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Laboratory Diagnosis in Thalassemia and Hemoglobinopathies

Laboratory Diagnosis in Thalassemia and Hemoglobinopathies. Ahmad Shihada Silmi Msc, FIBMS Staff Specialist in Hematology Medical Technology Department Islamic University of Gaza. Thalassemia and hemoglobinopathies.

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Laboratory Diagnosis in Thalassemia and Hemoglobinopathies

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  1. Laboratory Diagnosis inThalassemia andHemoglobinopathies Ahmad Shihada Silmi Msc, FIBMS Staff Specialist in Hematology Medical Technology Department Islamic University of Gaza

  2. Thalassemia and hemoglobinopathies • Disorders of globin chain production as a consequences of globin gene defects • As a results, hemoglobin productions are also affected. • Also, some properties of red blood cells are also affected. • Thus it can be recognized by various hematological laboratory tests.

  3. Making diagnosis of thalassemia • History retrieve • Physical examination • Laboratory investigations

  4. Laboratory Thalassemia Diagnosis • Red Cell Studies : CBC, One- Tube OF Test, DCIP Test • Hb Studies : Electrophoresis, Microcolumn chromatography, Alkali Denaturation Test, HPLC/LPLC, Imnunologic Detection, Acid elution test • DNA studies: Gene mapping, PCR, Nt sequencing, RFLP analysis

  5. Blood sample *Fresh venous blood sample stored in EDTA (3-5 ml) is enough. *This blood sample is used for both RBC studies, Hb studies and DNA studies.

  6. RBC studies

  7. CBC • CBC (automate is a must) for red blood parameters including Hb, Hct, RBC indicies and RBC morphology examination (already trained) • Hb H inclusion body test

  8. Hb and Hct • Low in thalassemia disease • Hb < 6 g% in thal major • Hb 6-10 g% in thal intermedia • Hb 10-12 g% in thal minor or thal trait • Normal or slightly low in heterozygote • ( Male ~15 g%, Female ~ 13 g%)

  9. MCV, MCH • Cut-off level : MCV 80 fl, MCH 20 pg • Low in thalassemia diseases and thalassemia trait • Normal or slightly low in a-thal-2 trait, HbE trait, Hb CS trait

  10. RBC Morphology • Thalassemia disease : Hypochromia, Anisocytosis, Poikilocytosis, Polychromasia, Target cells, Basophilic Stippling, NRBC • Thalassemia Trait and Homo E : Modest change in RBC morphology

  11. Normal or a-thal 2 trait

  12. a-thal 1 trait

  13. b-thal trait x400

  14. Hb E trait

  15. b-thal/HbE disease

  16. HbE/b-thal 400x 1000x

  17. Homozygous bo-thalassemia 250x (Hb6.3g/dl, Hct 20%, MCV62 fl ,Ret11.5%, NRBC10/100WBC)

  18. Hb H Disease

  19. Hb Bart’s Hydrops Fetalis

  20. Hb H inclusion body test Principle • Hb H (b4) is an unstable hemoglobin commonly seen in a-thalassemia. On incubation with some oxidative chemicals such as brilliant cresyl blue (BCB), HbH is oxidised, denatured and precipitated in the erythrocytes and seen as small, evenly-distributed, intra-erythrocytic blue dots which termed HbH inclusion bodies.

  21. Hb H inclusion body test Reagents : • As for reticulocyte count Step-by-step procedure : The stain and incubation are as for reticulocyte count But look for red blood cells containing HbH inclusion bodies and report as numbers of those red blood cells in certain amount of total red blood cells examined. If numerous HbH inclusion body containing eryhthrocytes are seen : Report in % If few or rare HbH inclusion body containing eryhthrocytes are seen : Report in actual numbers of those rbc/30,000 rbc

  22. Hb H inclusion body test • Homozygous b-thalassemia IB : Negative • HbE/b-thalassemia IB : Negative • AE Bart’s, EF Bart’s IB : 5-10% of total RBC • Hb H disease, Hb H-CS disease IB : 50-100%of total RBC • a-thalassemia 1 heterozygote IB : 1/30,000 RBC • a-thalassemia 2 heterozygote IB : Rare

  23. Picture of Hb H inclusion bodies Hb H disease a-thal 1 trait

  24. One-tube OF test Principle • At a constant hypotonic NaCl solution of 0.36% (w/v), hypochromic red blood cells are able to uphold certain amount of water and remain intact whereas the normal erythrocytes cannot and explode.

