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  1. 1. THALASSEMIAS ANDHEMOGLOBINOPATHIES Modified from an original presentation by Raymond L. Olesinski ©2001 University of Kentucky
  2. 2. Thalassemias and Hemoblobinopathies: Module ObjectivesAt the end of this module you should be able to• Explain the pathophysiology that causes thalassemia and hemoglobinopathies.• Explain how thalassemias are categorized.
  3. 3. Thalassemias and Hemoblobinopathies: Session Objectives• Correlate the results of laboratory testing with specific thalassemias and hemoglobinopathies.
  4. 4. Thalassemias and Hemoblobinopathies: Session ObjectivesFor the1. Dithionite tube test2. Hemoglobin electrophoresis3. Alkali denaturation test for fetal hemoglobin• Discuss specifics of specimen collection, handling, storage, and preparation
  5. 5. Thalassemias and Hemoblobinopathies: Session Objectives• Explain the physiologic theory relevant to the test/procedure.• Explain the principle of the test/procedure• Identify the disease manifestation/clinical correlation.• Differentiate or resolve technical, instrument, or physiologic causes of problems or unexpected test results.
  6. 6. Characteristics: Thalassemia• Hereditary disorders that can result in moderate to severe anemia• Basic defect is reduced production of selected globin chains
  7. 7. Demographics: Thalassemia• Found most frequently in the Mediterranean, Africa, Western and Southeast Asia, India and Burma• Distribution parallels that of Plasmodium falciparum
  8. 8. Classification & Terminology Alpha Thalassemia• Terminology • Silent carrier • Minima • Minor • Intermedia • Major
  9. 9. Symbolism Alpha Thalassemia• Greek letter used to designate globin chain: α
  10. 10. Symbolism Alpha Thalassemia / : Indicates division between genes inherited from both parents: αα/αα• Each chromosome 16 carries 2 genes. Therefore the total complement of α genes in an individual is 4
  11. 11. Symbolism Alpha Thalassemia- : Indicates a gene deletion: -α/αα
  12. 12. Classification & Terminology Alpha Thalassemia• Normal αα/αα• Silent carrier - α/αα• Minor -α/-α --/αα• Hb H disease --/-α• Barts hydrops fetalis --/--
  13. 13. Symbolism Other Thalassemia• Greek letter used to designate globin chain: β
  14. 14. Symbolism Other Thalassemia: Indicates diminished, but some+ production of globin chain by gene: β+
  15. 15. Symbolism Other Thalassemia0 :Indicates no production of globin chain by gene: β0
  16. 16. Symbolism Other ThalassemiaSuperscript T denotes nonfunctioning gene: αT
  17. 17. Classification & Terminology Beta Thalassemia• Normal β/β• Minor β/β0 β/β+• Intermedia β0/β+• Major β0/β0 β+/β+
  18. 18. Special Cases Thalassemia• Hb Lepore: δβ fusion seen in some types of δβ thalassemia• Hb Constant Spring • α chain with 31 additional amino acids • --/αcsα• Hereditary persistence of fetal hemoglobin (HPFH)
  19. 19. Special Cases: Thalassemia• Hb H • β4 tetramer • Associated with --/-α thalassemia
  20. 20. Special Cases: Thalassemia• Hb Barts & hydrops fetalis • Barts is a γ4 tetramer • Associated with --/-- • Lethal • High concentrations are capable of sickling
  21. 21. Primary Laboratory Investigation ThalassemiaVariable hemogram results proportional to the severity of the thalassemia
  22. 22. Primary Laboratory Investigation Thalassemia• Severe cases present with • Microcytosis • Hypochromia • Poikilocytosis • RBC counts higher than expected for the level of anemia
  23. 23. 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)
  24. 24. 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 & --/αcsα: Normal life span with chronic hemolytic anemia
  25. 25. 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
  26. 26. Characteristics: Hemoglobinopathies• Hereditary disorders that can result in moderate to severe anemia• Basic defect is production of an abnormal globin chain
  27. 27. Demographics Hemoglobinopathies• The demographics of hemoglobinopathies are varied.
