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

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Presentation on theme: "3 rd Basic Hematopathlogy Course 2013 Laboratory Investigations in Hemoglobinopathies Dr Sandeep Warghade Metropolis Healthcare Ltd."— Presentation transcript:

1 3 rd 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 - & -thalassemias – Alleles are classified into those producing no product ( 0, 0 ) and those producing reduced amounts of product ( +, +). 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 -globin molecule.

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

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8 Normal adult Haemoglobin HaemoglobinGlobin chains% Hb Aα 2 β Hb A2α 2 δ Hb Fα 2 γ

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 Commercial, widely available method used for many years. Gives an estimate of HbA2 level. Identifies some variant haemoglobins which are well characterized. 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).

14 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 Strengths

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16 Isoelectric Focusing Strength 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 Disadvantage Labor-intensive and time- consuming

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 Separation column Packing material Negatively charged carboxyl molecules bound to silica make up the cartridge matrix. Positively charge molecules (salt and hemoglobin) bind to the carboxyl groups.

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. Strong Weak Mobile phase Stationary phase

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

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

24 24 Separation Process and Chromatogram Output concentration Time Chromatogram

25 Chromatogram tRtR t0t0 Intensity of detector signal Time Peak t R : Retention time h A t 0 : Non-retention time A : Peak area h : Peak height

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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 Hemoglobin AgeHgb A1%Hgb A2%Hgb F% Month Months Months Months Months Months Months Months Months - Adult

32 CE-HPLC Interpretation HbA2 rangeInterpretation 2.0 – 3.3 %Normal. 3.8 – 7.0 %Beta thalessemia trait 3.4 – 3.7 % Fe deficiency in β thal trait; Δ chain variant with β thal trait. rare β thal mutations. HbS making measurement inaccurate > 7.0 % Exclude a structural variant. Can be due to rare β thal mutations. < 2.0 %Δ β thal (but HbF should be elevated). Alpha thal trait; Hb H disease Iron deficiency.

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

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

35 Patients detailsRBC IndicesHPLC Hb VariantsInterpretationAdvise RBC : 5.23HbF : 0.7Beta Thalassaemia Trait.- Zakiya NagoriHB : 9.9HbA2 : 4.5 Female / 26 yearsHCT : 31.4HbA : 94.8 MCV : 60.0 MCH : 18.8 MCHC : 31.3 RDW : 20.1 Beta Thalassaemia Trait

36 Patients detailsRBC IndicesHPLC Hb VariantsInterpretationAdvise RBC : 4.12HbF : 93.0Thalassaemia Syndrome.Family studies. Mohd Rehan SiddhiqueHB : 8.0HbA2 : 2.7 Beta Thalassaemia Major Child / 5 yearsHCT : 27.1HbA : 4.3 MCV : 65.7 MCH : 19.3 MCHC : 29.4 RDW : 36.4 Thalassaemia Syndrome

37 Patients detailsRBC IndicesHPLC Hb VariantsInterpretationAdvise RBC :HbF : 22.0Sickle cell disease.Family studies Rushi RathodHB :HbA2 : 3.3(Homozygous HbS) Child / 2.5 yearsHCT :HbA : 2.5 MCV :HbS : 72.2 MCH : MCHC : RDW : Sickle cell disease

38 Patients detailsRBC IndicesHPLC Hb VariantsInterpretationAdvise RBC : 5.30HbF : 0.8HbS TRAIT.- Jiji GeorgeHB : 14.8HbA2 : 3.1 Male/-HCT : 45.4HbA : 58.4 MCV : 85.6HbS : 37.7 MCH : 28.0 MCHC : 32.7 RDW : 16.1 HbS Trait

39 Patients detailsRBC IndicesHPLC Hb VariantsInterpretationAdvise RBC : 4.23HbF : 0.3HbD TRAIT.- Sonia GeorgeHB : 12.4HbA2 : 2.2 Female/-HCT : 37.6HbA : 60.7 MCV : 88.8HbD : 36.8 MCH : 29.2 MCHC : 32.9 RDW : 14.1 HbD Trait

