Thalassemia and
Hemoglobinopathies
Edna D’Souza
Product Specialist
Clinical Diagnostic Division
Hemoglobinopathies
Types of
defects
Sickle cell anemia
Thalassemia
Hb E
Hb D
Hb Q
Hb 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 D
HEMOGLOBIN E
3 - 50%
2%
5 -35 %
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- 9000-10000 (1-2 majors born every hour )
 Sickle Cell Disease-~5000
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
β thalassemia major
β thalassemia intermedia
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
Genotypically they are similar to thal majors.
However phenotypically they are not dependent on
regular transfusions.
clinical presentation
 b-thalassemia major: production of b-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.
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
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.
What test is required to
detect Thalassemia
•A complete Blood count test
•A Hemoglobin HPLC analysis to
estimate Hb A2 levels.
Percentage of hemoglobins
Hb A
a2 b 2
Hb F
a 2 g2
Hb A2
a2 d 2
Adult
~ 94-96 %
0- 1%
1.8-3.5%
Thal Minor
90-92%
1-5%
4-8%
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
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
None
Operator Variation
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
Sample Preparation
9 STEPS
to prepare hemolysate
Time taken
>40 MINUTES
PER SAMPLE
Chances of manual
error are high
On Bio-Rad VARIANT II
capped primary tubes are
directly loaded.
Time taken
1 STEP – 1 MINUTE PER SAMPLE
Complete automation
No manual error introduced
FEATURE
HPLC
ELECTROPHORESIS
Quantification
Yes, Objective
No, Subjective
ELECTROPHORESIS
V/S
HPLC
Accurate quantification of Hb A2
and Hb F
Reproducibility of results
Results required to be interpreted
by an experienced technician
Misinterpretation of bands is
possible resulting in incorrect
diagnosis
FEATURE
HPLC
ELECTROPHORESIS
Automation
Complete
Manual and Laborious

Primary tube sampling
ELECTROPHORESIS
V/S
HPLC

Automated bar-code reading
Time required to report results highly
reduced
Along with manual errors , the time
taken in reporting could be almost a day
FEATURE
HPLC
ELECTROPHORESIS
Data Storage
Convenient
Not convenient
LAN
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
V/S
HPLC
Electrophoresis strips information needs
to be manually fed into the report
For quantitation of bands additional
densitometer required
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
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
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 Variant
College of American Pathologists
Improved Hemoglobin
Analysis
by High-Performance Liquid
Chromatography

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.
β-thalassemia trait
Hb S trait
Hb E trait
Variant II Chromatogram Reports
Hb D-Punjab trait
New births of beta-thalassemia major can be prevented
urgent need to identify all carriers
screen for
screen
for
thalassemia
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