Thalassaemia: Pathogenesis and Lab Diagnosis Dr. M Sadequel Islam Talukder MBBS, M Phil (Pathology), MACP Assistant Professor Department of Pathology Dinajpur Medical College Presentation at Seminar on observation of World Thalassaemia Day 2009 Presentation: 10 May 2009, Dinajpur Medical College

Pathogenesis Heterogenous group of disorder Genetically determined Reduced synthesis of one or more types of normal haemoglobin polypeptide chain Reduced haemoglobin involving affected chain

Normal Haemoglobin HbA - α 2 β 2 HbA 2 - α 2 δ 2 HbF – α 2 γ 2

Each goblin chain have separate genetic control α –thalassaemia affect α-chain synthesis β –thalassaemia affect β -chain synthesis

β-Thalassaemia An absence or deficiency of β-chain synthesis of adult HbA β Chain synthesis Hb-A γ and δ chain Hb-A = α 2 β 2

On the basis of synthetic ability β-genes are designated as β gene – can synthesize normal amount of β-chain β + gene – can synthesize reduced amount of β-chain β 0 gene – cannot synthesize β-chain

β-thalassaemia major –Mutation of normal β-gene  β 0 -gene  absence HbA  increased HA 2 and HbF –genotype – β 0 β 0 β-thalassaemia intermedia –↑HbA 2 –↑HbF –↓HbA –Genotype β + β + or β 0 β β-thalassaemia minor –↑HbA 2 –HbA normal –HbF normal

Pathophysiology of β-Thalassaemia Various mutation in β-gene Complete or partial absence of β-chain Decreased adult HbA α-chain synthesis remain normal Free complementary α-chain – unstable and precipitate within normoblasts as insoluble inclusions Cell membrane damage & impaired DNA synthesis  apoptosis i.e. ineffective erythropoeisis

70-80% marrow normoblasts undergo apoptosis Inclusion bearing red cells undergo sequestration & destruction in spleen

Partial or lack of HbA synthesis  ↓MCHC & MCH  Hypochromia & microcytosis Normal Thalassaemia

Reticulocytes undergo intramedullary death Inadequate production + ineffective erythropoiesis + haemolysis  Anaemia

↑Haemolysis  ↑demands of phagocytic function  hyperplasia of phagocytes  Hepatosplenomegaly To compensate anaemia extramedullary haemopoiesis in liver, spleen & brain  Organomegaly

↑Erythropoiesis  marrow expansion & thinning of cortex of skull bone  Thalassaemia facies

α-Thassaemia An absence or deficiency of α-chain synthesis due to delation of α-genes.

Pathogenesis of α-Thalassaemia In normal individual HbA, HbA2 and HbF need α-chain for their formation. 4 genes of α-chain, each pair on short arm of chromosome 16 present with genotype α,α/α,α. In α-thalassaemia, delation of α-genes  reduction or absence of synthesis of α-chain depending on number of α-gene delation.

↓α-chain synthesis  free γ-chain in the fetus & β- chain in infant of 6 months, and continue in the rest of life. Complementary 4γ and 4 β are aggregated  Hb Bart ( 4γ ) and HbH (4 β ), respectively.

Variants of α-Thalassaemia Silent carrier –Delation of single α-gene –Genotype α/αα –Asymptomatic –Absence of RBC abnormality Thalasaemia trait –Delation of 2 α-genes –Genotype --/αα –Asymptometic, minimal or no anaemia –Minimal RBC abnormalities

Hb H disease –Delation of 3 α-genes –Genotype --/- α –75% reduction of α-chain –25% α-chain synthesis  small amount of HbF, HbA, & HbA 2 –Fetus can survive –Severe anaemia –Severe RBC abnormalites Hydrops fetalis –Delation of all α-genes –Genotype --/-- –Absence of α-chain synthsis –Only Hb Bart (γ 4 ) is produced (High affinity for O 2 and can not dissociate O 2 to tissue)

Inheritance

Laboratory Diagnosis of Thalassaemia

Laboratory Findings Hb concentration – Decreased ESR – Mild increased WBC – Neutrophilic leucocytosis or normal RBC count – Markedly decreased PCV – Markedly decreased MCV, MCH, MCHC – reduced Reticulocyte count – Increased Platelet count – May be increased

Peripheral Blood Film Normal

Morphology of PBF RBC –Marked anisocytosis, polikilocytosis, microcytosis, frequent target cells, basophilic stippling, fragmented cells or schistocytosis, polychromatic macrocytes and nucleated cells WBC –Whin normal or neutrophilic leucocytosis Platelets –Normal or increased

Thalassaemia slides

Haemoglobin Electrophoresis Different types of haemoglobins contain different surface charge which determine the elecrtrophoretic mobility and gives a specific bands on the electrophoretic papers when haemolysate is undergone eletrophoresis. HbF, HbA, HbA2, HbC, HbE, HbD, HbS, HbH and Hb Bart can be measured accurately by electrophoresis

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