Thalassemia and Hemoglobinopathies

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Thalassemia and Hemoglobinopathies

• Edna DSouza
• Product Specialist
• Clinical Diagnostic Division

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Hemoglobinopathies
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Types of defects
Thalassemia
Hb E Hb D Hb Q Hb J Hb C Hb Lepore Hb H
Sickle cell anemia
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Caste groups that have a higher carrier rate
Sindhis and Punjabis from Northern India,
Bhanushalis, Kutchis, Lohanas from Gujarat,
Mahars, Neobuddhists, Kolis and Agris from
Maharashtra, Gowdas and Lingayats from
Karnataka
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Scenario of Hb S carrier incidence in India
(Mohanty Colah et al, 2010)
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HEMOGLOBIN D
HEMOGLOBIN E
3 - 50
2
5 -35
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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

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Thalassemias

• Are a group of autosomal recessive disorders
  characterized by the complete absence or defect
  in the synthesis of the globin chains.
• ß- thalassemia presents itself in three forms

ß 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
ß thalassemia major
ß thalassemia intermedia
Genotypically they are similar to thal majors.
However phenotypically they are not dependent on
regular transfusions.
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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.

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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

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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.

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Percentage of hemoglobins
Hb A a2b2 Hb F a2g2 Hb A2 a2d2
Adult 94-96 0- 1 1.8-3.5
Thal Minor 90-92 1-5 4-8
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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.
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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 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
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Sample Preparation
9 STEPS to prepare hemolysate Time taken
gt40 MINUTES PER SAMPLE
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
Chances of manual error are high
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FEATURE HPLC ELECTROPHORESIS
Quantification Yes, Objective No, Subjective
ELECTROPHORESISV/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
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FEATURE HPLC ELECTROPHORESIS
Automation Complete Manual and Laborious

• Primary tube sampling

ELECTROPHORESISV/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
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FEATURE HPLC ELECTROPHORESIS
Data Storage Convenient Not convenient
LAN
ELECTROPHORESISV/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
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FEATURE HPLC ELECTROPHORESIS
Multianalyte More clinical info from each assay Requires both acid And alkaline ELP
ELECTROPHORESISV/S HPLC
Hb S/Hb D
Hb D
Hb S
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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 
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Electrophoresis with densitometry is NOT
RECOMMENDED

• CAP said
  in its 2003 survey
• Due to high CVs, densitometry from
  either
• alkaline electrophoresis or isoelectric
  focusing
• will not be reportable methods of HbA2
  quantitation

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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
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Hb S trait
Hb E trait
Hb D-Punjab trait
ß-thalassemia trait
Variant II Chromatogram Reports
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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
persons life but done the right way
THANK YOU