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

3rd Basic Hematopathlogy Course 2013

Laboratory Investigations in Hemoglobinopathies

Dr Sandeep WarghadeMetropolis Healthcare Ltd

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

• 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)

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.

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).

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

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

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

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.


• 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.


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).

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

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

Capillary Isoelectric Focusing.

• Hybrid technique combining capillary electrophoresis sensitivity with automated sampling and data acquisition of HPLC.

• Not commonly used

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.

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.

CE-HPLC

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

Comparing Chromatography to the Flow of a River...

Base

Water flow

Light leaf

Heavy stone

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

Separation Process and ChromatogramO

utpu

t co

ncen

trat

ion

Time

Chromatogram

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

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

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.

CE-HPLC Interpretation

• Age• Transfusion history• Ethnic origin• Clinical history• CBC

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

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.

HIGH Hb F

HOMOZYGOUS• Beta thalassaemia• HPFH• Delta-beta thalassaemia

(approx. 70 -90 %)

HETEROZYGOUS• HPFH• Delta-beta thalassaemia• Compound heterozygotes

(approx. 5 – 20 %)

Acquired causes of High Hb F

• Aplastic anemia• MDS• PNH• JMML• Acute Leukemia• Marrow recovery

• Hypoxia• Anemia• Pregnancy• Thyrotoxicosis• Renal failure

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


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


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


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


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


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


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


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


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

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

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

MOTHER

FATHER

1ST CHILD

2ND CHILD

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.

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.

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.

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.

IVS1-1 G-T

D-Punjab (beta 121 Glu-Gln GAA – CAA)

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

Hb-D punjab/beta-Thalassaemia

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

THANK YOU

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