THALASSAEMIA

1. THALASSAEMIA Alpha Thalassaemia Beta Thalassaemia Delta-Beta Thalassaemia

2. The underlying defect in thalassaemia is a reduction in the synthesis of one of the globin chains, resulting in an imbalance of available globin chains. Reduction of α or β chain synthesis causes α or β-thalassaemia respectively.

4. There are many variations at the gene level but the picture of hypochromasia and microcytosis is universal. The degree of chain imbalance determines the clinical picture from severe anaemia to a clinically silent form. Thalassaemia may involve other globin chains but the α and β chains are clinically of greater importance.

5. -THALASSAEMIA Particularly common in South East Asia and the Far East. Also frequent in Africa and the Mediterranean but is rare in northern Europe.

6. MOLECULAR CLASSIFICATION OF -THAL Man has a total of 4 α genes; 2 on each chromosome number 16. The normal genotype is written as αα/αα. α-Thalassaemia results from deletion of α globin genes. Gene mapping allows deletions to be identified. Loss of 1 of 2 genes on a single chromosome (- α/) = α+ thal. haplotype. Loss of both genes on a single chromosome (- -/) = α° thal. haplotype.

8. Non-deletional α-thal. Means gene mapping is normal (no deletions). e.g. Hb Constant Spring (ααCS/); 31 residues are added to the α chain due to defect in terminating the production of the α chain. Note: Hb Constant Spring is a slow moving band on cellulose acetate at pH 8.4. It runs behind Hb A2.

9. Decreased or absent α chain production results in excess γ chains during fetal life and excess β chains later. This causes the formation of stable tetramers, Bart's (γ4) and Hb H (β4). These stable, nonfunctional tetramers precipitate in older red cells to form inclusion bodies which interfere with membrane function. This results in decreased red cell survival and may induce a haemolytic crisis.

10. Golf ball appearance of Hb H (4) stained supravitally with brilliant cresyl blue.

11. Genes for α° are frequent in South East Asia and thus the total spectrum of clinical expression is seen. α+ occurs almost exclusively in Africa and the Mediterranean so the most severe clinical states of hydrops fetalis and H disease, which require deletion of four and three genes respectively, are relatively rare.

12. -THALASSAEMIA -Thalassaemia does not manifest itself until the switch from γ to β chain synthesis, several months after birth. Compensatory increased production of γ chains and δ chains, results in increased levels of Hb F and Hb A2. -Thalassaemia is broadly subdivided into β° and β+-thalassaemia:

13. β°-Thalassaemia results in complete absence of β chains. This is commonly found in: Mediterranean area Northern Italy Greece Algeria Saudi Arabia Southeast Asia

14. β+-Thalassaemia results in reduced numbers of β chains. Three different β genes have been described: Type 1 - (10% normal). Mediterranean region, Middle East, India and Southeast Asia. Type 2 - (50% normal). West Africa. Type 3 - (>50% normal). Italy, Greece and the Middle East.

15. The clinical syndromes: thalassaemia minima thalassaemia minor thalassaemia intermedia thalassaemia major reflect the affect on Hb A production.

17. CLINICAL FEATURES OF BETA-THAL Heterozygotes for β-thalassaemia do not usually present with serious signs or symptoms. Homozygotes often suffer from severe anaemia (Cooley's anaemia). In severe forms, it is difficult to maintain the Hb as high as 20-30 g/L without blood transfusions. Iron chelation therapy is required. Chronic ineffective erythropoiesis results in marrow hypertrophy during childhood. This produces classical facial features; frontal bossing of the skull, hypertrophy of the maxilla and mongoloid slant of the eyes.

18. Complications include: • massive hepatosplenomegaly • recurrent infections • spontaneous fractures • leg ulcers • dental /orthodontic problems • tumor masses / extramedullary erythropoiesis

19. DELTA-BETA THALASSAEMIAS result from deletions of δ and β genes (δβ-thalassaemia). Hb A2 is normal and Hb F is unusually high in the heterozygote. Hb A and Hb A2 are absent in the homozygote. The presence of Hb Lepore also leads to thalassaemia. This abnormal haemoglobin results from a fusion of δ and β chains produced by a crossing over between δ and β genes. There is partial deletion of δ and β genes.

20. A   Hb Lepore A     Hb anti-Lepore A   Normal 11 A   Normal 11

21. Thalassaemia Screening • FBC (red cell indices) • Peripheral blood film (morphology) • Iron status (serum ferritin) • Identification of Hb variants (electrophoresis) • Quantitation of Hb A2 • Quantitation of Hb F • Intracellular distribution of Hb F (Kleihauer) • Red cell inclusion bodies (Hb H) • DNA analysis (PCR)

22. Red Cell Indices Distinguishing thalassaemia from iron deficiency.

23. Peripheral Blood Film (PBF) • Hypochromasia / microcytosis • Anisocytosis • Target cells / leptocytes • Basophilic stippling / punctate basophilia • Poikilocytosis • Schistocytosis / red cell fragmentation • Nucleated red blood cells (NRBCs) • Red cell inclusions e.g. Fessas bodies

24. Iron Status Iron deficiency complicates the diagnosis of thalassaemia. e.g. Reduced Hb A2 in Fe deficiency can cause normal level in  thal minor. Methods: Ferritin Serum Iron (SI) TIBC Zinc protoporphyrin (ZPP)

25. Haemoglobin Separation Methods: • Electrophoresis • Cellulose acetate pH 8.2 – 8.6 • Cellulose acetate pH 7.0 • Citrate agar pH 6.0 • Iso-Electric Focusing (pH gradient of 6.0-8.0) • Polyacrylamide • Agarose gel • High Performance Liquid Chromatography (HPLC)

26. Interpretation of Haemoglobin Electrophoresis

27. Quantitation of Hb A2 • Methods: • Elution from cellulose acetate electrophoresis (pH 8.9) • Microcolumn chromatography • e.g. Diethylaminoethyl (DEAE) cellulose with Tris-HCL or glycine-KCN developers. • HPLC

28. Quantitation of Hb F • Methods: • Alkaline denaturation (Modified Betke Method) • HPLC • Radial Immuno Diffusion (RID) • Enzyme Linked Immuno-assay (ELISA)

29. Fetal cells Adult (ghost cells) Intracellular Distribution of Hb F Hetrozygous - thalassaemia = heterocellular African HPFH = pancellular distribution Methods: Kleihauer Acid elution (cytochemistry) Immunofluorescence using anti-Hb F

30. Red Cell Inclusions Hb H (precipitated  chain tetramers) 1.0% Brilliant cresyl blue or New methylene blue Fessas bodies (precipitated  chains) 0.5% Methyl violet

31. DNA Analysis (recombinant DNA technology) • Southern Blot Analysis • Useful for detection of large deletions, gene rearrangements and DNA polymorphisms. • Can detect the + gene deletion. • Difficult to detect point mutations of  thal. • Polymerase Chain Reaction (PCR) • Amplification of specific DNA fragments for ethidium bromide staining in agarose gel. • Method of choice for  thal mutations. • Can be used to detect common forms of °.

32. Prenatal Diagnosis Chorionic Villi Sampling (CVS) CVS can be taken between 9 and 12 weeks gestation. Fetal blood sample (FBS) can be taken at 16 to 22 weeks.