Lab 4: Thalassaemias and Bilirubin

This laboratory session covers the genetics and haematology of the thalassaemias and the biochemical basis of bilirubin measurement in clinical practice.

Thalassaemias are inherited disorders of haemoglobin synthesis in which mutations cause reduced or absent production of one or more globin chains. The resulting imbalance in globin chain synthesis leads to precipitation of unpaired chains, ineffective erythropoiesis, and haemolytic anaemia of varying severity.

Beta-thalassaemia results from mutations in the HBB gene encoding the β-globin chain. There are two broad mutation classes: β⁰ mutations produce no β-globin; β⁺ mutations produce reduced amounts. β-thalassaemia major (Cooley's anaemia) occurs in patients homozygous for β⁰/β⁰ or compound heterozygous for β⁰/β⁺ mutations. Severe anaemia presents in infancy as HbF (α₂γ₂) production declines and HbA (α₂β₂) fails to be produced. Without transfusion, extramedullary haematopoiesis expands in the skull and facial bones (thalassaemic facies), and the spleen massively enlarges. Patients require lifelong transfusion and iron chelation; curative treatment is allogeneic stem cell transplantation. HbF is markedly elevated. β-thalassaemia minor (trait) is the carrier state (one mutant allele); patients have mild microcytic anaemia with a raised HbA₂ (>3.5%), which is the hallmark of carrier status.

Alpha-thalassaemia results from deletions in HBA1/HBA2 genes on chromosome 16. Because there are four α-globin gene loci (two per chromosome 16), the severity depends on how many loci are deleted. Silent carrier (1 deletion): no anaemia. Alpha-thalassaemia trait (2 deletions): mild microcytosis, often misdiagnosed as iron deficiency. HbH disease (3 deletions): moderate haemolytic anaemia, splenomegaly; HbH (β₄ tetramers) forms and is detectable by haemoglobin electrophoresis or HPLC. Hydrops fetalis (4 deletions, Hb Bart's disease): Hb Bart's (γ₄ tetramers) has very high oxygen affinity, delivering no oxygen to tissues; fetus dies in utero or shortly after birth.

Haemoglobin electrophoresis separates haemoglobin variants by their electrical charge at defined pH. At alkaline pH: HbA migrates fastest, followed by HbF, HbS, HbC. HPLC is the modern standard, identifying HbA₂ elevation (β-thalassaemia trait), elevated HbF, HbH, or Hb Bart's.

Bilirubin. Haem from senescent red cells is catabolised in the spleen and liver: haem → biliverdin (by haem oxygenase) → unconjugated bilirubin (by biliverdin reductase). Unconjugated bilirubin is lipid-soluble, bound to albumin in plasma, and cannot be filtered by the kidney. In the liver, UDP-glucuronosyltransferase conjugates bilirubin with glucuronic acid, making it water-soluble (conjugated bilirubin). Conjugated bilirubin is excreted in bile, converted to urobilinogen by intestinal bacteria, and partly reabsorbed (enterohepatic circulation) or excreted as stercobilin in faeces or urobilin in urine.

In rhesus (Rh) disease, maternal anti-D IgG crosses the placenta and causes haemolysis of fetal red cells. Amniotic fluid bilirubin concentration, measured spectrophotometrically by absorbance at 450 nm (OD450, using the Beer-Lambert law), was historically used to assess haemolysis severity in the fetus. The ΔOD450 (the deviation from the expected spectrophotometric baseline) correlates with the degree of haemolysis and guides the need for intrauterine transfusion. This has been largely superseded by middle cerebral artery Doppler measurements.

Gilbert's syndrome is a benign genetic condition (reduced UGT1A1 activity due to a TATAA repeat polymorphism in the UGT1A1 promoter) causing mild unconjugated hyperbilirubinaemia (typically 20–50 µmol/L), often noticed during fasting or illness. No treatment is required.