Thalassaemia hemoglobinopathies dr.neela-feb_2012


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Thalassaemia hemoglobinopathies dr.neela-feb_2012

  1. 1. February11,2012 1 THALASSAEMIA – HEMOGLOBINOPATHIES Presented by: Dr. Neela Ferdoushi
  2. 2. 2 February11,2012 THALASSEMIAS  Thalassemias are a heterogenous group of genetic disorders Individuals with homozygous forms are severely affected and die early in childhood without treatment Heterozygous individuals exhibit varying levels of severity The disorders are due to mutations that decrease the rate of synthesis of one of the two globin chains (α or β). The genetic defect may be the result of:
  3. 3. 3 February11,2012 THALASSEMIAS  A mutation in the noncoding introns of the gene resulting in inefficient RNA splicing to produce mRNA, and therefore, decreased mRNA production  The partial or total deletion of a globin gene  A mutation in the promoter leading to decreased expression  A mutation at the termination site leading to production of longer, unstable mRNA  A nonsense mutation Any of these defects lead to:  An excess of the other normal globin chain  A decrease in the normal amount of physiologic hemoglobin made  Development of a hypochromic, microcytic anemia
  5. 5. 5 February11,2012 THALASSEMIAS Beta (β) thalassemia  The disease manifests itself when the switch from γ to β chain synthesis occurs several months after birth  There may be a compensatory increase in γ and δ chain synthesis resulting in increased levels of hgb F and A2.  The genetic background of β thalassemia is heterogenous and may be roughly divided into two types:  β0 in which there is complete absence of β chain production. This is common in the Mediterranean.  β+ in which there is a partial block in β chain synthesis. At least three different mutant genes are involved:  β+1 – 10% of normal β chain synthesis occurs  β+2 – 50% of normal β chain synthesis occurs  β+3 - > 50% of normal β chain synthesis occurs
  6. 6. 6 February11,2012 ALPHA AND BETA THALASSEMIAS
  7. 7. 7 February11,2012 THALASSEMIAS  The clinical expression of the different gene combinations (1 from mom and 1 from dad) are as follows:  β0 /β0 , β+1 / β+1 , or β0 / β+1,+2,or +3 = thalassemia major, the most severe form of the disease.  Imbalanced synthesis leads to decreased total RBC hemoglobin production and a hypochromic, microcytic anemia.  Excess α chains precipitate causing hemolysis of RBC precursors in the bone marrow leading to ineffective erythropoiesis  In circulating RBCs, α chains may also precipitate leading to pitting in the spleen and decreased RBC survival via a chronic hemolytic process.  The major cause of the severe anemia is the ineffective erythropoiesis.
  8. 8. 8 February11,2012 THALASSEMIAS  The severe, chronic anemia early in life leads to marked expansion of the marrow space and skeletal changes due to the increased erythropoiesis.  Untreated individuals die early, usually of cardiac failure (due to overwork and hemochromatosis).  Individuals may have massive splenomegaly leading to secondary leukopoenia and thrombocytopenia. This can lead to infections and bleeding problems.  Lab findings include: - hypochromic, microcytic anemia - marked anisocytosis and poikilocytosis - schistocytes, ovalocytes, and target cells - basophilic stippling from α chain precipitation - increased reticulocytes and nucleated RBCs
  9. 9. 9 February11,2012 THALASSEMIAS - serum iron and ferritin are normal to increased and there is increased saturation - chronic hemolysis leads to increased bilirubin and gallstones - hemoglobin electrophoresis shows increased hgb F, variable amounts of hgb A2, and no to very little A
  10. 10. 10 February11,2012 THALASSEMIA MAJOR
  11. 11. 11 February11,2012 THALASSEMIAS  Therapy – transfusions plus iron chelators to prevent hemochromatosis and tissue damage from iron overload; Gene therapy?  β +2, or 3 homozygous = thalassemia intermedia  Heterozygosity of β0 , or β+ = thalassemia minor  Mild hypochromic, microcytic anemia  Patients are usually asymptomatic with symptoms occurring under stressful conditions such as pregnancy  β thalassemia may also be found in combination with any of the hemoglobinopathies (S, C, or E) leading to a mild to severe anemia depending upon the particular combination.
