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Hemoglobin Electrophoresis Dr Nisha S Ahmad Chief of Lab Services Metropolis Health Services.

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Presentation on theme: "Hemoglobin Electrophoresis Dr Nisha S Ahmad Chief of Lab Services Metropolis Health Services."— Presentation transcript:

1 Hemoglobin Electrophoresis Dr Nisha S Ahmad Chief of Lab Services Metropolis Health Services

2 Agenda Brief overview of hemoglobin The globin genes The Thalassemias Structural hemoglobinopathies Testing

3 Hemoglobin 4 Heme groups 4 polypeptide chains

4 B B A A heme Hemoglobin structure

5 A A A AA A BGD BDG HbAHbFHbA %~1% <3.5% Hemoglobins in normal adults

6 The Globin Genes


8 Hemoglobin Type NameComponents AdultA 2 2 A2 2 2 FetalF 2 2 EmbryonicPortland 2 2 Gower Gower AbnormalH 4 Barts 4

9 Hemoglobin TypesQuantity Alpha Thal HbA (α 2 β 2 ) >95%HbH HbA 2 (α 2 δ 2 ) <3.5% - HbF (α 2 γ 2 ) <2.0%Hb Barts

10 An inherited mutation of the globin genes leading to a quantitative or qualitative abnormality of globin synthesis Hemoglobinopathy

11 Thalassemia - Defined A family of genetic anemias characterized by a reduced rate of production of 1 or more globin subunits of hemoglobin (Hb) Symptoms are caused by the deleterious effects of the normally produced subunits that are now in excess

12 Pathophysiology Excess alpha chains precipitate and form inclusion bodies that associate with the RBC cell membrane Cause membrane damage and shortened cell survival Large scale destruction of precursor cells in bone marrow Decreased B production causes increased δ production and an elevated A 2 (α2δ 2)

13 Types of B-globin mutations B 0 – No B-globin chains are produced B + - some beta chains produced Decreased

14 Alpha thalassemia AA / AANormal AA / A -Mild microcytosis (Silent Carrier) AA / - - A - / A - Mild microcytosis (Trait or Carrier) (cis vs trans) A - / - -Hemoglobin H disease- clinically variable - - / - -Hydrops Fetalis (Alpha Thal Major)

15 α- and β-thalassemia Alpha Thalassemia Deletions of alpha- globin gene (s) Symptoms can begin in fetal life Complicated inheritance – 4 alpha genes Beta Thalassemia Nonsense, splice and frameshift mutations in beta-globin gene Symptoms begin in infancy/childhood Simple AR inheritance; genotype-phenotype correlation

16 Structural variant - Defined Abnormal globin protein that is produced at a normal rate, with varying consequences Oxygen affinity, stability and function

17 Normal

18 NormalThalassemia

19 NormalStructural Variant

20 Laboratory Investigation CBC-MCV,MCH,RDW Tests Hemoglobin electrophoresis Cellulose acetate: Alkaline pH Citrate agar: Acid pH Capillary Electrophoresis HPLC IEF DNA

21 Preanalytical EDTA sample Age H/O Transfusion Area of Residence E/D/C


23 Manual System

24 Cellulose Acetate pH-8.6 In a alkaline solution,Hb molecules have a net negative charge and move towards the anode

25 Cellulose Acetate Hb Electrophoresis - A 2 F A + C/E/O S/D/G BTT NORMAL

26 Cellulose Acetate Hb Electrophoresis - A 2 /C S F A+ Normal Hb AS Hb SS

27 Cellulose Acetate Hb Electrophoresis - A 2 /C/E S /D F A+ Normal Hb AS HB AD

28 Citrate Agar Electrophoresis pH-6.4 Citrate agar electrophoresis at an acid Ph provides ready separation of hemoglobins that migrate together on cellulose acetate S from D and G C from E and O

29 Citrate Agar Hb Electrophoresis + C S A F _ Normal

30 Citrate Agar Hb Electrophoresis + C S A F _ Normal Sickle trait

31 HPLC °Separation based on interaction between Stationary Phase & Mobile Phase °Stationary Phase is Analytical Cartridge; Mobile Phase is Buffer °Compounds are separated to target analytes according to physical properties: - size, shape, charge, hydrophobicity & affinity for other molecules °Bound analytes elute off the stationary phase by manipulating the mobile phase

32 Contd… Desorption is then brought about by increasing the salt concentration or by altering the pH of the mobile phase Charged particles (matrix) bind reversibly to sample molecules (proteins, etc.)

33 HPLC Automated system;precalibrated column and gradient

34 Stationary Phase: Cation Exchange Cartridge Carboxyl groups attached to a resin base Direction of flow Detector

35 Hemoglobin Introduction Positively charged hemoglobin fragments in the hemolysate attach to the carboxyl groups at varying strengths.

36 Starting Gradient: Low Ionic Strength Buffer The gradient starts with a low % of Buffer B (high % Buffer A) At this gradient, hemoglobin fragments with an ionic strength lower than the buffer gradient, such as A and F, are displaced from the cartridge and pass into the detector

37 Ending Gradient: High Ionic Strength Buffer As the % of the High Ionic Strength Buffer B increases, the more hemoglobin fragments will be displaced Once the gradient is 100% Buffer B all remaining hemoglobin fragments, including any variant hemoglobins such as S, D and C, will be removed

38 CHROMATOGRAMSOutputTime Peak RT Area


40 Normal HPLC Graph

41 Hb O Hb C Hb H Hb J Hb Köln Hb Q

42 A2 Suggests <3.5%Normal %BTT /(Megaloblastic anemia) 11-15%Hb Lepore 20-40%HbE 30-48%HbD


44 Hb Lepore

45 HbE

46 HbD

47 Hb EE/DD

48 HbF F Suggests <2%Normal <5% HPFH/Pregnancy/ Aplastic anemia 2-15%HPFH BTT Sickle 10-15%Delta Beta Thalassemia >50%Beta Thal Major HPFH (homozygous) Delta Beta Thalassemia (homo)

49 Hb F 1.Delta beta Thal Trait 2.HPFH+IDA 1.Iron Studies 2.DNA Analysis

50 Hb F= 100% Hb A 2 = 0% HbA0 =0% CBC: Hb = 6.8 g/dl MCV = 67.3 Fl MCH = 21.3 pg RDW = 24.7%

51 Hb F = 7.0% HbA 2 = 2.6% HbA0 = 90.4% CBC: Hb = 11.9 MCV = 64.7 MCH = 21.1 Impression: S/o Delta beta thal trait

52 HbF = 8.0% HbA 2 = 2.8% HbA0 = 89.2% CBC: Hb = 13.4 MCV = 68.8 MCH = 22.4 Impression S/O Delta beta thal trait


54 Low A2 Iron Deficiency Anemia Alpha Thalassemia trait HbH Disease Delta Thalassemia Delta –Beta Thalassemia

55 Peak before 1 min Sample Integrity Icteric sample HbH

56 Age-8 yrs Hb-7.8g/dl MCV-61 fl MCH-17.8pg

57 HbH

58 Extended Retic stain HbH inclusions

59 Capillary Electrophoresis Advantages HbH HbE vs HbD Disadvantages Preanalyticals (Cap piercing model) Interpretation

60 Limitations…. Silent carriers Alpha Thalassemia DNA analysis-gold standard

61 DNA analysis Silent carriers Prenatal period 5 common mutations 23 Mutations

62 Conclusion….. Clinical Suspicion CBC PBS Reticulocyte count Specific tests Electrophoresis HPLC Capillary Electrophoresis DNA analysis-Pre natal/gold standard

63 Questions ?

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