hemoglobinopathies

hemoglobinopathies

1. Hemoglobinopathies

2. Hemoglobinopathy The hemoglobinopathies are a group of disorders passed down through families (inherited) in which there is abnormal production or structure of the hemoglobin molecule. They fall into 2 broad categories: o Sickle cell disease o Thalassemias

3. Structure of Hemoglobin Hemoglobin is a tetrameric protein composed of 2 pairs of globin chains (4 globin polypeptides) complexed with 4 heme groups. Each globin chain, or subunit, is associated with a heme group in its center. Globin chains are designated as α, β, γ, and δ

4. Classification of hemoglobinopathy  QUANTITATIVE DISORDERS OF GLOBIN CHAIN SYNTHESIS/ACCUMULATION The thalassemia syndromes A. ) β- Thalassemia β- Thalassemia minor or trait β- Thalassemia major β- Thalassemia intermedia γ- Thalassemia Lepore fusion gene δβ- Thalassemia β- Thalassemia with other variants: HbS / β- thalassemia HbE / β- thalassemia

5. B .) α- Thalassemia Deletions of α- globin genes: One gene: α + - thalassemia Two genes in  cis : α - thalassemia Three genes: HbH disease Four genes: Hydrops fetalis

6. De novo and acquired α- thalassemia α- Thalassemia with mental retardation syndrome (ATR): Due to large deletions on chromosome 16 involving the α- globin genes Due to mutations of the ATRX transcription factor gene on chromosome X α- Thalassemia associated with myelodysplastic syndromes (ATMDS): Due to mutations of the ATRX gene

7. II. QUALITATIVE DISORDERS OF GLOBIN STRUCTURE Sickle cell disorders SA, sickle cell trait SS, sickle cell anemia/disease SC, HbSC disease S/ β thal , sickle β- thalassemia disease S with other Hb variants: D, O-Arab Hemoglobins with decreased stability (unstable hemoglobin variants) Mutants causing congenital Heinz body hemolytic anemia Acquired instability—oxidant hemolysis : Drug-induced, G6PD deficiency

8. C. Hemoglobins with altered oxygen affinity High oxygen affinity states: Fetal red cells Decreased 2,3-BPG Carboxyhemoglobinemia , HbCO Low oxygen affinity states: Increased RBC 2,3-BPG

9. D Methemoglobinemia Congenital methemoglobinemia Cytochrome b5 reductase deficiency Acquired (toxic) methemoglobinemia E  Posttranslational modifications Nonenzymatic glycosylation Amino-terminal acetylation Amino-terminal carbamylation Deamidation

10. Thalassemia

11. Inherited abnormality of globin production Defect lies in the rate of production of α or β chains. Clinical and hematologic features occur due to reduced hemoglobin production and accumulation of α or β globin chain .

12. Classification β Thalassemia Commonest Reduced synthesis of β chains of globin α Thalassemia Reduced synthesis of α chains of globin Misc Thalassemia syndromes Combination of β, α genes with other structurally abnormal Hb

13. β Thalassemia Syndromes Thalassemia Major Homozygous Most severe form Thalassemia Intermedia Double heterozygous Milder form of Thalassemia major Thalassemia Minor Heterozygous state Asymptomatic

14. Geogrophic Distribution

15. Genetics of β Thalassemia Autosomal Recessive disorder Point mutations on the globin gene (a) promotor region and chain terminator mutants (b) mutations affecting m-RNA processings

16. >300 mutations of β globin gene 30 seen in india - 6 of these account for 90-95% cases Mutatiions Frequency IVS 1-5 48% Commonest 619 bp deletion 18% IVS 1-1 9% FR 41/42 9% FR8/9 5% CODON 15 5% Others 6%

17. β / β + Thalassemia – some chains formed . HbA is present β / β Thalassemia - absence/ marked reduction of beta chains . Nearly total Hb is HbF

18. PATHOPHYSIOLOGY a. Accumulation of free a-chains alfa -chain synthesis is not affected – accumulation in developing normoblasts and red cells forming intracellular inclusions which result in membrane damage. Such normoblasts fail to mature and die in the marrow (apoptosis). ineffective erythropoiesis - anemia

19. PATHOPHYSIOLOGY b. Extravascular hemolysis Red cells which do get formed from abnormal normoblasts have membrane abnormalities and free a-chain inclusions. P ass through the spleen -sequestered resulting in hemolytic anemia.

20. PATHOPHYSIOLOGY c. Marrow and bone changes Development of anemia stimulates erythropoietin (EPO) production which acts on bone marrow leading to marked erythroid hyperplasia. There is expansion of medullary cavities of the bones - widening of diploe of the skull and long bones.

