3 rd basic hematopathlogy course 2013 laboratory investigations in hemoglobinopathies dr sandeep...
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3rd Basic Hematopathlogy Course 2013
Laboratory Investigations in Hemoglobinopathies
Dr Sandeep WarghadeMetropolis Healthcare Ltd
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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
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• 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)
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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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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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Normal adult Haemoglobin
Haemoglobin
Globin chains
%
Hb A α2 β2 96-98
Hb A2 α2 δ2 1.7-3.6
Hb F α2 γ2 0.1-2.0
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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
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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
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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.
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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.
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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).
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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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Isoelectric FocusingStrength
• 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
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Capillary Isoelectric Focusing.
• Hybrid technique combining capillary electrophoresis sensitivity with automated sampling and data acquisition of HPLC.
• Not commonly used
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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.
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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.
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CE-HPLC
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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
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Comparing Chromatography to the Flow of a River...
Base
Water flow
Light leaf
Heavy stone
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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.
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24
Separation Process and ChromatogramO
utpu
t co
ncen
trat
ion
Time
Chromatogram
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Chromatogram
tR
t0
Inte
nsity
of
dete
ctor
sig
nal
Time
Peak tR : Retention time
h
A
t0 : Non-retention time
A : Peak areah : Peak height
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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
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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.
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CE-HPLC Interpretation
• Age• Transfusion history• Ethnic origin• Clinical history• CBC
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CE-HPLC InterpretationHemoglobin
Age Hgb A1% Hgb A2% Hgb F%
0 - 1 Month 17.7 - 54.0 0.0 - 1.3 46.0 - 81.0
2 Months 37.1 - 70.6 0.4 - 1.9 29.0 - 61.0
3 Months 41.0 - 84.0 1.0 - 3.0 15.0 - 56.0
4 Months 68.2 - 88.6 2.0 - 2.8 9.4 - 29.0
5 Months 74.9 - 95.6 2.1 - 3.1 2.3 - 22.0
6 - 8 Months 83.5 - 95.8 1.9 - 3.5 2.3 - 13.0
9 - 12 Months 91.7 - 96.7 2.0 - 3.3 1.3 - 5.0
13 - 24 Months 94.5 - 98.2 1.6 - 3.5 0.2 - 2.0
25 Months - Adult 94.3 - 98.5 1.5 - 3.7 0.0 - 2.0
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CE-HPLC InterpretationHbA2 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.
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HIGH Hb F
HOMOZYGOUS• Beta thalassaemia• HPFH• Delta-beta thalassaemia
(approx. 70 -90 %)
HETEROZYGOUS• HPFH• Delta-beta thalassaemia• Compound heterozygotes
(approx. 5 – 20 %)
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Acquired causes of High Hb F
• Aplastic anemia• MDS• PNH• JMML• Acute Leukemia• Marrow recovery
• Hypoxia• Anemia• Pregnancy• Thyrotoxicosis• Renal failure
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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
Beta Thalassaemia Trait
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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
Thalassaemia Syndrome
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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 :
Sickle cell disease
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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
HbS Trait
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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
HbD Trait
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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
HbS - D disease
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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
HbH disease
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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
HbE - S Disease
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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
HbS - C Disease
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Sid. No. – 100053882
HPLC findings- ? Bet Thal Major or? Beta Thal Intermediate
Mutation screening- IVS 1-5 Homozygous Mutant
Internal control
IVS 1-5- Mut
DN
A Lad
der
IVS 1-1
WT M
IVS 1-5
WT M
Cd 8/9
WT M
Cd 41/42
WT M
Hbe
WT M
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Sid. No. – 900834752
Mutation screening- IVS 1-5 Homozygous Mutant
Internal control
IVS 1-5- Mut
DN
A Lad
der
IVS 1-1
WT M
IVS 1-5
WT M
Cd 8/9
WT M
Cd 41/42
WT M
Hbe
WT M
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MOTHER
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FATHER
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1ST CHILD
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2ND CHILD
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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.
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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.
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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.
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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.
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IVS1-1 G-T
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D-Punjab (beta 121 Glu-Gln GAA – CAA)
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• Mutations: IVS1-1 G-T/ D-Punjab (beta 121 Glu-Gln GAA – CAA) – Compound Heterozygous
Hb-D punjab/beta-Thalassaemia
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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
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THANK YOU