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

Hemoglobinopathies

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

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

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

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.

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.

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

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 a2b2 Hb F a2g2 Hb A2 a2d2 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 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

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

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

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

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

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

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 

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 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

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

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