21. PATHOPHYSIOLOGY d. Extramedullary hemopoiesis Hyperfunction of the spleen and also development of extramedullary hemopoietic foci in liver and spleen , leads to hepatosplenomegaly . e. Synthesis of HbF Synthesis of some gamma-chains continues even after 6 months of age, which combine with a-chains resulting in increased level of HbF which varies from 20-95%.

22. Clinical features T halassemia major present within the first year. At birth, the baby is asymptomatic because HbF level is high. After 3 months, HbF production wanes, beta-chains are not formed, so anemia develops.

23. Failure to thrive, intermittent infection poor feeding progressive increase in pallor. protuberant abdomen because of progressive hepatosplenomegaly . Frontal bossing and overgrowth of zygomatic bones which makes the cheeck bones prominent - thalassemia facies .

24. Myocardial hemosiderosis : Arrhythmias and congestive cardiac failure. Iron overload : (a) increased hemolysis of red cells, (b) increased absorption of iron from GIT and (c) repeated transfusions given to these patients (each unit= 200 mg of iron). Excessive iron deposition occurs in liver, bone marrow, pituitary, thyroid, parathyroids , myocardium .

25. Hematologic Findings Hb % MCV MCH β Thalassemia Major <7 gm/dl 50-70 fl 12-20 pcg /cell β Thalassemia Intermedia 7-10 gm/dl 60-80 fl 15-25 pcg /cell β Thalassemia Minor 9-11 gm/dl 65-70 fl 25-30 pcg /cell

26. Anisopokilocytosis Rbc - microcytic , hypochromic Target cells Basophilic stippling Nucleated RBC (mainly late normoblast )- 5-40/100 RBC

27. Tear drop cells, elliptical cells, fragmented RBCs Howell jolly bodies . Reticulocyte count <2% - does not correspond to severity of anemia.

28. PBF Microcytic hypochromic red cellls , tear drop cells, target cells, anisopoikilocytosis

29. PBF Microcytic hypochromic red cellls , tear drop cells, nucleated RBCs

30. Iron status: S. Ferritin is usually > 1000pg/L and transferrin saturation is markedly increased to 55% to 90% (normal 30-35%). Total iron binding capacity TIBC is reduced to 250-300 pg% (normal 320-360pg%). S. iron is increased.

31. Bone Marrow: Markedly hypercellular Erythroid hyperplasia M:E ratio reversal (1:1 to 1:2) Erythropoiesis is normoblastic . Normoblasts demonstrate pink inclusions of free alpha-chains and Basophilic stippling in intermediate and late normoblast Gaucher like cells may be present. Myelopoiesis and megakaryopoiesis are normal

32. Bone marrow Hypercellular bone marrow, erythroid hyperplasia.

33. LABORATORY TESTS FOR DIAGNOSIS 1. Hb electrophoresis, HPLC and capillary Electrophoresis. 2. Mutation studies- DNA analysis 3. Amplification Refractory Mutation System (ARMS). 4. Polymerase chain reaction (PCR)

34. HPLC beta thal major- shows HbF 91.2%

35. Thalassemia intermedia : Clinical entity in which patients have a clinical spectrum intermediate between thalassemia trait and thalassemia major, Patients are anemic, but are not transfusion dependent. Includes cases of interaction of beta, alfa , HbE,HbD , HbS genes. HbE-Thalassemia in paticular results in picture of thalassemia intermedia . Thalassemia Minor: These are heterozygotes for thalassemia gene These carriers are asymptomatic. Clinically normal.

36. Clinical picture Hemat / Pbf findings Thalassemia Intermedia Pallor -mild to moderate. Splenomegaly is mild. Growth and development may be normal or retarded. Mild skeletal and facial changes and frontal bossing. Develop iron overload because of the increased iron absorption from GIT. Iron stores are increased Hb being 7-10 gm/dl. Moderate degree of anisopoikilocytosis with microcytic hypochromic red cells, target cells and basophilic stippling . HbF is 10-30% Marrow iron stores -increased. Thalassemia Minor / trait Clinically normal Hb 10-l 2gm/dl HbA is 90-93% and HbA2 is 3.9-8% and is diagnostic The Mentzer index is <13 is suggestive of thalassemia trait Mild anisopoikilocytosis with microcytosis , hypochromia , few target cells S iron and S ferritin - normal

37. HPLC- Thal intermedia HPLC- Thal trait

38. Differential diagnosis Beta Thalassemia trait (BTT) is differentiated from iron deficiency anemia (IDA) by high RBC count, target cells and stippled cells in BTT ; while RDW is increased in IDA and MCHC is Low. S. ferritin and bone marrow iron is normal or increased in BTT in contrast to IDA. In addition , HbA2 > 3.5% is a useful criterion, being 1-3.5% in IDA.

