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3rd Basic Hematopathlogy Course 2013

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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.

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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).

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8 Normal adult Haemoglobin
Globin chains % Hb A α2 β2 96-98 Hb A2 α2 δ2 Hb F α2 γ2

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

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16 Isoelectric Focusing Strength Disadvantage
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 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
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 Liquid chromatography can be categorized by shape of separation field into column-shaped and planar types. A representative type of chromatography that uses a column-shaped field is “column chromatography”, which is performed using a separation column consisting of a cylindrical tube filled with packing material. Another type is “capillary chromatography”, which is performed using a narrow hollow tube. Unlike column chromatography, however, capillary chromatography has yet to attain general acceptance. (In the field of GC, however, capillary chromatography is a commonly used technique.) Types of chromatography that use a planar (or plate layer) field include “thin layer chromatography”, in which the stationary phase consists of a substrate of glass or some other material to which minute particles are applied, and “paper chromatography”, in which the stationary phase consists of cellulose filter paper. 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 In chromatography, the field of separation is divided into two phases. One phase, called the “stationary phase”, does not move. The other phase, called the “mobile phase”, moves at a constant speed in one direction. The stationary phase and mobile phase make contact via an interface. They do not intermingle, and are kept in a steady state of equilibrium. In the river analogy, the riverbed corresponds to the stationary phase and the flowing water corresponds to the mobile phase. Let us suppose that some substance has been introduced into the flow of the mobile phase and led to the separation site. If this substance contains a component that is only weakly attracted by the stationary phase and a component that is strongly attracted by the stationary phase, the former component will be pulled along quickly by the flow of the mobile phase whereas the latter component will stick to the stationary phase and only move slowly. In this way, differences in the properties of the various components contained in the sample being analyzed give rise to differences in speed. This makes it possible to separate components from each other. Incidentally, in the river analogy, the interaction that determines the speed of motion is based on gravity (and buoyancy in water). In chromatography, various physical and chemical properties, such as solubility and the degree of adsorption, determine the dynamics of separation. Stationary phase

22 Comparing Chromatography to the Flow of a River...
Light leaf Water flow Heavy stone Chromatography can be often compared to the flow of a river. A river consists of a stationary riverbed and water that continuously moves in one direction. What happens if a leaf and a stone are thrown into the river? The relatively light leaf does not sink to the bottom, and is carried downstream by the current. On the other hand, the relatively heavy stone sinks to the bottom, and although it is gradually pulled downstream by the current, it moves much more slowly than the leaf. If you stand watch at the mouth of the river, you will eventually be able to observe the arrival of the leaf and the stone. However, although the leaf will arrive in an extremely short time, the stone will take much longer to arrive. This analogy represents the components of chromatography in the following way: River: Separation field Leaf and stone: Target components of sample Standing watch at the river mouth: Detector 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. The solutes interact with the stationary and mobile phases. These interactions are the most important contributing factor behind separation. Representative examples of the types of interactions that take place in liquid chromatography are given below. (They are not based on strict classifications.) Adsorption Distribution Hydrophobic interaction Ion exchange Ion pair formation Osmosis and exclusion Affinity Stationary phase Mobile phase

24 Separation Process and Chromatogram
The separation process for column chromatography is shown in the above diagram. After the eluent is allowed to flow into the top of the column, it flows down through the spaces in the packing material due to gravity and capillary action. In this state, a sample mixture is placed at the top of the column. The solutes in the sample undergo various interactions with the solid and mobile phases, splitting up into solutes that descend quickly together with the mobile phase and solutes that adsorb to the stationary phase and descend slowly, so differences in the speed of motion emerge. At the outlet, the elution of the various solutes at different times is observed. A detector that can measure the concentrations of the solutes in the eluate is set up at the column outlet, and variations in the concentration are monitored. The graph representing the results using the horizontal axis for times and the vertical axis for solute concentrations (or more accurately, output values of detector signals proportional to solute concentrations) is called a “chromatogram”. Output concentration Chromatogram Time

25 Chromatogram tR tR : Retention time t0 t0 : Non-retention time h
Peak Intensity of detector signal t0 t0 : Non-retention time h A : Peak area A Usually, during the time period in which the sample components are not eluted, a straight line running parallel to the time axis is drawn. This is called the “baseline”. When a component is eluted, a response is obtained from the detector, and a raised section appears on the baseline. This is called a “peak”. The components in the sample are dispersed by the repeated interactions with the stationary and mobile phases, so the peaks generally take the bell-shape form of a Gaussian distribution. The time that elapses between sample injection and the appearance of the top of the peak is called the “retention time”. If the analytical conditions are the same, the same substance always gives the same retention time. Therefore, the retention time provides a means to perform the qualitative analysis of substances. The time taken for solutes in the sample to go straight through the column together with the mobile phase, without interacting with the stationary phase, and to be eluted is denoted as “t0”. There is no specific name for this parameter, but terms such as “non-retention time” and “hold-up time” seem to be commonly used. Because the eluent usually passes through the column at a constant flow rate, tR and t0 are sometimes multiplied by the eluent flow rate and handled as volumes. The volume corresponding to the retention time is called the “retention volume” and is notated as VR. The length of a straight line drawn from the top of a peak down to the baseline is called the “peak height”, and the area of the raised section above the baseline is called the “peak area”. If the intensities of the detector signals are proportional to the concentrations or absolute quantities of the peak components, then the peak areas and heights are proportional to the concentrations of the peak components. Therefore, the peak areas and heights provide a means to perform the quantitative analysis of sample components. It is generally said that using the peak areas gives greater accuracy. h : Peak height Time

