (colloid osmotic) pressure, transports drugs and bilirubin
- Globulins (α, β, γ) — transport proteins (α and β), antibodies (γ-globulins = immunoglobulins)
- Fibrinogen — essential for blood clotting (converted to fibrin in the coagulation cascade)
• Electrolytes: Na⁺, K⁺, Ca²⁺, Cl⁻, HCO₃⁻
• Nutrients: glucose, amino acids, lipids
• Waste products: urea, creatinine, bilirubin
• Hormones, enzymes, dissolved gases

Plasma vs serum: Plasma contains fibrinogen; serum is plasma minus fibrinogen and clotting factors (what remains after blood clots).

2. Formed elements (45% of blood volume) — the cells
• Red blood cells (erythrocytes) — 99% of formed elements. 4.5–5.5 million/µL in males, 3.8–4.8 million/µL in females
• White blood cells (leucocytes) — 4,000–11,000/µL. Five types: neutrophils, lymphocytes, monocytes, eosinophils, basophils
• Platelets (thrombocytes) — 1.5–4.0 lakh/µL (150,000–400,000/µL). Cell fragments from megakaryocytes

Haematocrit (packed cell volume, PCV): The percentage of blood volume occupied by red cells. Normal: 40–54% in males, 36–48% in females. A high haematocrit (polycythaemia) increases blood viscosity; a low haematocrit indicates anaemia.

Buffy coat: When blood is centrifuged, a thin whitish layer appears between the RBC sediment and the plasma — this is the buffy coat, containing WBCs and platelets (< 1% of blood volume).

Haematopoiesis — The Blood Cell Factory (PY2.2)

All blood cells originate from a single type of cell — the pluripotent haematopoietic stem cell (HSC) — residing in the red bone marrow. This process is called haematopoiesis.

Figure: Haematopoiesis — The Blood Cell Factory (PY2.2)

Sites of haematopoiesis change with age:
• Fetal life: Yolk sac (first 2 months) → liver and spleen (2nd trimester) → bone marrow (from 5th month)
• After birth: Bone marrow of virtually all bones in infants
• Adults: Red marrow in flat bones (sternum, ribs, iliac crest, vertebrae) and proximal ends of long bones (femur, humerus). The rest converts to yellow (fatty) marrow.

The HSC gives rise to two main lineages:
• Myeloid lineage → RBCs, platelets, granulocytes (neutrophils, eosinophils, basophils), monocytes
• Lymphoid lineage → lymphocytes (T cells, B cells, NK cells)

Erythropoiesis — making red blood cells:
The sequence: HSC → committed erythroid progenitor (BFU-E → CFU-E) → proerythroblast → basophilic erythroblast → polychromatic erythroblast → orthochromatic erythroblast (nucleus expelled here) → reticulocyte (still has some RNA, released into blood) → mature erythrocyte (after 1–2 days in circulation).

Regulation of erythropoiesis — erythropoietin (EPO):
The kidney is the oxygen sensor. When renal oxygen delivery falls (hypoxia, anaemia, high altitude), the peritubular interstitial cells of the kidney release erythropoietin (EPO). EPO travels to the bone marrow and stimulates:
1. Proliferation of erythroid progenitors
2. Faster maturation (shortening the transit time from proerythroblast to reticulocyte)
3. Early release of reticulocytes into the circulation

Clinical connection: Patients with chronic kidney disease (CKD) develop anaemia because their kidneys can't produce enough EPO. This is treated with recombinant EPO (synthetic erythropoietin). Athletes have abused EPO for blood doping — more RBCs = more oxygen delivery = better endurance.

Requirements for erythropoiesis:
• Iron — for haemoglobin synthesis (most important, covered in PY2.6)
• Vitamin B₁₂ and folate — for DNA synthesis during rapid cell division
• Vitamin B₆ (pyridoxine) — cofactor in haem synthesis
• Vitamin C — promotes iron absorption
• Proteins — amino acids for globin chains

Red Blood Cells — Structure and Function (PY2.3)

The red blood cell (erythrocyte) is the most abundant cell in your body — about 25 trillion in total. It is uniquely designed for one job: carrying oxygen.