39. NESTROF TEST Naked eye single tube red cell osmotic fragility test 5ml of 0.35% saline – 2 test tubes (one test and one control) Add 0.02ml of patients blood ( in test) and 0.02ml of normal person’s blood in (in control) After 30mins – put a white paper with a black line behind test tubes . In control tube black line is clearly seen In positive cases – not clearly seen in control tube . This test is widely used in india and other developing countries in thalassemia screening programmes . Cases found positives- subjected to HbA2 estimation.

40. Alpha thalassemias Alpha thalassemias are the hemolytic anemias in which the a-chains of globin are not/partly synthesized. alfa -chains are required for both HbA and HbF synthesis Lack of synthesis of alfa -chains results in accumulation of gamma4 chains ( Hb Bart's) in intrauterine life and beta4 chains ( HbH ) after birth. Majority of the a- thalassemia cases result from gene deletions because of tandemly duplicated sequences in the cluster of alfa -genes.

41. Hb Barts / hydrops fetalis All 4 α - gene deletion IUD Pale and bloated Placenta is edematous Moderate to massive hepatomegaly Hb 4-10 gm/dl MCV increased Severe Anisopoikilocytosis Hypochromia Nucleated RBCs HbH disease β 4 variation in clinical severity. Children develop progressive anemia Jaundice,splenomegalymoderate skeletal modifications Hb 6-10 gm/dl MVC 55-65fl MCH 20pg Moderate anisopoikolocytosis Microcytosis Hypochromia Target cells Reticulocyte prepration - HbH inclusions α - thalaseemia trait Deletion of 2 gene heterozygous . Clinically normal 9-12 gm/dl MCV and MCH low Confirmation of diagnosis – DNA analysis

42. Reticulocyte prepration – HbH inclusion HPLC – showing shap peak before start of integration in the first minute of elution

43. Misc thal syndromes Clinical picture HbS thal syndrome Double heterozygote HbS and beta thal Similar to thal intermedia Prognosis better than thal major and sickle cell anemia Increased HbF and HbS level HbE thal Beta thal trait and Hb E gene Similar to thal intermedia HbF and HbE increased HbD thal Beta thal trait and Hb D gene Similar to thal intermedia HbF and HbD increased δβ- Thalassemia heterozygous Normal alfa chain with δβ fusion – Hb leproe Similat to thal trait Hb F (5-20%) HbA2 normal/low δβ- Thalassemia Homozygous Deletion of both delta and beta gene Similar to thal intermedia HbF >95%

44. Sickle cell disorders.

45. Substitution of valine in place of glutamic acid acid in 6 th position of beta chain – HbS . Alters solublity and characterstics of Hb . Presence of HbS imaprts sickle shape to cells in reduced Oxygen tension. When Oxygen tension returns to normal , polymers dissolves and normal shape is recovered

46. Sickle cell anemia Homozygous SS State Manifest early HbS >70% in RBCs Sickle cell trait Heterozygous AS state HbS 25-40% in RBCs Sickle cell Disease All disease states in which atleast one gene is HbS , other may be Beta thalassemia , HbD , Hb E

47. PHYSIOLOGIC DETERMINANTS OF POLYMERIZATION Oxygen Hemoglobin S Concentration Other Hemoglobins - Hb A and Hb F have an inhibitory effect

48. MEMBRANE ALTERATIONS Reversibly & Irreversibly Sickled Cells Red cell sickling is associated with reversible membrane changes. With repeated cycles of sickling and unsickling , aberrations in membrane function and structure become increasingly pronounced. This leads to fixation of the membrane in the sickled configuration.

49. Pathogenesis of Hemolysis Intravascular hemolysis : Results from the lysis of complement-sensitive red cells Extravascular hemolysis : (a) monocyte and macrophage recognition and phagocytosis of red cells that have undergone sickling (b)oxidation-induced membrane changes and physical entrapment of compromised red cells

50. Clinical features sickle cell anaemia SS state Majority cases present between 3month- 1 st year of life . Chronic hemolytic anaemia Vaso -occlusive events

51. Gowth and development retarded After 6 month- splenomegaly , multiple infarcts, autosplenectomy Recurrent leg ulcers Avascular necrosis of femoral head Dactylitis (hand-foot syndrome)- first 4 years of life Acute infections- pneumonia , meningitis (S. pneumoniae ) , osteomyelitis (salmonella)

