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Hemoglobin Electrophoresis

Hemoglobin Electrophoresis. Dr Nisha S Ahmad Chief of Lab Services Metropolis Health Services. Agenda. Brief overview of hemoglobin The globin genes The Thalassemias Structural hemoglobinopathies Testing. Hemoglobin. 4 Heme groups 4 polypeptide chains. Hemoglobin structure. A. B.

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Hemoglobin Electrophoresis

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  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. Hemoglobin structure A B B A heme

  5. Hemoglobins in normal adults A B A G A D B A G A D A HbA HbF HbA2 95-98% ~1% <3.5%

  6. The Globin Genes

  7. Hemoglobin

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

  9. 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

  10. 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 A2 (α2δ2)

  11. Types of B-globin mutations • B0 – No B-globin chains are produced • B+ - some beta chains produced Decreased

  12. Alpha thalassemia

  13. 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 α- and β-thalassemia

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

  15. Normal

  16. Normal Thalassemia

  17. Normal Structural Variant

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

  19. Preanalytical • EDTA sample • Age • H/O Transfusion • Area of Residence E/D/C

  20. SCREENING ANTENATAL PROFILE HbA1c ANEMIA PROFILE PRE MARITAL NEONATAL PROFILE

  21. Manual System

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

  23. Cellulose Acetate Hb Electrophoresis - A2 F A + C/E/OS/D/G NORMAL BTT

  24. Cellulose Acetate Hb Electrophoresis - A2/C S F A+ Normal Hb AS Hb SS

  25. Cellulose Acetate Hb Electrophoresis - A2/C/E S /D F A+ Normal Hb AS HB AD

  26. 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

  27. Citrate Agar Hb Electrophoresis + C S A F _ Normal

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

  29. 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

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

  31. HPLC Automated system;precalibrated column and gradient

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

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

  34. 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

  35. 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

  36. Output Time CHROMATOGRAMS Peak Area RT

  37. Normal HPLC Graph HbA F A2

  38. Hb C Hb H HbF HbS Hb J Hb Köln HbE Hb O HbD Hb Q

  39. A2

  40. Hb Lepore

  41. HbE

  42. HbD

  43. Hb EE/DD

  44. HbF

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

  46. HPLC GRAPH OF CHILD Hb F= 100% Hb A2= 0% HbA0 =0% CBC: Hb = 6.8 g/dl MCV = 67.3 Fl MCH = 21.3 pg RDW = 24.7%

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