1. Thalassemia and Hemoglobinopathies
Edna D’Souza
Product Specialist
Clinical Diagnostic Division

2. Hemoglobinopathies

4. Types of defects Thalassemia Hb E Hb D Sickle cell anemia Hb Q Hb J
Hb C
Hb Lepore
Hb H
Sickle cell anemia

5. 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
High Incidence of Thal & Sickle , HBE towards eastern and north eastern belts ,Alpha thalassaemia – Eastern and Western
Mapped by InDian Council of Medical Reseafch
Madhuri Competition persepctive : Sebia – using HBE / Barts pick uo –pushing the ystem

6. Scenario of Hb S carrier incidence in India
(Mohanty & Colah et al, 2010)

7. HEMOGLOBIN D
HEMOGLOBIN E
3 - 50%
2 %
5 -35 %

8. Thalassemia –National Problem
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 (1-2 majors born every hour )
Sickle Cell Disease-~5000

9. β thalassemia intermedia
Thalassemias
Are 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
The patient suffers from the disorder.
Is unable to synthesize hemoglobin and requires blood transfusion to survive beginning as early as 6 months of age
β thalassemia major
β thalassemia intermedia
Genotypically they are similar to thal majors.
However phenotypically they are not dependent on regular transfusions.

10. clinical presentation
b-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.

11. 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 –
NORMAL
50 % chance: children –
CARRIERS
HOMOZYGOTES

12. 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.
What test is required to detect Thalassemia
A complete Blood count test
A Hemoglobin HPLC analysis to estimate Hb A2 levels.

13. Percentage of hemoglobins
Hb A
a2b2
Hb F
a2g2
Hb A2
a2d2
Adult
~ %
0- 1% %
Thal Minor
90-92%
1-5%
4-8%

14. 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.

15. Why HPLC ??? FEATURE HPLC Electrophoresis Quantification
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 to
Operator Variation
None
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

16. Chances of manual error are high
Sample Preparation
9 STEPS
to prepare hemolysate
Time taken
>40 MINUTES
PER SAMPLE
On Bio-Rad VARIANT II capped primary tubes are directly loaded.
Time taken
1 STEP – 1 MINUTE -
PER SAMPLE
Chances of manual error are high
Complete automation
No manual error introduced

17. ELECTROPHORESIS V/S HPLC
FEATURE
HPLC
ELECTROPHORESIS
Quantification
Yes, Objective
No, Subjective
ELECTROPHORESIS V/S
HPLC
Results required to be interpreted by an experienced technician
Misinterpretation of bands is possible resulting in incorrect diagnosis
Accurate quantification of Hb A2 and Hb F
Reproducibility of results

18. ELECTROPHORESIS V/S HPLC
FEATURE
HPLC
ELECTROPHORESIS
Automation
Complete
Manual and Laborious
Primary tube sampling
ELECTROPHORESIS V/S
HPLC
Automated bar-code reading
Along with manual errors , the time taken in reporting could be almost a day
Time required to report results highly reduced

19. ELECTROPHORESIS V/S HPLC
FEATURE
HPLC
ELECTROPHORESIS
Data Storage
Convenient
Not convenient
LAN
ELECTROPHORESIS V/S
HPLC
LAN
Lab Network
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
Electrophoresis strips information needs to be manually fed into the report
For quantitation of bands additional densitometer required

20. ELECTROPHORESIS V/S HPLC
FEATURE
HPLC
ELECTROPHORESIS
Multianalyte
More clinical info from each
assay
Requires both acid
And alkaline ELP
ELECTROPHORESIS V/S
HPLC
Hb S/Hb D
Hb D
Hb S

21. Would you want to use a technique with a higher imprecision????
Technology
HPLC
ELECTROPHORESIS CV
4.3
33.6
Can the diagnosis by electrophoresis
be 100 % accurate if it has a CV of 33%
Would you want to use a technique with a higher imprecision????
CAP reference for  HPLC comparison with electrophoresis with densitometry
Lafferty.J.
College of American Pathologists Survey 1999 

22. 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…”

23. Evaluation of Variant College 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

26. Variant II Chromatogram Reports
β-thalassemia trait
Hb S trait
Hb E trait
Hb D-Punjab trait
Variant II Chromatogram Reports

27. urgent need to identify all carriers
New births of beta-thalassemia major can be prevented
urgent need to identify all carriers
screen for
thalassemia
screen for
thalassemia
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