HbF<2% 95-98% H b A %

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5 HbF<2% 95-98% H b A %

6 Hemoglobinopathies Molecular Pathology of Hemoglobinopathies A genetic defect that results in abnormal structure of given hemoglobin or chains Structural mutations Altered exterior (HbS) Altered active site (HbM) Altered third structure (Hb Gunhill) Altered quaternary structure Oxygen low affinity Oxygen high affinity

7 Thalassemia A genetic defect that results in production or abnormally low quantity of a given hemoglobin or chains Molecular l Pathology Alpha Thalassemia Deletional mutations (Hb H) Nondeletional mutations ( Constant Spring) Molecular Pathology Beta Thalassemia 1. RNA synthesis and processing is impaired 2. Premature e chain termination 3. Frame shift mutation 4. Noncoding intron mutation

8 Hemoglobin variants Over 1000 Hb variants have been documented few are common and clinically significant Abnormalities lead to change in molecule structure or charge that can be detected with appropriate methodologies

9 β, α and δ Chain Variants Hb S HbC β 6Glu Glu Val β 6Glu Glu Lys Altered Exterior Altered Exterior Hb D β 126 HbE β Glu Gln 26Glu Lys Hb O Arab β 121Glu Gu Hb G α 68 HbA2 ' δ Glu Lys 68Asp Lys chain variant

10 Methods for the Identification of Hemoglobinopathies&Thalassemia Cellulose Acetate electrophoresis in Alkaline ph Citrate agar electrophoresis in ph Acidic Isoelectricfocusing HPLC Capillary electrophoresis Complementary e test Solubility Test Heat instability MetHb percentages Chain synthesis DNA Analysis

11 Cellulose Acetate Electrophoresis ph 8.4 Surface charge is affected by amino acid substitution Hb variant can be quantitated by densitometry It is not accurate for HbA2 and HbF percentages HbS and HbG and Hb D migrate at the same position HbC,HbE b and Hb A2 migrate at the same position o

12 Fast Hbs

13 Citrate Agar Electrophoresis (ph 6.2) Hb Molecules separate based on their charge and ability combine with agar Used to differenciate variants that comigrate in alkaline ph on cellulose acetate tt HbC from E and S from Dand G Can not distinguish between HbD or G Qualitative method - + A2 +

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15 IEF Separates Hb according to their isoelectric point on the gel. The gel containing ph gradient. Hb will migrate in electerical field until it encounters part that corresponds to its pi. Excellent separation but lengthy procedure Qualitative ti

16 IEF

17 Anion Exchange Chromatography Microcolumn Chromatography

18 HPLC Cation exchange chromatography ph 6.4 Amino Acid substitution will alter the Hb retention time relative to HbA The eluting solution competes with Hb s attach to resin,absorbed Hb molecules l are reabsorbed b df from the column. Detectors records OD changes over time and outcome is a graph Negativ e

19 HPLC Diagram The sample mixture is introduced into the system at the injection port and a mobile phase liquid flowing through the system carries the compounds into the column at an acid ph. solvent column Chrom atogra m chroma togram injector pump sample detect or waste

20 HPLC Improved separation of variants in comparison to gels Accurate measurement of Hb A2 and HbF, in absence of Hb variants HbS and HbC glycated fractions interfere with HbA2 quantification HbA2 and HbE do not separate HbH and Hb Bart s can be detected in the chromatogram but not quantitated Hb Lepore and HbA2 do not separate

21 Anylate Identification Windows

22 HPLC Migration of Hemoglobin Variants Hemoglobin HbA2 Result HbF Comments S Falsely elevated Coelution S 1c with A2 E Falsely elevated Coelution of Hb E with A2 G philadelphia Falsely elevated Coelution of HbG with A2 Lepore Falsely elevated Coelution of Hb Lepore with A2 HbA1c Falsely elevated

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26 Capillary Electrophoresis Diagram Separation is achieved in a small glass tube Capillary filled with alkaline buffer (ph 9.4) A few volume (nl) of sample injected

27 Principle of Hbs Separation by Capillary Electrophoresis Mixtures separated due to Hb Charge and electroosmotic flow Electroosmotic flow is stronger than migration based on the charge

28 As a result all hemoglobins are carried towards the cathodic end of the capillary

29 Electrophoregrams were recorded with the location of specific hemoglobins in specific zones. Z15 Z14 Z13 Z12 Z11 Z10 Z9 Z8 Z7 Z6 Z5 Z4 Z3 Z2 Z1 H N or J Bart A F D G Lepor e S E A2 C CS A2

30 Hemoglobin Control

31 HbH,HbA,HbA2

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34 Hemoglo bin CZE Migration of Hemoglobin Variants Interferes with HbA2 Result HbA2: zone 3 HbF: zone 3 Comments S E D No No No Hb S: zone5 Hb E :zone 4 HbD: zone6 G philadel phia Lepore Hb C No No Sometimes HbG :zone 6 Hb lepore: zone 6 Hb C: zone 2

35 Normal Pattern

36 High Hemoglobin F

37 Hemoglobin Control

38 Final goal HPLC vs. Capillary Electrophoresis HPLC Capillary Electrophoresis Separating compound in a mixture Separating compound in a mixture Sample separated inside of Column filled with solvent Capillary filled with buffer Deriven by Mixture separated due to pressure Cation -exchange Electrical field Electrophoretic mobility &electroosmotic flow

39 Screening For Thalassemia RBC Indices MCH> 27pg MCH>27 pg MCH< 27 pg HbA2< 3.3% Hb A2<1.7 % Hb A2< 3.3% Hb A2 > 3.5% Normal Delta -Thal Further Investigation Minor Beta=Thal

40 Determination of HbA2 in the Presence of Common Hemoglobin Variants by CZE vs. HPLC and CAE

41 Normal Patterns

42 HbA2 Percentages CZE vs HPLC Normal samples Samples with HbS

43 Measurement of HbA2 and HbS&C in Measurement of HbA2 and HbS Samples Containing HbS&HbC in Samples Containing HbS

44 Measurement of HbA2 and HbE in Samples Containing HbE

45 Measurment of HbA2 and HbC in the Samples Containing HbC

46 Conclusion The first group of tests should include red cell indices determination, Hb A2 and HbF quantitation, Hb variant detection by traditional or novel methods. In individuals with hypochromia microcytosis and normal iron values and normal HbA2 indicate the presence of a thalassemia determinant (α-thal, thal,δβ-thal,mild β-thal,double heterozygotesity for α + δ or α+ β-thal). Discrimination between these genotypes is carried out by globin chain synthesis s and α, β,,a and dδ-globin gob gene ea analysis. ayss Finally,it is recommended to perform DNA sequencing.