Figure: Red Blood Cells — Structure and Function (PY2.3)

Shape: Biconcave disc — thinner in the centre, thicker at the edges (like a doughnut without a complete hole). Diameter: 7–8 µm, thickness: 2.5 µm at the edge, 1 µm in the centre.

Why biconcave? Three reasons:
1. Maximises surface area — the biconcave shape gives a surface area of ~136 µm² (30% more than a sphere of the same volume). More surface = faster gas exchange.
2. Flexibility — the cell can fold and squeeze through capillaries as narrow as 3 µm (half its own diameter). This is crucial for microcirculation.
3. Uniform diffusion distance — no point inside the cell is far from the membrane, so O₂ and CO₂ can diffuse rapidly throughout.

No nucleus, no mitochondria:
Mature RBCs have no nucleus (expelled during erythropoiesis) and no mitochondria. This means:
• They cannot divide or repair themselves
• They generate ATP only by anaerobic glycolysis (they don't consume the oxygen they carry — a clever design)
• Their lifespan is limited: 120 days on average

RBC membrane — the spectrin-actin skeleton:
The lipid bilayer is anchored to an internal mesh of spectrin proteins linked by actin and ankyrin (connecting to band 3 protein) and protein 4.1 (connecting to glycophorin). This cytoskeleton gives the RBC its deformability. Defects cause hereditary spherocytosis (spherical, rigid RBCs that get trapped and destroyed in the spleen).

Functions of RBCs:
• Oxygen transport — haemoglobin carries O₂ from lungs to tissues
• CO₂ transport — carries ~23% of CO₂ as carbaminohaemoglobin; the enzyme carbonic anhydrase inside RBCs converts CO₂ + H₂O → H₂CO₃ → H⁺ + HCO₃⁻ (the chloride shift)
• Buffering — haemoglobin is a powerful buffer, absorbing H⁺ ions

Red cell indices (used to classify anaemias — covered in PY2.7):
• MCV (Mean Corpuscular Volume) = Haematocrit/RBC count. Normal: 80–100 fL
• MCH (Mean Corpuscular Haemoglobin) = Hb/RBC count. Normal: 27–33 pg
• MCHC (Mean Corpuscular Haemoglobin Concentration) = Hb/Haematocrit. Normal: 32–36 g/dL

Haemoglobin — Types and the Oxygen-Dissociation Curve (PY2.4)

Haemoglobin (Hb) is the oxygen-carrying protein in RBCs. Normal concentration: 13–17 g/dL in males, 12–15 g/dL in females. Each RBC contains approximately 280 million haemoglobin molecules.

Structure: Haemoglobin is a quaternary protein with 4 subunits, each containing:
• A globin chain (polypeptide) — determines the Hb type
• A haem group — a porphyrin ring with a central iron atom (Fe²⁺) that binds one O₂ molecule

Each Hb molecule can bind 4 molecules of O₂ (one per haem group) = fully oxygenated = oxyhaemoglobin (bright red). Deoxygenated Hb = deoxyhaemoglobin (dark red/blue).

Types of haemoglobin:
• HbA (α₂β₂) — 96–98% of adult Hb. Two α-chains + two β-chains
• HbA₂ (α₂δ₂) — 1.5–3% of adult Hb
• HbF (α₂γ₂) — fetal haemoglobin. Predominant in fetal life, < 1% in adults. HbF has a higher affinity for O₂ than HbA — this allows the fetus to 'steal' oxygen from maternal blood across the placenta
• HbS — sickle haemoglobin (β-chain mutation: glutamate → valine at position 6). Causes sickle cell disease

The Oxygen-Haemoglobin Dissociation Curve:
This S-shaped (sigmoid) curve plots % O₂ saturation of Hb (y-axis) against partial pressure of O₂ (PaO₂) (x-axis).

