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Thalassemia

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Thalassemia

  1. 1. Thalassemia Dr. Bahoran Singh
  2. 2. Thalassemia • Defective production of globin portion of hemoglobin molecule. • Globin chains structurally normal but have imbalance in production of two different types of chains. • Two major types of thalassemia: • Alpha (α) - Caused by defect in rate of synthesis of alpha chains. • Beta (β) - Caused by defect in rate of synthesis in beta chains.
  3. 3. Normal Human Haemoglobins
  4. 4. BETA THALASSEMIA • Beta thalassemia usually caused by genes mutation on chromosome 11. • Specifically, it is characterized by a genetic deficiency in the synthesis of beta- globin chains. • It is classified into 4 types- • Thalassemia Major • Thalassemia intermedia • Thalassemia minor • Thalassemia minima- which is clinically undetectable.
  5. 5. CLASSIFICATION OF β THALASSEMIA CLASSIFICATION GENOTYPE CLINICAL SEVERITY β thal minor/trait β/β+, β/β0 Silent β thal intermedia β+ /β+, β+/β0 Moderate β thal major β0/ β0 or β+ /β+, Severe
  6. 6. INHERITANCE • Autosomal recessive • Beta thal - point mutations on chromosome 11. • The most common mutation is point mutation. Following are the types • Splicing mutations- Most common cause of β+ thalassemia, mutations lie with in exon so destroy the normal RNA splice junction. • Promoter region mutations - these reduce the transcription by 75-80%. • Chain terminator mutation- most common cause of β0 thalassemia. This create a new stop codon with in the axon.
  7. 7. Thalassemia mutation in India Mutataions Frequency IVS 1-5 (G C) 48 % 619 bp deletion 18% IVS 1-1 ( G T) 9% FR 41/42 (TCTT) 9% FR 8/9 (+G) 5% Codon 15 (G A) 5% Others 6%
  8. 8. Beta Thalassemia Major • Also known as Cooley’s anemia • Most common in Mediterranean country, part of Africa, and Southest Asia. • Infants are well developed at birth. • After 6 months develop following symptoms • Moderate to severe anemia • Failure to thrive • Hepatosplenomegaly • Bone changes.
  9. 9. Clinical features : - Anemia (Hb < 7 g/dl) - Bone deformity - Marked splenomegaly - Osteoporosis - Cholelithiasis (4 – 23% cases) - Thrombotic complication - Mild jaundice of hemolytic type - Growth and development - Short stature, large head, delayed puberty, increased susceptibility to infection
  10. 10. ACCUMULATION OF IRON • Deposition in pituitary - endocrine disturbance - short stature, delayed puberty, poor sec. sexual characteristics • Hemochromatosis - cirrhosis of liver • Cardiomyopathy (cardiac hemosiderosis) -cardiac failure, sterile pericarditis, arrythmias, heart block • Deposition in pancreas -diabetes mellitus
  11. 11. Hematological Findings • Hemoglobin- 3- 8 gm % • RBCs are microcytic hypochromic • MCV- 50-70 fl, MCHC- 22-33%, MCH- 12-20 pg • Target cells, Basophilic stippling, nucleated RBCs • Tear drop cells, elliptocytes, fragmented red cells and occasional Howell Jolly body. • Reticulocyte count < 2%
  12. 12. • Iron Status • Serum ferritin is raised (>1000 µg/L) • Transferrin saturation Raised to 55% • TIBC reduced • Serum iron increased. • Bone marrow • Hypercellular • Erythroid hyperplasia with M:E ratio 1:1 to 1:2 ( normoblastic erythropoiesis) • Myelopoiesis and megacaryopoiesis is normal.