40 Patients detailsRBC IndicesHPLC Hb VariantsInterpretationAdvise RBC : 2.83HbF : 24.1HbS - D disease- B/O Sonia GeorgeHB : 7.9HbA2 : 1.8Parents studies show father HbS Trait Child / -HCT : 23.9HbA : 26.4and mother HbD Trait. MCV : 84.6HbS : 16.6 MCH : 27.9HbD : 31.1 MCHC : 32.9 RDW : 22.3 HbS - D disease

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43 Patients detailsRBC IndicesHPLC Hb VariantsInterpretationAdvise RBC : 4.44HbH : 9.6HbH disease. (Alpha Thalassaemia)Family studies. VALSAHB : 8.72 : 2.1(Capillary's Haemoglobin Electrophoresis) Female / -HCT : 30.7HbA : 87.4 MCV : 69.2HbA2 : 0.9 MCH : 19.6 MCHC : 28.4 RDW : 21.5 HbH disease

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46 Patients detailsRBC IndicesHPLC Hb VariantsInterpretationAdvise RBC : 4.31HbF : 7.5HbE - S Disease.Family studies. Mast AbdielHB : 10.8HbA2 : 33.9 (A2+E) Child / 3.6 yearsHCT : 33.7HbS : 54.3 MCV : 78.3HbA : 4.3 MCH : 25.0 MCHC : 31.9 RDW : 15.9 HbE - S Disease

47 Patients detailsRBC IndicesHPLC Hb VariantsInterpretationAdvise RBC : 3.61HbF : 1.0HbS - C Disease.Family studies Ganiath YayaHB : 10.4HbA2 : 3.9 F / 35 yearsHCT : 31.0HbA : 2 MCV : 85.6HbS : 46.9 MCH : 28.8HbC : 46.2 MCHC : 33.6 RDW : 17.9 HbS - C Disease

48 Sid. No. – HPLC findings- ? Bet Thal Major or? Beta Thal Intermediate Mutation screening- IVS 1-5 Homozygous Mutant Internal control IVS 1-5- Mut 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

49 Sid. No. – Mutation screening- IVS 1-5 Homozygous Mutant Internal control IVS 1-5- Mut 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

50 MOTHER

51 FATHER

52 1 ST CHILD

53 2 ND CHILD

54 DNA Analysis. Indicated when the hemoglobinopathy not confirmed by other methods or when the underlying mutation important to management. For genetic counseling defining the particular mutation or deletion is often required – this is achieved by a variety of molecular techniques.

55 DNA Analysis DNA from WBCs, amniocytes, or chorionic tissue may be utilized for diagnosis of various α and β globin chain abnormalities. PCR amplifies globin genes and utilizes allele specific primers to detect known globin chain mutations eg HbS, E, D, O + several β thal.

56 DNA Analysis PCR can be used to detect unknown mutations. Aims to separate amplified DNA on gels or with HPLC on the principle that different amino acids migrate differently. 3 primary methods – mutation analysis, DNA scanning and DNA sequencing.

57 DNA Sequencing. DNA sequencing is now standard practice for looking for mutations in the beta and alpha globin genes. Indicated if mutations are not detectable with the preliminary screening and in difficult cases eg N HbA2 beta thal or silent beta thalassaemia. Difficult cases best delineated by direct gene sequencing because a number of causative mutations result in the observed phenotype.

58 IVS1-1 G-T

59 D-Punjab (beta 121 Glu-Gln GAA – CAA)

60 Mutations: IVS1-1 G-T/ D-Punjab (beta 121 Glu-Gln GAA – CAA) – Compound Heterozygous Hb-D punjab/beta-Thalassaemia

61 CONCLUSION CE-HPLC is the preferred methodology for Hemoglobinpathies screening Combination of technologies (HPLC & capillary electrophoresis) is recommended for diagnosing common & some rare hemoglobinopathies DNA studies (PCR or Sequencing) should be utilized for difficult and rare cases

62 THANK YOU


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