  25. One-tube OF test • Reagent: 0.36% Buffered Saline Solution (BSS) Procedure • Mix 20 ul EDTA blood with 5 ml 0.36% BSS • Stand at RT for 5 min. • Visualize for hemolysis -If clear red solution is observed : negative -If turbid red solution is observed : positive

  26. One-tube OF test (0.36% BSS) Negative Positive

  27. One Tube OF Test Thalassemia diseases + b-thal trait + a-thal-1 trait + a-thal-2 trait +/- HbE trait +/- Homo E +/-

  28. DCIP precipitation test Principle • HbE (a2bE2) has loose contact between a1 and b1-globin chains. When it is incubated with dichlorophenol indophenol (DCIP); oxidizing agent, it will be denatured and precipitated. The reaction is stopped by adding ascorbic acid and the denatured HbE precipitates.

  29. DCIP test • Reagent : DCIP reagent Procedure • Mix 20 ul packed red cell with 5 ml DCIP reagent in 13x100 test tube • Incubate the mixture at 37C water bath for 60 min. • Look for precipitation before or after addition of 5% ascorbic acid • Report

  30. DCIP test Ways to report • Negative : No precipitation • Positive : Precipitation seen

  31. DCIP test After adding 5% ascorbic acid Pos Pos Neg Before adding 5% ascorbic acid Blk Neg Pos Pos

  32. DCIP Test Normal Negative Homo E 3+-4+ HbE trait 1+-2+ b-thal/HbE disease1+-2+ HbH disease 1+-2+ Report as positive or negative

  33. Hb studies

  34. Hemolysate preparation • Centrifuge EDTA blood at 3000-5000 rpm and remove plasma • Wash packed red cell with NSS for three time and remove supernatant as much as possible at the last washing round • Add DW 1.5 time the volume of PRC and mix vigorously • Add CCl4 to the half of the volume of lysed red cells and mix vigorously • Centrifuge 3000 -5000 rpm and collect the upper red portion which is “Hemolysate or Hemoglobin solution)

  35. Hemoglobin electrophoresis at alkali pH • Hb: Amphotericmolecule • Molecular net charge depends on pH of the medium. • pH > pI (Iso-electric point) : Molecular net charge is negative. • pH < pI : Molecular net charge is positive. • pI (Iso-electric point) is the pH where molecular net charge of hemoglobin is zero.

  36. Hemoglobin electrophoresisat alkali pH • Principle • In alkali medium, Hbs will gain negative net charge. • Different Hbs have different molecular negative net charge. • Being placed between cathode and anode, Hbs will move away from the anode. • The velocity of the movement depends solely on the molecular net charge. • Pattern from cathode to anode is : A2/E, F, A, Bart’s, H

  37. Hemoglobin electrophoresisat alkali pH Reagent : Tris-EDTA-Borate (TBE) pH 8.4-8.6

  38. Equipment • 1. Power supply for 500 V • 2. Electrophoretic chamber • 3. Cellulose acetate plate • 4. Sample applicator • 5. Stain box • 6. Large filter paper or • blotter

  39. Equipment Sample applicator Aligning base Sample preparation well

  40. Equipment Cellulose acetate plate Blotter

  41. Equipment Power supply Cellulose acetate plate Electrophoretic chamber http://sun.science.wayne.edu/~hhmi/gifs/elec1.jpg

  42. Equipment

  43. Specimens • Hb in the solution or “hemolysate”.

  44. Procedure • Hemolysate in wells • Serum applicator dipped and applied on soaked cellulose acetate plate • Place cellulose acetate, face-down, in electrophoretic chamber. • Run elctophoresis at 300 volts for 10-20 min. • Stained with Ponceau S

  45. Ponceau S staining • Dip cellulose acetate plate in the stain and leave for 5 min • Wash with destaining solution (5% HOAc) twice and 5 min each time or until background becomes white • Read Hb bands

  46. During electrophoresis

  47. Ponceau S and Destaining solution

  48. Hb electrophoretic pattern

  49. Hb pattern on CAE with TBE pH 8.6

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