Rama Univ. J. Med Sci 2015;1(1):6-9 Nitu et al (2015)

Original Research

HPLC as a classical tool for screening of β-Thalassemia andHemoglobinopathies in Kanpur

Nigam.N, Mishra.A, Nair.N, Nigam.S, Rao.Y, Singh,S. KAbstractBackground: Hemoglobinopathies and beta- thalassemia is one of the most common autosomal disordersworldwide different molecular mechanisms, most of which are base substitution or small deletions or insertionsof one or two nucleotides in the globin genes. It has been found that hemoglobinopathies and β-thalassemiamutations are relatively populations specific; each ethnic group has its own set of common mutations. Aim:High Performance liquid chromatography (HPLC) forms an important tool for accurate and speedy diagnosis ofvarious haemoglobin disorders. About 50 cases have been studied for various haemoglobin variants fromKanpur and adjoining area. Materials and Methods: The study was performed Agilent 1220 Infinity LC(Agilent Technologies) a High Performance Liquid Chromatography (HPLC) using beta thalassemia program.Results: Abnormal haemoglobin fractions on HPLC were seen in 50 cases. Of this beta-thalassemia was thepredominant. There were 16 (32%) cases of beta-thalassemia major and 34 (68%) cases of beta-thalassemiacarriers possessing HbA1c(0.16), HbF(0.72),HbA0(0.5), HbE(0.44), HbD(0.36), HbS(0.5), HbA2(0.46), andHbC(0.42). Automated HPLC and beta thalassemia program is an appropriate approach for the screening andpresumptive identification of patients as well as carrier of beta-thalassemia prior to DNA studies for definitivediagnosis.

Keywords: HPLC, Thalassemia, MCV, Beta Thalassemia

Introduction

India is an ethnically diverse country with anapproximate population of 1.2 billion. Theinherited disorders of hemoglobin are the single-gene disorders commonly encountered in humans.They fall into three wide but overlapping groups:structural variants; thalassemias characterized byreduced rate of synthesis of one or more globinchains; and conditions in which fetal hemoglobinsynthesis persists beyond the neonatal period,collectively known as hereditary persistence offetal haemoglobin. Of these, thalassemiasyndromes particularly beta thalassemia major andcertain alpha thalassemia are serious and a majorcause of morbidity [1].

Thalassemia is a single gene hereditaryhaemoglobin disorder in human. It has beenreported that nowadays approximately 1 out of 14people are carriers for thalassemia [2]. WHOfigures state that about 370,000 severely affectedhomozygotes or compound heterozygotes ofthalassemia are born every year [3]. The frequencyof β-thalassemia in India ranges from 3.5 to 15%in general population [4]. There are certainmutations responsible for >90% of thalassemiacases and about 10% mutations which are unknownto us and or considered as the rare ones [2]. Indeveloping countries like India,hemoglobinopathies increase at an alarming rate

due to lack of proper health care and knowledge.About 10% of total world thalassemia patientsbelong to Indian sub-continent out of which 3-4%are carriers. In India 32,400 infants are born withhemoglobinopathies [5]. The carrier rate for β-thalassemia gene varies from 1 to 3% in SouthernIndia and 3 to 15% in Northern India [6,7,8]. Thefrequency of beta-thalassemia trait ( TT) hasβvariously been reported from <1% to 17% and anaverage of 3.3% [9]. In India where there is highmortality from infections and malnutrition in thefirst year of life, many of the hemoglobinopathiesgo unrecognized. Thalassemia major can cause lifethreatening situation and chronic ill health. Theypose economical and psychological burden on theaffected individual and his/her family, and thesociety as a whole. Hence, the population needs tobe screened for haemoglobin.

