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Laboratory Diagnosis in Thalassemia and Hemoglobinopathies Ahmad Shihada Silmi Msc, FIBMS Staff Specialist in Hematology Medical Technology Department.

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Presentation on theme: "Laboratory Diagnosis in Thalassemia and Hemoglobinopathies Ahmad Shihada Silmi Msc, FIBMS Staff Specialist in Hematology Medical Technology Department."— Presentation transcript:

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

3 3 Thalassemia and hemoglobinopathies n Disorders of globin chain production as a consequences of globin gene defects n As a results, hemoglobin productions are also affected. n Also, some properties of red blood cells are also affected. n Thus it can be recognized by various hematological laboratory tests.

4 4 Making diagnosis of thalassemia n History retrieve n Physical examination n Laboratory investigations

5 5 Laboratory Thalassemia Diagnosis n Red Cell Studies : CBC, One- Tube OF Test, DCIP Test n Hb Studies : Electrophoresis, Microcolumn chromatography, Alkali Denaturation Test, HPLC/LPLC, Imnunologic Detection, Acid elution test n DNA studies : Gene mapping, PCR, Nt sequencing, RFLP analysis

6 6 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.

7 7 RBC studies

8 8 CBC n CBC (automate is a must) for red blood parameters including Hb, Hct, RBC indicies and RBC morphology examination (already trained) n Hb H inclusion body test

9 9 Hb and Hct n Low in thalassemia disease –Hb < 6 g% in thal major –Hb 6-10 g% in thal intermedia –Hb g% in thal minor or thal trait n Normal or slightly low in heterozygote –( Male ~15 g%, Female ~ 13 g%)

10 10 MCV, MCH n Cut-off level : MCV 80 fl, MCH 20 pg n Low in thalassemia diseases and thalassemia trait Normal or slightly low in  -thal-2 trait, HbE trait, Hb CS trait

11 11 RBC Morphology n Thalassemia disease : Hypochromia, Anisocytosis, Poikilocytosis, Polychromasia, Target cells, Basophilic Stippling, NRBC n Thalassemia Trait and Homo E : Modest change in RBC morphology

12 12 Normal or  -thal 2 trait

13 13  -thal 1 trait

14 14  -thal trait x400

15 15 Hb E trait

16 16  -thal/HbE disease

17 17 HbE/  -thal 400x 1000x

18 18 Homozygous  o -thalassemia 250x (Hb6.3g/dl, Hct 20%, MCV62 fl,Ret11.5%, NRBC10/100WBC)

19 19 Hb H Disease

20 20 Hb Bart’s Hydrops Fetalis

21 21 Hb H inclusion body test Principle Hb H (  4) is an unstable hemoglobin commonly seen in  -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.

22 22 Hb H inclusion body test Reagents : n 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

23 23 Hb H inclusion body test Homozygous  -thalassemia IB : Negative HbE/  -thalassemia IB : Negative n AE Bart’s, EF Bart’s IB : 5-10% of total RBC n Hb H disease, Hb H-CS disease IB : % of total RBC  -thalassemia 1 heterozygote IB : 1/30,000 RBC  -thalassemia 2 heterozygote IB : Rare

24 24 Picture of Hb H inclusion bodies  -thal 1 trait Hb H disease

25 25 One-tube OF test Principle n 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.

26 26 One-tube OF test n Reagent : 0.36% Buffered Saline Solution (BSS) Procedure n Mix 20 ul EDTA blood with 5 ml 0.36% BSS n Stand at RT for 5 min. n Visualize for hemolysis -If clear red solution is observed : negative -If turbid red solution is observed : positive

27 27 One-tube OF test (0.36% BSS) Negative Positive

28 28 One Tube OF Test Thalassemia diseases +  -thal trait +  -thal-1 trait +  -thal-2 trait +/- HbE trait +/- Homo E +/-

29 29 DCIP precipitation test Principle HbE (  2  E 2 ) has loose contact between  and  -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.

30 30 DCIP test n Reagent : DCIP reagent Procedure n Mix 20 ul packed red cell with 5 ml DCIP reagent in 13x100 test tube n Incubate the mixture at 37C water bath for 60 min. n Look for precipitation before or after addition of 5% ascorbic acid n Report

31 31 DCIP test Ways to report n Negative : No precipitation n Positive : Precipitation seen

32 32 DCIP test Before adding 5% ascorbic acid After adding 5% ascorbic acid Pos Pos Neg Blk Neg Pos Pos

33 33 DCIP Test Normal Negative Homo E HbE trait  -thal/HbE disease HbH disease Report as positive or negative

34 34 Hb studies

35 35 Hemolysate preparation Centrifuge EDTA blood at 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 rpm and collect the upper red portion which is “Hemolysate or Hemoglobin solution)

36 36 Hemoglobin electrophoresis at alkali pH Hb: Amphoteric molecule 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.

37 37 Hemoglobin electrophoresis at 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 : A 2 /E, F, A, Bart’s, H

38 38 Hemoglobin electrophoresis at alkali pH Reagent : Tris-EDTA- Borate (TBE) pH

39 39 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

40 40 Equipment Sample preparation well Aligning base Sample applicator

41 41 Equipment Blotter Cellulose acetate plate

42 42 Equipment Power supply Electrophoretic chamber Cellulose acetate plate

43 43 Equipment

44 44 Specimens Hb in the solution or “hemolysate”.