Why is it sigmoid? Because of cooperative binding — when one haem group binds O₂, it changes the shape of the molecule, making it easier for the next haem group to bind O₂. The first O₂ is hardest to bind; the fourth is easiest.

Key points on the curve:
• At PaO₂ 100 mmHg (lungs): Hb is ~98% saturated — fully loaded with O₂
• At PaO₂ 40 mmHg (tissues): Hb is ~75% saturated — has unloaded ~25% of its O₂
• At PaO₂ 26 mmHg: Hb is 50% saturated — this is the P₅₀ (the PaO₂ at which Hb is half-saturated)

Shifts of the curve:
• Right shift (decreased affinity — Hb releases O₂ more easily to tissues):
Caused by: ↑ temperature, ↑ PCO₂, ↑ H⁺ (↓ pH), ↑ 2,3-DPG
Think: exercising muscle is hot, acidic, CO₂-rich — it needs MORE O₂ delivered
The effect of pH/CO₂ is the Bohr effect

• Left shift (increased affinity — Hb holds onto O₂, releases less to tissues):
Caused by: ↓ temperature, ↓ PCO₂, ↓ H⁺ (↑ pH), ↓ 2,3-DPG, HbF, CO poisoning
HbF left-shifts because fetal Hb doesn't bind 2,3-DPG well

2,3-DPG (2,3-bisphosphoglycerate): Produced by RBCs during glycolysis. It binds to deoxyhaemoglobin and stabilises the T (tense) state, reducing O₂ affinity. Levels increase in chronic hypoxia and anaemia (adaptive response — helps deliver more O₂ to tissues).

Haemoglobin Synthesis and Breakdown — The Bilirubin Pathway (PY2.5)

Haemoglobin synthesis occurs in developing erythroblasts in the bone marrow. It requires two components:

1. Haem synthesis — occurs partly in mitochondria, partly in cytoplasm:
Succinyl CoA + Glycine → δ-aminolevulinic acid (ALA) [rate-limiting step, enzyme: ALA synthase] → porphobilinogen → uroporphyrinogen → coproporphyrinogen → protoporphyrin IX + Fe²⁺ → Haem [enzyme: ferrochelatase]

1. Globin synthesis — on ribosomes. The type of globin chain (α, β, γ, δ) determines the Hb type.

Haem + globin assemble into Hb subunits → 4 subunits form the complete Hb tetramer.

Haemoglobin breakdown — when RBCs die (after 120 days):
Old or damaged RBCs are recognised and destroyed by macrophages in the spleen (primary site), liver, and bone marrow — this is called extravascular haemolysis.

The breakdown sequence:
1. Haemoglobin → Globin (recycled to amino acid pool) + Haem
2. Haem → haem oxygenase removes iron → Biliverdin (green pigment)
3. Biliverdin → biliverdin reductase → Unconjugated (indirect) bilirubin — this is lipid-soluble, bound to albumin in blood, and cannot be excreted by the kidneys
4. In the liver, unconjugated bilirubin is conjugated with glucuronic acid by UDP-glucuronyl transferase → Conjugated (direct) bilirubin — water-soluble
5. Conjugated bilirubin is secreted into bile → reaches the intestine
6. In the intestine, bacteria convert it to urobilinogen → most becomes stercobilin (gives faeces their brown colour). Some urobilinogen is reabsorbed (enterohepatic circulation) and excreted by kidneys as urobilin (gives urine its yellow colour).

Clinical connection — jaundice:
• Pre-hepatic (haemolytic): Excessive RBC destruction → ↑ unconjugated bilirubin. Causes: haemolytic anaemias, malaria, sickle cell crisis
• Hepatic: Liver cannot conjugate bilirubin → ↑ both types. Causes: hepatitis, cirrhosis, Gilbert syndrome
• Post-hepatic (obstructive): Bile duct blocked → ↑ conjugated bilirubin, pale stools (no stercobilin), dark urine (conjugated bilirubin in urine). Causes: gallstones, pancreatic head tumour