  28. 28. 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. Terminology HemoglobinopathyAbnormal hemoglobins discovered earlier have been given letter designations: Hb S
  30. 30. Terminology HemoglobinopathyMore recently discovered hemoglobins have been named by the city or location of discovery: Hb C-Harlem
  31. 31. Amino Acid Substitution HemoglobinopathyGreek letter designates affected globin chain β
  32. 32. Amino Acid Substitution HemoglobinopathySuperscript number designates affected amino acid(s), e.g., β6
  33. 33. Amino Acid Substitution HemoglobinopathyLetters and numbers in parentheses designate the helical segment and amino acid sequence in that segment affected (sometimes omitted), e.g., β6(A3)
  34. 34. Amino Acid Substitution HemoglobinopathyAmino 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
  35. 35. Classification: Hemoglobinopathy• Functional Abnormality • Aggregation • Polymerization • Crystallization • Unstable hemoglobins • Methemoglobin • Oxygen affinity
  36. 36. 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
  37. 37. Primary Laboratory Investigation Heterozygous & Other Disorders• AS• S-Thal• Other hemoglobinopathies, e.g., SC• Hb C
  38. 38. Morphologic FindingsHb SS vs. Hb SC vs. Hb CC + =Hb S Hb C Hb SC
  39. 39. Morphologic FindingsHb SS vs. Hb SC vs. Hb CC + =Hb S Hb C Hb SC + =
  40. 40. Where Do Sickle Cells Come From? Sheared inmicrocirculation Irreversible Sickle Cell
  41. 41. Sickle Cells
  42. 42. Secondary Laboratory Investigation• Hemoglobin electrophoresis • Major test for identifying thalassemia and hemoglobinopathy • Types • Cellulose acetate: Alkaline pH • Citrate agar: Acid ph
  43. 43. Secondary Laboratory Investigation• Patterns of mobility (see handout)
  44. 44. Secondary Laboratory Investigation Cellulose Acetate Hb Electrophoresis - A2/C S F A+Normal
  45. 45. Secondary Laboratory Investigation Cellulose Acetate Hb Electrophoresis - A2/C S F A+NormalHb SS
  46. 46. Secondary Laboratory Investigation Cellulose Acetate Hb Electrophoresis - A2/C S F A+NormalHb SSHb AS
  47. 47. Secondary Laboratory Investigation Cellulose Acetate Hb Electrophoresis - A2/C S F A+NormalHb SSHb ASHb SC
  48. 48. Secondary Laboratory Investigation Cellulose Acetate Hb Electrophoresis - A2/C S F A+NormalHb SSHb ASHb SCHb CC
  49. 49. Secondary Laboratory Investigation Cellulose Acetate Hb Electrophoresis - A2/C S F A+NormalHb SSHb ASHb SCHb CCHB AD
  50. 50. 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
  51. 51. 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
  52. 52. 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
  53. 53. Alkali Denaturation for Hemoglobin FHgb F, % Diff. Between Duplicates, % <5 0.5 5-15 1.0 >15 2.0
  54. 54. Alkali Denaturation for Hemoglobin F• Sources of error • Too short or too long an incubation time • Filtrate turbidity • Outdated reagents • Incorrect reagent concentrations • Poor quality filter paper
  55. 55. 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
  56. 56. 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
  57. 57. 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
  58. 58. Course and TreatmentIn both thalassemia and hemoglobinopathy therapy is usually supportive rather than curative
  59. 59. Course and Treatment• Blood transfusion is used to • Control severe anemia • Reduce the risk of complications of sickle hemoglobinopathies (cerebrovascular accident, hypersplenism, etc.)
  60. 60. 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
  61. 61. Course and Treatment• Excess iron can cause the appearance of sideroblastic conditions• Transfusion interferes with the typical laboratory findings for the disorder
  62. 62. Course and Treatment• Alternative treatment • Activation of fetal hemoglobin genes • Bone marrow transplantation
  63. 63. WWW Sites of Interest Joint Center for Sickle Cell and Thalassemic Disorders: http://www- (Overview of sickle cell disease, thalassemia and iron kinetics) The Sickle Cell Information Center, Emory University: (Includes PowerPoint presentations on sickle cell disease)