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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 Age Hgb A1% Hgb A2% Hgb F% 0 - 1 Month 2 Months 3 Months 4 Months 5 Months 6 - 8 Months Months Months 25 Months - Adult

32 CE-HPLC Interpretation
HbA2 range Interpretation 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 HETEROZYGOUS Beta thalassaemia HPFH
Delta-beta thalassaemia (approx %) 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 Beta Thalassaemia Trait
Patients details RBC Indices HPLC Hb Variants Interpretation Advise RBC : 5.23 HbF : 0.7 Beta Thalassaemia Trait. - Zakiya Nagori HB : 9.9 HbA2 : 4.5 Female / 26 years HCT : 31.4 HbA : 94.8 MCV : 60.0 MCH : 18.8 MCHC : 31.3 RDW : 20.1

36 Thalassaemia Syndrome
Patients details RBC Indices HPLC Hb Variants Interpretation Advise RBC : 4.12 HbF : 93.0 Thalassaemia Syndrome. Family studies. Mohd Rehan Siddhique HB : 8.0 HbA2 : 2.7 Beta Thalassaemia Major Child / 5 years HCT : 27.1 HbA : 4.3 MCV : 65.7 MCH : 19.3 MCHC : 29.4 RDW : 36.4

37 Sickle cell disease Patients details RBC Indices HPLC Hb Variants
Interpretation Advise RBC : HbF : 22.0 Sickle cell disease. Family studies Rushi Rathod HB : HbA2 : 3.3 (Homozygous HbS) Child / 2.5 years HCT : HbA : 2.5 MCV : HbS : 72.2 MCH : MCHC : RDW :

38 HbS Trait Patients details RBC Indices HPLC Hb Variants Interpretation
Advise RBC : 5.30 HbF : 0.8 HbS TRAIT. - Jiji George HB : 14.8 HbA2 : 3.1 Male/- HCT : 45.4 HbA : 58.4 MCV : 85.6 HbS : 37.7 MCH : 28.0 MCHC : 32.7 RDW : 16.1

39 HbD Trait Patients details RBC Indices HPLC Hb Variants Interpretation
Advise RBC : 4.23 HbF : 0.3 HbD TRAIT. - Sonia George HB : 12.4 HbA2 : 2.2 Female/- HCT : 37.6 HbA : 60.7 MCV : 88.8 HbD : 36.8 MCH : 29.2 MCHC : 32.9 RDW : 14.1

40 HbS - D disease Patients details RBC Indices HPLC Hb Variants
Interpretation Advise RBC : 2.83 HbF : 24.1 HbS - D disease - B/O Sonia George HB : 7.9 HbA2 : 1.8 Parents studies show father HbS Trait Child / - HCT : 23.9 HbA : 26.4 and mother HbD Trait. MCV : 84.6 HbS : 16.6 MCH : 27.9 HbD : 31.1 MCHC : 32.9 RDW : 22.3

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43 HbH disease Patients details RBC Indices HPLC Hb Variants
Interpretation Advise RBC : 4.44 HbH : 9.6 HbH disease. (Alpha Thalassaemia) Family studies. VALSA HB : 8.7 2 : 2.1 (Capillary's Haemoglobin Electrophoresis) Female / - HCT : 30.7 HbA : 87.4 MCV : 69.2 HbA2 : 0.9 MCH : 19.6 MCHC : 28.4 RDW : 21.5

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46 HbE - S Disease Patients details RBC Indices HPLC Hb Variants
Interpretation Advise RBC : 4.31 HbF : 7.5 HbE - S Disease. Family studies. Mast Abdiel HB : 10.8 HbA2 : 33.9 (A2+E) Child / 3.6 years HCT : 33.7 HbS : 54.3 MCV : 78.3 HbA : 4.3 MCH : 25.0 MCHC : 31.9 RDW : 15.9

47 HbS - C Disease Patients details RBC Indices HPLC Hb Variants
Interpretation Advise RBC : 3.61 HbF : 1.0 HbS - C Disease. Family studies Ganiath Yaya HB : 10.4 HbA2 : 3.9 F / 35 years HCT : 31.0 HbA : 2 MCV : 85.6 HbS : 46.9 MCH : 28.8 HbC : 46.2 MCHC : 33.6 RDW : 17.9

48 Mutation screening- IVS 1-5 Homozygous Mutant
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. – HPLC findings- ? Bet Thal Major or? Beta Thal Intermediate Mutation screening- IVS 1-5 Homozygous Mutant

49 Mutation screening- IVS 1-5 Homozygous Mutant
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. – Mutation screening- IVS 1-5 Homozygous Mutant

50 MOTHER

51 FATHER

52 1ST CHILD

53 2ND 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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