Thalassemia and Hemoglobinopathies

Edna D’SouzaProduct Specialist

Clinical Diagnostic Division

Hemoglobinopathies

Types of defects Thalassemia

Sickle cell anemia Hb EHb DHb QHb J Hb C

Hb Lepore Hb H

Caste groups that have a higher carrier rate

Sindhis and Punjabis from Northern India,

Bhanushali’s, Kutchis, Lohana’s from Gujarat,

Mahar’s, Neobuddhist’s, Koli’s and Agri’s from Maharashtra,

Gowda’s and Lingayat’s from Karnataka

Scenario of Hb S carrier incidence in India

(Mohanty & Colah et al, 2010)

HEMOGLOBIN E

3 - 50%

5 -35 %

HEMOGLOBIN D

2 %

India: Average Incidence of thalassemia carriers -3.9% (varying from 1-

17%) 1 in 25 Carriers in India!!!! 30-40 million carriers.

Affected births/yr Thalassemia major- 9000-10000 (1-2 majors born every hour ) Sickle Cell Disease-~5000

Thalassemia –National Problem

ThalassemiasAre a group of autosomal recessive disorders characterized by the

complete absence or defect in the synthesis of the globin chains.

β- thalassemia presents itself in three forms:

β thalassemia trait asymptomatic condition wherein there is mild microcytic , hypochromic anemia

β thalassemia major

β thalassemia intermedia

The patient suffers from the disorder. Is unable to synthesize hemoglobin and requires blood transfusion to survive beginning as early as 6 months of ageGenotypically they are similar to thal majors. However phenotypically they are not dependent on regular transfusions.

thalassemia major: production of -globin chains is severely impaired

Patients with thalassemia major need blood transfusions every 3-4 weeks to maintain their hemoglobin levels

Due to transfusions they are at a risk of: Blood transfusion related infections

like hepatitis C, hepatitis B , HIV

Iron overload with a damage to all vital organs like heart, lung , liver , kidney etc.

The survival of individuals who have been well transfused and treated with appropriate chelation extends beyond 30 years.

clinical presentation

Inheritance of Hemoglobinopathies

In a marriage between a carrier and

a normal individual: 50 % chance: children

- CARRIERS

50 % chance: children - NORMAL

In a marriage between 2 carriers: 25 % chance: children

–

NORMAL50 % chance: children

–

CARRIERS 25 % chance: children

–

HOMOZYGOTES

How to avoid baby with Thalassemia major Follow only 2 simple steps Step 1: Get your partner and yourself tested for

thalassemia before marriage. Step 2: If both your partner and you are

thalassemia minors, consult your doctor for prenatal diagnostic test.

•A complete Blood count test

•A Hemoglobin HPLC analysis to estimate Hb A2 levels.

What test is required to detect Thalassemia

Hb A

Hb F

Hb A2

Adult~ 94-96

%0- 1%

1.8-3.5%

Thal Minor

90-92% 1-5% 4-8%

Percentage of hemoglobins

Variant II hemoglobin testing system

Fully automated, High-throughput

hemoglobin analyzer Providing an integrated

method for sample preparation, separation and determination of the relative percent of specific hemoglobins in whole blood.

Why HPLC ???FEATURE HPLC ElectrophoresisQuantification Yes, Objective No, Subjective

Automation Yes Manual and Laborious

Data Storage Convenient Not convenient

Optimization Calibrators and QC Sera Not optimised

Multianalyte More clinical info from each assay

Requires both acid and alkaline ELP

Operator toOperator Variation

None Yes

Time Taken 6.5 min/sample Couple of hours to entire day.

Sample capacity

100, continuous sample loading facility

Fixed depending on the wells

Number of steps

One Many

Reporting format

Printable Chromatogram with complete information

Electrophoresis strip only shows band separations

9 STEPS to prepare hemolysate

Time taken >40 MINUTES

PER SAMPLE

On Bio-Rad VARIANT II capped primary tubes are directly loaded.

Time taken1 STEP – 1 MINUTE -

PER SAMPLE

Sample Preparation

Chances of manual error are

high

Complete automation No manual error introduced

Results required to be interpreted by an experienced technician

Misinterpretation of bands is possible resulting in incorrect diagnosis

FEATURE HPLC ELECTROPHORESIS

Quantification Yes, Objective No, Subjective

ELECTROPHORESISV/S

HPLC

Accurate quantification of Hb A2 and Hb F

Reproducibility of results

Along with manual errors , the time taken in reporting could be almost a day

FEATURE HPLC ELECTROPHORESIS

Automation Complete Manual and Laborious

Primary tube sampling

Automated bar-code reading

ELECTROPHORESISV/S

HPLC

Time required to report results highly reduced

Electrophoresis strips information needs to be manually fed into the report

For quantitation of bands additional densitometer required

FEATURE HPLC ELECTROPHORESIS

Data Storage Convenient Not convenient

LAN

LAN

Lab Network

ELECTROPHORESISV/S

HPLC

All the information of the sample chromatogram is directly transferred onto the report

Complete information of all the percentages of the various hemoglobins on the report

FEATURE HPLC ELECTROPHORESIS

Multianalyte More clinical info from each

assay

Requires both acid

And alkaline ELP

Hb S/Hb D

Hb D

Hb S

ELECTROPHORESISV/S

HPLC

Technology HPLC ELECTROPHORESIS

CV 4.3 33.6

Would you want to use a technique with a higher imprecision????

Can the diagnosis by electrophoresis

be 100 % accurate if it has a CV of 33%

CAP reference for  HPLC comparison with electrophoresis with densitometry Lafferty.J. College of American Pathologists Survey 1999 

Electrophoresis with densitometry is ‘NOT RECOMMENDED’

CAP said in its 2003 survey

“Due to high CV’s, densitometry from either

alkaline electrophoresis or isoelectric focusing

will not be reportable methods of HbA2 quantitation…”

Evaluation of VariantCollege of American Pathologists

Analyzed 1,370 consecutive samples over a 1-year period using an automated Bio-Rad HPLC system and compared the results with standard methods

HPLC analysis detected 3 abnormal Hb patterns without corresponding gel abnormalities

HPLC is more sensitive than the standard methods for the detection of Hb variants and can be considered for routine use by hospital or clinical reference laboratories.

Improved Hemoglobin Analysis

by High-Performance Liquid Chromatography

β-thalassemia trait

Hb S trait

Hb E trait Hb D-Punjab trait

Variant II Chromatogram Reports

screen forthalassemia

New births of beta-thalassemia major can be prevented

do it the right way

The screening test needs to done only once in a person’s life

but done the right way THANK YOU

urgent need to identify all carriers

screen forthalassemia