45 45 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 min. Stained with Ponceau S

46 46 Ponceau S staining n Dip cellulose acetate plate in the stain and leave for 5 min n Wash with destaining solution (5% HOAc) twice and 5 min each time or until background becomes white n Read Hb bands

47 47 During electrophoresis

48 48 Ponceau S and Destaining solution

49 49 Hb electrophoretic pattern

50 50 Hb pattern on CAE with TBE pH 8.6

51 51 Hb pattern on CAE with TBE pH 8.6 A 2 A A 2 FA A 2 SF EF FA Carbonic anhydrase

52 52 A2AA2A AE A2AA2A Carbonic anhydrase

53 53 Portland Bart’s

54 54 A2AA2A A2AA2A EE

55 55 Microcolumn chromatography Principle In basic (alkali) solution, net charge of Hbs becomes negative. Different Hbs have different negative net charge. On passing through a colume packed with DEAE-cellulose or sephadex resin, different Hbs bind resin at different strength.

56 56 Microcolumn chromatography Principle On passing Cl - ion through column, it will elute Hb off the resin. Order of Hbs being eluted is dependent on negative net charge.

57 Hb A HbA2 Hb F Hb E Hb H Hb Bart’s Cl - Microcolumn chromatography Principle

58 58 Microcolumn chromatography Procedure n Fill pre-swollened DEAE-Sephadex A-50 into Pastuer pipette n Dilute 200 ul hemolysate with 20 ml THK buffer pH 8.5 n Apply 10 ml diluted hemolysate into the column, overlayer column with the resin n Equilibrate with 5 ml THK pH 8.5 n Move column to new tube and elute with 30 ml THK pH 8.2 n OD415 vs DW = OD A2

59 59 Microcolumn chromatography Procedure n Mix 10 ml remaining diluted hemolysate with 20 ml DW n OD415 vs DW = OD Total Calculate n HbA 2 =(OD A2 / OD Total x 5) x 100

60 60 Microcolumn chromatography

61 61 Aim of microcolumn chromatography n To quantify HbA 2 or HbE Benefit: To make diagnosis of  -thalassemia heterozygote or HbE n If 10% : HbE HbA ±0.88% =  -thalassemia trait n HbE 25±5.5% = HbE trait n HbE 87.2±6.9 = Homo E HbE 41.3±7.9 = HbE/  -thal

62 62 Alkali denaturation test Principle n Hb F is resistant to alkali treatment while other Hbs are not and denatured.

63 63 Alkali denaturation test Reagent: n Cyanide solution, 1.2 N NaOH, Sat Amm. Sulfate Equipmet : n Test tubes (13x100), Stop watch, Funnel, Whatman No 1 filter paper, Spectrophotometer

64 64 Alkali denaturation test Procedure n Mix 200 ul hemolysate with 3.8 ml cyanide solution  CyanHb n Treat 2.8 ml cyanHb with 200 ul 1.2N NaCl for 2 min exactly n Stop reaction with 2.0 ml Sat. Amm.Sulfate and filter through Whatman No1 n Read OD540 of filtrate = OD Filtrate n Mix 400ul CyanHb with 6.75ml DW n Read OD540 of total = OD Total n Calculate HbF(%) = OD Filtrate x 100/OD Total x n OR HbF(%) =[ OD Filtrate /OD Total ] x 10

65 65 Alkali Denaturation Test n Normal % High in  -thalassemia disease, HPFH,  -thalassemia Normal or slightly high in  -thal trait : HbE trait and Homo E n Hb Bart’s is also resistant to alkali denaturation.

66 66 Alkali denaturation test

67 67

68 68 Final product (OD540) Filtrate Total

69 69 Acid Elution Test HbF also resists to acid treatment while others (except Hb Bart’s) do not. F cell : normal or slightly high in  -thal trait and heterocellular HPFH F cell : % in  -thalassemia

70 70 n Make a thin blood film, let it air-dry n Fix the smear in 80% EtOH for 2 min exactly n Dip the fixed smear into 0.1% Amido Black solution in 80% EtOH pH for 2 min exactly n Wash the stained blood smear with flushing tap water n Let it air-dry n Look for F cell (stained deep blue) under oil- immersion power n Report in % (in 1,000 rbc examined), but if few F cells are seen, report as number per HPF Acid Elution Test

71 71 F Cells Non-F cell

72 72 Hb identification by HPLC Principle n Hb is amphoteric molecule and changes net charge according to pH of medium. n If pH < PI, net charge becomes positive (cation ) and different Hbs have different positive charge. n HPLC separation of Hbs is based on cation exchange chromatography n Stationary phase is negatively charged by functional group, e.g. polyaspatic acid. n Mobile phase is buffer with pH lower than pI of Hbs n Order of Hbs : Bart’s, H, F, A, A 2 /E according to RT

73 73 Normal or  -thal trait  -thal trait

74 74 Homo E HbE trait

75 75 Hb H disease in newborn HbE/   -thalassemia

76 76 Immunologic Demonstration of Hemoglobin Polyclonal and monoclonal Abs for Hbs are produced aiming to detect Hbs in heterozygous state. Anti-Hb Bart’s for  -thalassemia 1 heterozygote Anti-Hb Bart’s plus anti  -globin chain for  - thalassemia 1 heterozygote (SEA type) Anti HbE for HbE heterozygote Anti HbA 2 for  -thalassemia heterozygote Detection techniques : RIA, ELISA, Immunochromatography (Strip test) Immunofluorescence staining and examine using fluoromicroscope or flow cytometer.

77 DNA Analysis DNA was released from nucleated cells ; white blood cells. Polymerase Chain Reaction (PCR) to amplify globin gene fragment Mutation detection by: electrophoresis, hybridization or gene sequencing

78 Thank You ALL


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