  12. 12. 12 February11,2012 THALASSEMIA MINOR
  13. 13. 13 February11,2012 THALASSEMIAS  Alpha (α) thalassemia  The disease is manifested immediately at birth  There are normally four alpha chains, so there is a great variety in the severity of the disease.  At birth there are excess γ chains and later there are excess β chains. These form stable, nonfunctional tetramers that precipitate as the RBCs age leading to decreased RBC survival.  The disease is usually due to deletions of the α gene and occasionally to a functionally abnormal α gene.
  14. 14. 14 February11,2012 THALASSEMIAS  The normal haploid genotype is α/ α  If one gene is deleted, the haploid phenotype is α thal 2  If both genes are deleted, the haploid phenotype is α thal 1  Since one gets two genes from each parent, there are four types of α thalassemia:  α/ α thal 2 = silent carrier  α/ α thal 1, or α thal 2/ α thal 2 = α thal trait with mild anemia  α thal 1/ α thal 2 = hemoglobin H disease (β4 = hgb H) Hgb H has a higher affinity for O2 and precipitates in older cells. Anemia may be chronic to moderate to severe.
  15. 15. 15 February11,2012 THALASSEMIAS  α thal 1/ α thal 1 = hydrops fetalis which is fatal with stillbirth or death within hours of birth. Hemoglobin Barts (γ4) forms and has such a high affinity for O2 that no O2 is delivered to the tissues.  Hgb S/ α thalassemia – symptomless to moderate anemia
  16. 16. 16 February11,2012 ALPHA THALASSEMIAS
  17. 17. 17 February11,2012 THALASSEMIAS Delta/beta (δ/β) thalassemia – both δ and β chains are absent with no or little compensatory increase in γ chain synthesis. This leads to 100% hgb F and mild hypochromic, microcytic anemia Hereditary persistence of hgb F – are a group of heterogenous disorders with the absence of δ and β chain synthesis which is compensated for by an increase in γ chain synthesis leading to 100% hgb F. Since hgb F has an increased affinity for O2, this results in polycythemia.
  18. 18. 18 February11,2012 Primary Laboratory Investigation Thalassemia Variable hemogram results proportional to the severity of the thalassemia
  19. 19. 19 February11,2012 Primary Laboratory Investigation Thalassemia • Severe cases present with • Microcytosis • Hypochromia • Poikilocytosis • RBC counts higher than expected for the level of anemia
  20. 20. 20 February11,2012 Primary Laboratory Investigation Thalassemia • Findings in severe cases can mimic those seen in other microcytic/hypochromic anemias • Results of the reticulocyte count are variable • NRBCs may be present (contrast with iron deficiency anemia)
  21. 21. 21 February11,2012 Course and Treatment Thalassemia • Time of presentation • Related to degree of severity • Usually in first few years of life • Untreated severe α thalassemia • --/--: Prenatal or perinatal death • --/-α & --/αcs α: Normal life span with chronic hemolytic anemia
  22. 22. 22 February11,2012 Course and Treatment Thalassemia • Untreated β thalassemia • Major: Death in first or second decade of life • Intermedia: Usually normal life span • Minor/Minima: Normal life span
  23. 23. Haemoglobinopathy
  24. 24. 24 February11,2012 Characteristics: Hemoglobinopathies • Hereditary disorders that can result in moderate to severe anemia • Basic defect is production of an abnormal globin chain
  25. 25. 25 February11,2012 Definition: The haemoglobinopathies are inherited disorders of haemoglobin synthesis (thalassaemias) or structure (sickle cell disorders) that are responsible for significant morbidity and mortality allover the world. They are seen mainly in individuals who originate from Africa, the Middle East,, the Mediterranean, Asia and the Far East. However, the increased mobility of the world’s population and inter-ethnic mixing lead to prevailing of these conditions within any region of the world.
  26. 26. 26 February11,2012 These disorders result in errors in oxygen-carrying capacity of haemoglobin . Diseases linked to genetic predisposition are not only kill prematurely, but result in long years of ill health and disability, loss of work and income, possible poverty, loneliness and depression.
  27. 27. 27 February11,2012 Sickle cell and thalassaemia are inherited disorders that are passed on from parents to children through unusual haemoglobin genes. People only have these disorders if they inherit two unusual haemoglobin genes – one from their mother, and one from their father. People who inherit just one unusual gene are known as ‘carriers’. (Some people call this having a ‘trait’.) Carriers are healthy and do not have the disorders.
