THALASSAEMIA

Alpha Thalassaemia Beta Thalassaemia Delta-Beta Thalassaemia

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.

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

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

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

Hb H <40% Hb A 0%.Genotype Genes Features normal  /   4 normal hetero +  / –  -thal-2 hetero °  / – – -thal-1 homo + -thal-1 – / –  3 2 2 1 0 essentially normal micro / hypo mild anaemia Bart’s 2-8% (at birth) Hb H <2% moderate anaemia Barts <5%. Bart’s 70-80% Portland 10-15% + + ° – / – – H Disease homo ° Hydrops ––/–– .

4. 31 residues are added to the α chain due to defect in terminating the production of the α chain. e. It runs behind Hb A2. Note: Hb Constant Spring is a slow moving band on cellulose acetate at pH 8. .g. Means gene mapping is normal (no deletions).Non-deletional α-thal. Hb Constant Spring (ααCS/).

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

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

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

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

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

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

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

0 Hb F 0-6 0-6 + / + ° / ° ° / + intermedia major major 25-65 0 <20 1-4 1-4 1-4 30-70 >95 >75 .Genotype + /  ° /  Syndrome Hb A Hb A2 minima minor 95 95 3.5-8.0 3.5-8.

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

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

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

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

Peripheral blood film (morphology) 3. Identification of Hb variants (electrophoresis) 5. Quantitation of Hb A2 6. Red cell inclusion bodies (Hb H) 9.Thalassaemia Screening 1. Quantitation of Hb F 7. FBC (red cell indices) 2. Intracellular distribution of Hb F (Kleihauer) 8. Iron status (serum ferritin) 4. DNA analysis (PCR) .

6 .4 RDW <1530 <0 <14.8 Fe def. >13 >3.6 >1530 >0 >14. Equation MCV  RBC MCH  RBC Thal.8 (MCV2  MCH)  100 MCV – RBC – (Hb  5) – 3. <13 <3.Red Cell Indices Distinguishing thalassaemia from iron deficiency.

Fessas bodies .g.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.

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

0) Polyacrylamide Agarose gel •High Performance Liquid Chromatography (HPLC) .0 Citrate agar pH 6.0-8.0 •Iso-Electric Focusing (pH gradient of 6.Haemoglobin Separation Methods: •Electrophoresis Cellulose acetate pH 8.6 Cellulose acetate pH 7.2 – 8.

Interpretation of Haemoglobin Electrophoresis .

Quantitation of Hb A2 Methods: •Elution from cellulose acetate electrophoresis (pH 8.g. Diethylaminoethyl (DEAE) cellulose with Tris-HCL or glycine-KCN developers. •HPLC .9) •Microcolumn chromatography e.

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

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

0% Brilliant cresyl blue or New methylene blue Fessas bodies (precipitated  chains) 0.Red Cell Inclusions Hb H (precipitated  chain tetramers) 1.5% Methyl violet .

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 °.DNA Analysis (recombinant DNA technology) •Southern Blot Analysis Useful for detection of large deletions.

.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.

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