  13. 13. Lab test for diagnosis • HbF levels are high, 30-90%, higher in β0 thalassemia than in β+ thalassemia. • Hb F is demonstrated by acid elution test. • Hb electrophoresis and HPLC • It demonstrate band of both Hb A and Hb F in β+ thalassemia • In β0 thalassemia Hb F is >90% • Globin chain synthesis • α: β globin chain synthesis ratio is altered to 2-30:1 ( N is 1:1)
  14. 14. Difference between iron deficiency anemia and thalassemia Character Iron deficiency anemia Thalassemia major Etiology Deficiency of iron Reduced β chain synthesi Laboratory finding RBC count Decreased Increased (>5million/cumm) Peripheral smear • RBC type • Anisopoikilocytosis • Target cells • Microcytic hypochromic • Mild to moderate • Absent • Microcytic hypochromic • Severe • present Bone marrow iron Decreased Markedly Increased Serum iron profile • Serum ferritin • Serum iron • TIBC • Reduced <15µg/L • Reduced • Increased • Increased (300-1000 µg/L) • Increased • Normal Foetal hemoglobin (HbF) Normal (0-1%) Markedly increased (30-90%) RDW Increased Normal Clinical features Age Any age <2 years of age Growth and development Normal Retarded Hepatosplenomegaly Absent Present X- Ray findings Nil Hair on end appearance
  15. 15. Thalassemia intermedia • It include • Double heterozygote for mild β+ thalassemia alleles • β+ thalassemia with α + / α 0 thalassemia • Hb E/ β thalassemia • HbD / β thalassemia • HbS / β thalassemia • Hb Q india / β thalassemia • Hb Lepore/ β thalassemia • δ- β thalassemia • Hb Lepore ( homozygous)
  16. 16. CLINICAL FEATURES (THAL INTERMEDIA) • Moderate pallor, usually maintains Hb >6gm% • Anemia worsens with pregnancy and infections (erythroid stress) • Less transfusion dependant • Skeletal changes present, progressive splenomegaly • Growth retardation • Iron deposition in liver parenchyma • Longer survival than Thal major • HPLC
  17. 17. Thalassemia Minor ( β thalassemia trait) • Heterozygote for thalassemia gene. • Clinical feature • Usually ASYMPTOMATIC • Mild pallor, no jaundice • No growth retardation, no skeletal abnormalities, no splenomegaly • MAY PRESENT AS IRON DEFICIENCY ANEMIA (Hypochromic microcytic anemia) • Unresponsive/ refractory to Fe therapy • Normal life expectancy
  18. 18. Diagnosis • Hemoglobin- 10-12 gm%, Red cell count elevated >5.2 million/cumm • RBC indices – MCV & MCH,MCHC reduced, RDW normal • Microcytic hypochromic • Anisopoikilocytosis, target cells, nucleated RBC, leptocytes, basophilic stippling, tear drop cells. • Reticulocyte count increased. • Osmotic fragility test (NESTROF)- increased resistance to hemolysis • Haptoglobulin and hemopexin – depleted • B.M. study: hyperplastic erythropoesis
  19. 19. NESTROF TEST • Used to test the osmotic fragility of RBC’s • Method • Take 5 ml of 0.35% of saline solution in two test tube • Then add 0.02 ml of patient blood(test) and normal person (control) • After half an hour put a white paper with black line behind both the tube.
  20. 20. Diffrenece between iron deficiency anemia and thalassemia minor Thalassemia minor Iron deficiency anemia 1. RDW Normal Increased Mentzer’s index = MCV/RBC count million/cumm <13 >13
  21. 21. • Electrophoresis • HbA is 90-93%, HbA2 is 3.6-8 % • In cases with HbA2 3.3-3.7% needs iron status • Iron overload assessment • S. Ferritin • Urinary Fe excretion • Liver biopsy • Chemical analysis of tissue Fe • Endomyocardial biopsies • Myocardial MRI indexes • Ventricular function – ECHO, ECG HPLC Chromatogram
  22. 22. Management • Blood transfusion at 4-6 weeks interval ( Hb 9-11) • CHELATION THERAPY – start when serum ferritin >1200 µg/L • Desferrioxamine • Deferiprone • Combination of desferrioxamine and deferiprone • Deferasirox • Splenectomy- when patients develop pancytopenia. • BONE MARROW TRANSPLANTATION
  23. 23. Newer therapies: • GENE MANIPULATION AND REPLACEMENT • Remove defective β gene and stimulate γ gene • 5-azacytidine increases γ gene synthesis • Hb F AUGEMENTATION • Hydroxyurea • Myelaran • Butyrate derivatives • Erythropoetin in Thal intermedia
  24. 24. Prevention of thalassemia major • Thalassemia trait in parents • Assess mother during antenatal checkup by HbA2 level (3.6-8%) • Also assess father- if father is also trait – chorionic villous sampling should be carried out <12 week or amniotic fluid sampling (12-20 weeks) • Antenatal chorionic villous sampling • Done at 9-10 week of gestation • RFLP analysis/ PCR analysis is done on foetal DNAto identify the foetus is homozygous or heterozygous.