  28. 28. 28 February11,2012 Hemoglobinopathy Genetics • Homozygous: Inheritance of two genes from each parent coding for the same type of abnormal hemoglobin, e.g., Hb SS • Heterozygous: Inheritance of genes from each parent which code for a different type of abnormal hemoglobin each, e.g., Hb SC
  29. 29. 29 February11,2012 Terminology Hemoglobinopathy Abnormal hemoglobins discovered earlier have been given letter designations: Hb S
  30. 30. 30 February11,2012 Amino Acid Substitution Hemoglobinopathy Greek letter designates affected globin chain β
  31. 31. 31 February11,2012 Amino Acid Substitution Hemoglobinopathy Superscript number designates affected amino acid(s), e.g., β6
  32. 32. 32 February11,2012 Amino Acid Substitution Hemoglobinopathy Letters and numbers in parentheses designate the helical segment and amino acid sequence in that segment affected (sometimes omitted), e.g., β6(A3)
  33. 33. 33 February11,2012 Amino Acid Substitution Hemoglobinopathy Amino acid substitutions are denoted by the three letter abbreviation for the normally occurring amino acid followed by an arrow followed by the three letter abbreviation for the substituted amino acid: β6(A3)Glu → Val
  34. 34. 34 February11,2012 Classification: Hemoglobinopathy • Functional Abnormality • Aggregation • Polymerization • Crystallization • Unstable hemoglobins • Methemoglobin • Oxygen affinity
  35. 35. 35 February11,2012 Individuals with Haemoglobinopathy are: either healthy carriers (trait ) i.e. unaware of their carrier status unless specifically screened. If a couple both carry a haemoglobinopathy trait there is a 1 in 4 chance with each pregnancy that their child will inherit a clinically manifested haemoglobinopathy. or having clinically manifested haemoglobinopathy
  36. 36. 36 February11,2012 The diagram below shows the chances (for each pregnancy) of two carrier parents having a child with a sickle cell or thalassaemia disorder.
  37. 37. 37 February11,2012 If the mother is anemic & the father is healthy carrier 50% of the off springs are carriers and 50% is anaemic
  38. 38. 38 February11,2012 Sickle Cell is a condition that affects the normal oxygen carrying capacity of red blood cells. When the cells are de-oxygenated and under stress in sickle cell conditions, they can change from round flexible disc-like cells to elongated sickle or crescent moon shape. The effect of these changes is that the cells do not pass freely through small capillaries and form clusters, which block the blood vessels. This blockage prevents oxygenation of the tissues in the affected areas resulting in tissue hypoxia and consequent pain (known as sickle cell crisis pain) other symptoms of sickle cell disorders include severe anaemia, susceptibility to infections and damage to major organs.
  39. 39. 39 February11,2012 The term sickle-cell disease is preferred because it is more comprehensive than sickle-cell anaemia.
  40. 40. 40 February11,2012
  41. 41. 41 February11,2012
  42. 42. 42 February11,2012 In children, sickle-shaped red blood cells often become trapped in the spleen, leading to a serious risk of death before the age of seven years from a sudden profound anaemia associated with rapid splenic enlargement or because lack of splenic function permits an infection. Affected children may present with painful swelling of the hands and/or feet (hand-foot syndrome). Survivors may suffer recurrent & severe painful crises, as well as “acute chest syndrome” (pneumonia or pulmonary infarction), bone or joint necrosis, or renal failure.
