3 rd Basic Hematopathlogy Course 2013

1. 3rd Basic Hematopathlogy Course 2013 Laboratory Investigations in Hemoglobinopathies Dr Sandeep Warghade Metropolis Healthcare Ltd

2. Hemoglobinopathies occupy a special place in human genetics for many reasons: • They are by far the most common serious Mendelian diseases on a worldwide scale • More mutations and more diseases are described for hemoglobins than for any other gene family

3. World Health Organization (WHO) figures estimates that 7% of world population is carrier for hemoglobin disorders. (World Health Organization 2008) • Population screening has identified the prevalence of β-thalassemia carrier status as high as 17% in certain communities in India. (Indian Journal of Public Health 2012)

4. Two groups of hemoglobinopathies • Thalassemias are generally caused by inadequate quantities of the polypeptide chains that form hemoglobin. • The most frequent forms of thalassemia are therefore the a- & b-thalassemias • Alleles are classified into those producing no product (a0, b0) and those producing reduced amounts of product (a+, b+). • Abnormal hemoglobins (Variants) with amino acid changes cause a variety of problems, of which sickle cell disease is the best known. • In sickle cell disease, a missense mutation (glutammic acid to valine at codon 6) replaces a polar by a neutral amino acid on the outer surface of the b-globin molecule.

6. Chromosomal locations of globin genes Chromosomal distribution of the genes for the a family of globins on chromosome 16 and the b family of globins on chromosome 11 in humans. Gene structure is shown by black bars (exons) and colored bars (introns).

8. Normal adult Haemoglobin

9. METHODOLOGIES FOR INVESTIGATIONS • CBC • Kleihauer-Betke for fetal Hb • Sickling/solubility test • Electrophoresis • IEF • CE-HPLC – most widely used primary technology • Combinations • Molecular techniques – PCR/DNA Sequencing

10. CRITERIAS FOR SELECTION OF METHODOLOGY • Provisionally identify all the common, diagnostically important, normal & variant haemoglobins. • Quantification of Hb A2 & HbF must be precise & accurate • Easy to perform- preferably automation

11. Electrophoresis - Gel • Separation of haemoglobins with electrophoresis at pH 8.4 (alkaline) and pH 6.2 (acid). • Scanning allows quantification of the hemoglobin present, bands are seen by staining. • At alkaline pH Hb C, E, A2 and O migrate together to form a single band, Hb S, D and G also co migrate.

12. Electrophoresis - Gel • At acid pH Hb C separates from E and O and Hb S separates from D and G. • Hb E and O cannot be separated by electrophoresis neither can Hb D and G.

13. Electrophoresis - Gel Strengths Disadvantages Labor-intensive. Inaccurate in quantification of low-concentration variants (HbA2) and in detection of fast variants (HbH, Hb Barts). The precision and accuracy for Hb A2 using scanning of electrophoretic gels is poor (in comparison to HPLC). • Commercial, widely available method used for many years. • Gives an estimate of HbA2 level. • Identifies some variant haemoglobins which are well characterized.

14. Strengths Capillary Electrophoresis • Utilizes 8 silica glass capillary tubes instead of agarose gel • Easy to perform, automated • Processed at very high voltage - Better resolution than gel electrophoresis • Accurate quantification of HbA2 in HbS & HbD cases

16. Isoelectric Focusing Strength Disadvantage Labor-intensive and time-consuming • Equilibrium process in which Hb migrates in a pH gradient to a position of 0 net charge  can be used to separate and quantify Hb. • Excellent resolution allowing precise and accurate Hb quantification. • The migration order is the same as with alkaline electrophoreses however HbC and E separate as do HbO and S and HbD and G

17. Capillary Isoelectric Focusing. • Hybrid technique combining capillary electrophoresis sensitivity with automated sampling and data acquisition of HPLC. • Not commonly used

18. HPLC Principle • Cation-exchange HPLC can be preformed on an automated instrument that can quantify Hb A2, Hb F, Hb A, Hb S, and Hb C. • Studies show equivalence or superiority over electrophoresis in terms of identification of variant haemoglobins and quantification of HbA2 level.

19. HPLC – High Performance Liquid Chromatography • Negatively charged carboxyl molecules bound to silica make up the cartridge matrix. • Positively charge molecules (salt and hemoglobin) bind to the carboxyl groups. Separation column Packing material

20. CE-HPLC

21. Mobile Phase / Stationary Phase • A site in which a moving phase (mobile phase) and a non-moving phase (stationary phase) make contact via an interface that is set up. • The affinity with the mobile phase and stationary phase varies with the solute. Separation occurs due to differences in the speed of motion. Mobile phase Weak Strong Stationary phase

22. Comparing Chromatography to the Flow of a River... Light leaf Water flow Heavy stone Base

23. Interaction Between Solutes, Stationary Phase, and Mobile Phase • Differences in the interactions between the solutes and stationary and mobile phases enable separation. Solute Degree of adsorption, solubility, ionicity, etc. Stationary phase Mobile phase

24. Separation Process and Chromatogram Chromatogram Output concentration Time

25. Chromatogram tR tR : Retention time Peak t0 Intensity of detector signal t0: Non-retention time h A: Peak area A h: Peak height Time

28. HPLC Strengths. • Method of choice for screening for Hb variants; for quantification of HbA2 + HbF concentrations and in neonatal screening. • Quicker and more sensitive than standard techniques for detecting HbF (in diagnosis of HPFH and monitoring sickle cell anemia). • Established role in the diagnosis of thalassaemia and haemoglobinopathies, including with cord blood samples

29. HPLC Disadvantages • HbE, HbD, and HbG co-elute with Hb A2, making quantification Hb A2 impossible when these variants present. • The measurement of Hb A2 is complicated in individuals with Hb S because the Hb A2 is falsely increased by the presence of Hb S adducts. • Capillary zone Electrophoretic method can be used to quantify Hb A2 in the presence of Hb S by eliminating interference from these adducts.

30. CE-HPLC Interpretation • Age • Transfusion history • Ethnic origin • Clinical history • CBC

31. CE-HPLC Interpretation

32. CE-HPLC Interpretation

33. HIGH Hb F HOMOZYGOUS HETEROZYGOUS HPFH Delta-beta thalassaemia Compound heterozygotes (approx. 5 – 20 %) • Beta thalassaemia • HPFH • Delta-beta thalassaemia (approx. 70 -90 %)

34. Acquired causes of High Hb F Hypoxia Anemia Pregnancy Thyrotoxicosis Renal failure • Aplastic anemia • MDS • PNH • JMML • Acute Leukemia • Marrow recovery

35. Beta Thalassaemia Trait

36. Thalassaemia Syndrome

37. Sickle cell disease

38. HbS Trait

39. HbD Trait

40. HbS - D disease

43. HbH disease

46. HbE - S Disease

47. HbS - C Disease

48. DNA Ladder IVS 1-1 WT M IVS 1-5 WT M Cd 8/9 WT M Cd 41/42 WT M Hbe WT M Internal control IVS 1-5- Mut Sid. No. – 100053882 HPLC findings- ? Bet Thal Major or? Beta Thal Intermediate Mutation screening- IVS 1-5 Homozygous Mutant

49. DNA Ladder IVS 1-1 WT M IVS 1-5 WT M Cd 8/9 WT M Cd 41/42 WT M Hbe WT M Internal control IVS 1-5- Mut Sid. No. – 900834752 Mutation screening- IVS 1-5 Homozygous Mutant

50. MOTHER