  25. 25. Thalassemia screening • All mothers with Hb <11 gm% during first trimester • Checked for Hb, MCV, MCH, MCHC and NESTROF test • If patient with MCV <70 fl, MCH<23 and NESTROF +ve • Further evaluate for HbA2 estimation • If HbA2is borderline – need iron study • Non invasive approach for prenatal diagnosis • Isolation of foetal cells from maternal blood by using density grediaents and isolation of small no of foetal cells by magnetic cell sorting (MACS) and fluorescent activated cell sorting (FACS)
  26. 26. Th. Major Th. Intermedia Th. Minor 1. Anemia Severe (<7g/dl (7-735g/dl) Mild/absent 2. Hb electrophoresis (a) HbA2 (b) HbF  (N)    (50-98%)  (N)    (4-8%) (N)/slightly  3. MVC (92  9 fl    4. MCH (29.5  2.5 pg)    5. RDW-CV (9-12%)   Slightly  (N) 6. Red cell Inclusion (- inclusion) (+) (+) (+) 7. Serum Bilirubin    - 8. Osmotic fragility   
  27. 27. - THALASSEMIA • Alpha thalassemia usually caused by gene mutation on chromosome 16 • Normally, people have four (4) genes for alpha globin with two (2) genes on each chromosome (αα/αα). • Deletion on alpha globin locus on Chr 16 • Defective synthesis of α-globin chain • Excess of ‫-ץ‬ chains - in the fetus (Hb Bart- ‫ץ‬4) • Excess of β-chains in the adult (Hb H- β4)
  28. 28. (A) Deletion • (i) Reciprocal recombination - Chromosome of only one - gene • -  3.7 kb rightward deletion • -  4.2 kb leftward deletion • (ii) Non-reciprocal cross over •   anti 3.7 •   anti 4.2
  29. 29. (b) Non-Deletion 1. RNA splicing mutation 2. Poly (A) signal mutation 3. Frame shift mutation 4. Non-sense mutation
  30. 30. Pathophysiology
  31. 31. ALPHA THALASSEMIA - CLASSIFICATION CLINICAL CLASSIFICATION GENOTYPE NO. OF GENES PRESENT Silent carrier αα/- α 3 genes α thalassemia trait - α/- α or αα/- - 2 genes Hemoglobin H disease -α/- - 1 gene Hb Barts / Hydrops fetalis - -/- - 0 genes
  32. 32. • Highest prevalence in Thailand • α chains shared by fetal as well as adult life. Hence manifests both times • These thalassemias don’t have ineffective erythropoesis because β and γ are soluble chains and hence not destroyed always • α Thalassemia trait mimics Fe deficiency anemia • Silent carrier – silent – not identified hematologically, diagnosed when progeny has Hb Barts/ Hb H
  33. 33. Hb Bart / hydrops fetalis • Homozygous state (- - / - - ) deletion of all four genes. • Clinical features : 1. Still born/die after birth 2. Anemia – severe (Hb  3 - 8g/dl) 3. Placenta is edematous 4. Moderate to massive hepatomegaly 5. Hb Bart (γ4) has high affinity for oxygen therefore, oxygen does not dissociate from Hb.
  34. 34. Hb H disease (β4) • Both O & + Thalassemia inherited (- - / - ) • Clinical features -Progressive Anemia – moderate (Hb  6 – 10 g/dl) - Jaundice - Hepato splenomegaly - Moderate skeletal malformation • Reticulocyte count- 4-10% • RBC- Microcytic hypochromic, anisopoikilocytosis, and target cells • Hb electrophoresis demonstrate fast moving HbH band in the range of 5-35% HbH disease A. PS- Micro, hypo, and target cells, B. Retic stain show tiny HbH inclusion(golf ball HbH( ), C. Electrophoresis show fast moving HbH and Hb Bart , D. HPLC show sharp peak before the start of integration in the first minute of elution.
  35. 35. O Thalassemia trait • Clinical features : • Asymptomatic • Anemia – very mild/absent • HbH and Hb Bart are not demonstrable • Adult-Diagnosis difficult should exclude other causes of microcytic hypochromic anemia. • Definitive diagnosis - Globin chain synthesis • - Genetic analysis
  36. 36. DIAGNOSIS • Hb electrophoresis: • CBC, PS, BM study • Heinz bodies in HbH disease – brilliant cresyl blue • Hb electrophoresis – for HbH and Hb Barts • α/β chain ratio decreased
  37. 37. Treatment: • Generally not required • Blood transfusion , iron chelation therapy – For transfusion dependent cases • Avoidance of oxidant drugs • Prompt treatment of infections • Folic acid supplementation • Splenectomy • BM transplantation, gene therapy
  38. 38. Hb Bart HbH -Th. trait Silent carrier 1. Anemia Severe (Hb3-8g/dl) Moderate (Hb8-9g/dl) Mild/absent (Hb N/) Absent 2. Reticulocyte count  Mildly  -- -- 3. MCV   N/ N 4. MCH   N/ N 5. HbA2 Absent N/ N/ N/ 6. Hb Bart (4) 80 – 100% NB 20 – 40% NB 5 – 15% NB 1 – 2% NB 7. Hb H (4) -- 1 – 40% (adult) (N) (N) 8. HbH inclusion -- (+) (+) (+) (+) -- 9. Globin chain synthesis ratio 0 0.2- 0.4 0.6 0.8
  39. 39. Thank you

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