  43. 43. 43 February11,2012 Primary Laboratory Investigation Hemoglobinopathy • Variety of hemogram findings depending on • Type • Severity of the specific disorder • Only sickle hemoglobinopathies and Hb C will be described here
  44. 44. 44 February11,2012 Primary Laboratory Investigation Heterozygous & Other Disorders • AS • S-Thal • Other hemoglobinopathies, e.g., SC • Hb C
  45. 45. 45 February11,2012 Morphologic Findings Hb SS vs. Hb SC vs. Hb CC =+ Hb S Hb C Hb SC
  46. 46. 46 February11,2012 Morphologic Findings Hb SS vs. Hb SC vs. Hb CC =+ Hb S Hb C Hb SC + =
  47. 47. 47 February11,2012 Where Do Sickle Cells Come From? Sheared in microcirculation Irreversible Sickle Cell
  48. 48. 48 February11,2012 Sickle Cells
  49. 49. 49 February11,2012 Secondary Laboratory Investigation • Hemoglobin electrophoresis • Major test for identifying thalassemia and hemoglobinopathy • Types • Cellulose acetate: Alkaline pH • Citrate agar: Acid ph
  50. 50. 50 February11,2012 Secondary Laboratory Investigation Cellulose Acetate Hb Electrophoresis - A2/C S F A + Normal
  51. 51. 51 February11,2012 Secondary Laboratory Investigation Cellulose Acetate Hb Electrophoresis - A2/C S F A + Normal Hb SS
  52. 52. 52 February11,2012 Secondary Laboratory Investigation Cellulose Acetate Hb Electrophoresis - A2/C S F A+ Normal Hb SS Hb AS
  53. 53. 53 February11,2012 Secondary Laboratory Investigation Cellulose Acetate Hb Electrophoresis - A2/C S F A+ Normal Hb SS Hb AS Hb SC Hb CC
  54. 54. 54 February11,2012 Secondary Laboratory Investigation Cellulose Acetate Hb Electrophoresis - A2/C S F A+ Normal Hb SS Hb AS Hb SC Hb CC HB AD
  55. 55. 55 February11,2012 Secondary Laboratory Investigation • Solubility testing-Dithionite tube test • Alkali denaturation test for quantification of fetal hemoglobin • Acid elution test for fetal hemoglobin distribution • Unstable hemoglobin testing for Heinz bodies
  56. 56. 56 February11,2012 Alkali Denaturation for Hemoglobin F • Recommended assay for hgb F in the range of 2- 40% • Principle • Other hemoglobins are more susceptible than hgb F to denaturation at alkaline pH • Denaturation stopped by addition of ammonium sulphate • Denatured hemoglobin precipitates
  57. 57. 57 February11,2012 Alkali Denaturation for Hemoglobin F • Remaining hemoglobin (F) can be measured spectrophotometrically • Specimen: EDTA anticoagulated whole blood • QC: Normal and elevated controls should be used with each batch of specimens
  58. 58. 58 February11,2012 Alkali Denaturation for Hemoglobin F Hgb F, % Diff. Between Duplicates, % <5 0.5 5-15 1.0 >15 2.0
  59. 59. 59 February11,2012 Acid Elution for Fetal Hemoglobin • Indication of distribution of fetal hemoglobin in a population of RBC • Homogeneous distribution: hereditary persistence of fetal hemoglobin • Heterogeneous distribution: thalassemia
  60. 60. 60 February11,2012 Course and Treatment Sickle Cell Disease • Sickle cell disease • Asymptomatic at birth • Symptoms appear as percentage of fetal hemoglobin decreases during first year of life • Untreated crises increase morbidity and early death
  61. 61. 61 February11,2012 Course and Treatment Sickle Cell Disease • Sickle cell disease • Asymptomatic at birth • Symptoms appear as percentage of fetal hemoglobin decreases during first year of life • Untreated crises increase morbidity and early death
  62. 62. 62 February11,2012 Course and Treatment Sickle Cell Disease • Life span can be significantly increased with early and effective treatment • Studies of natural populations reveal that individuals with sickle cell disease are capable of normal life spans
  63. 63. 63 February11,2012 Course and Treatment In both thalassemia and hemoglobinopathy therapy is usually supportive rather than curative
  64. 64. 64 February11,2012 Course and Treatment • Blood transfusion is used to • Control severe anemia • Reduce the risk of complications of sickle hemoglobinopathies (cerebrovascular accident, hypersplenism, etc.)
  65. 65. 65 February11,2012 Course and Treatment • Chronic blood transfusion • Results in iron overload of major organs resulting in increased morbidity • Laboratory monitoring • Necessitates the use of chelating agents to remove excess iron
  66. 66. 66 February11,2012 Course and Treatment • Excess iron can cause the appearance of sideroblastic conditions • Transfusion interferes with the typical laboratory findings for the disorder
  67. 67. 67 February11,2012 Course and Treatment • Alternative treatment • Activation of fetal hemoglobin genes • Bone marrow transplantation