52. Jaundice , liver enlargment - 1 st year of life Pigment gall stones Acute abdominal pain- visceral infarction Priapism Acute chest syndrome- fever , chest pain, pulmonary infarcts. Sickle cell retinopathy –” salmon patches” intraretinal hemorrhages

53. Crises in sickling syndrome A) Sickling crisis ( vaso-occlusice crisis): blockage of microcirculation by sickled cells-acute abdominal pain Precipated by fever , dehydration , infection. B) Hemolytic crisis: Further increase in hemolysis with sudden lowering of Hb , reticulocytosis , jaundice. Concurrent G6PD Deficiency

54. C) Aplastic crisis: Sudden aplasia of erythroid precursosrs Parvovirus B19 D)Sequestration crisis: Massive sequestration of sikled red cells in spleen and liver with rapid organomegaly Reduced blood volume- hypovolemic shock

55. Laboratory Features Hb : 6 – 10 g/dl RED BLOOD CELLS: moderate to severe normocytic , normochromic anemia manifests by 3 months of age and persists throughout life. PBF- sickled forms, target cells and ovalocytes , polychromatophilia , basophilic stippling, howell jolly bodies and normoblastosis . WBC: Both total and segmented leukocyte numbers increase during vasoocclusive crises and infections PLATELETS: The platelet count is increased

56. Sickle Cells Erythroblasts Howell-Jolly Body Sickle Cell Anemia – blood film

57. Diagnostic tests 1) Sickling test A strong reducing agent, sodium metabisulphite 2% is mixed with blood specimen, cause red cell to become sickle shaped due to deoxygenation . 2) Hb electrophoresis- Hbs slow moving Hb 3) HPLC- Retention time of 4.30-4.50 min

58. Sickle cell trait The heterozygous state for the Hb S gene. The red cells of such individuals contain both Hb A and Hb S, but there is always more Hb A than Hb S. Clinical Features Individuals have no anemia , and red cell morphology is normal. Growth and development proceed normally. Diagnosis Electrophoretic pattern containing both Hb A and Hb S, with more Hb A than Hb S.

59. Comparison of hemat findings of Sickle cell anemia and Sickle cell trait Sickle cell anemia Sickle cell trait Hb 5-7 gm/dl 10-13 gm/dl Anisopoikilocytosis +++ +/- Target cells +++ +/- Sickle cells + to ++ -- Howell jolly bodies ++ -- Basophilic stippling ++ -- Reticulocyte count Mild increase Normal Sickling test + ve immediately or within 30 mins + ve within 4 hours Hb electrophoresis Band of HbS , HbF , HbA Band of HbS and HbA

60. HPLC- sickle cell anemia 2 peaks of HbS (75%) And HbF (19.9%) HPLC- Sickle cell trait – significant peaks in A And S window . HbS 33.8% ( <50% of total Hb )

61. Other Sickling syndromes Hb S beta thalassemia Hb SE disease Hb SD disease Hb S alfa thalassemia

62. Other hemoglobinopathies Hb D punjab – at 121 position of beta globin chain glutamic acid->glutamine Hb D trait ( Hb AD) HbD disease ( HbDD ) Hb D beta thalassemia HbD iran HbD leproe disease Hb M Hb Saskatoon Hb C trait Hb E disorder Hb C disease

63. Hb E Disorders Substitution of glutamic acid by lysine on 26 position of beta globin chain forms Hb E On electrophoresis HbE is slow moving , migrates in same position as HbC and Hb A2 Hb E trait: Clinically normal PBF- Microcytosis (MCV 60-80 fl) Hb E Disease: HbEE Mild anemia and splenomegaly , mild jaundice PBF – marked anisocytosis and microcytosis , target cells , hypochromia .

64. Hb C Disorders Substitution of glutamic acid by lysine on 6 position of beta globin chain forms Hb C Hb C Trait: Clinically normal PBF- Target cells Hb C Disease ( HbCC ): Anemia with mild splenomegaly Recurrent arthralgias , mild jaundice PBF- anisocytosis , marked microcytosis , many target cells.

65. HbC is less soluble than Hb A If present in sufficient amounts it forms crystals within Red cell These crystals can be seen in a wet prepration or after incubation of blood in 3% NaCl soln at 37 C . On electrophoresis HbC is slow moving , migrates in same position as HbE and Hb A2

66. Hb D disease Hb D trait: Clinically normal PBF- Target cells Hb D disease: Hb DD Mild anemia +/- splenomegaly Hb - 7-10 gm/dl PBF- Mild anisopoikilocytosis , few target cells. Hb Electrophoresis – 90-95% HbD rest is HbA2 and HbF On Alkaline elctrophoresis HbD moves in same region as Hb S – Sickling test or HPLC diffrentitates .

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