2a. Material and MethodsThis was a prospective study carried by theDepartment of Paediatrics, G.S.V.M MedicalCollege Kanpur and Central Research Lab, RamaMedical College & Research Center, RamaUniversity Mandhana, Kanpur for 6 monthsperiod. A total of 50 cases were screened forpresence of thalassemia or any structural variant.These included all cases of microcytichypochromic anaemia (MCV < 80 fl, MCH <27pg, and RBC count > 5million/cubmm) notresponding to conventional treatment, clinically

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Correspondence Address : Dr. Nitu Nigam, Rama Medical College, Kanpur

ISSN NO:2395-0757 HPLC in β-Thalassemia and Hemoglobinopathies

suspected cases of hemoglobinopathy, antenatal,and other cases coming to the department forthalassemia screening. A 5 ml. intravenous bloodsample was collected in EDTA anticoagulant

Haemoglobin , red blood cell counts and red cellindices were measured on an automated bloodcounter. Hematological parameters : Hb, PCV,MCV, MCH, MCHC, RBC count and WBC countwere measured using. Peripheral smear wasevaluated for features of red cell morphology [10].Classical red cell indices for β-thalassaemia traitare indicated by a MCV <75 fl and MCH <27 pg[11]. We used the Agilent 1220 Infinity LC (AgilentTechnologies) a High Performance LiquidChromatography (HPLC) under the experimentalconditions specified by the manufacturer.

2b. Principle and sample collection : Agilent1220 Infinity LC (Agilent Technologies ) a HighPerformance Liquid Chromatography (HPLC) usesa double beam photometer for detection in therange of 190–600 nm. This cationic exchangecolumn chromatography enables qualitativedeterminations of HbA2, Hb F and abnormalhaemoglobins. Here we used β-thalassemia kit byGordion Diagnostik Ltd., Turkey. The kitcontained Buffer A, Buffer B, Hemolysis Reagent,and one analytical column for β-thalassemia. Only10 µl hemolysis mixture prepared by diluting 5 µlof whole blood with 1 ml hemolysis reagent wasinjected into HPLC system. A flow rate of 1.5ml/min, with an analytical run time of 6.5 min asrecommended in kit protocol, was set. Achromatrogram of a case with beta-thalassaemiatrait detected on the Agilent 1220 Infinity LC isshown in figure 1.2c. Interpretation of ReportsReports and chromatograms generated werestudied and interpreted by observing HbA2 andHbF concentration for beta thalassemia andretention time and area percentage of other peaksand windows for structural variants. Eachchromatogram shows peaks of Hb A0, A2, and HbF along with C window, D window, S window, and

two minor peaks, P2 and P3. Several hemoglobinvariants elute same window; they wereprovisionally diagnosed by retention time and areapercentage keeping in mind the ethnicity of thepatients.

Figure 1:

Mi nut es0 1 2 3 4 5 6

650

700

750

650

700

750

VWD: Signal A, 415nmRetentionTime

Table2: Distribution of Hemoglobin variants.

Result:About fifty beta-thalassemia samples were studiedin which sixteen samples were thalassemia majorand thirty four samples were thalassemic carriers inwhich HbA1c(0.16%), HbF (0.72%), HbAo(0.5%)

HbA (0.46%), and HbC (0.42%) were predominant.2

Presumptive identification of haemoglobin variantswas made primarily by retension time (RT)windows and area percent; however geographicalfactors, ethnicity and clinical presentation weretaken into consideration. Distribution ofhaemoglobin variants identified is shown inas expected and prenatal study and diagnosis wasdone to find out carrier status.

Discussion:India is an ethnically diverse country with markedregional variation. This diversity is reflected in thepresence of different hemoglobin variants indifferent ethnic groups. Due to migration, there isconstant mixing of peoples from different regions.Many of these abnormal variants are of littleclinical significance in heterozygous state, butwhen combined with other variants they may giverise to severe disease. The hemoglobinopathies andthalassemia are the most commonest inheritedsingle gene disorder in India. Therefore there isalways a need for a screening method which candetect maximum variants. HPLC has the advantage

Hemoglobin Variants Number(%)HbA1cHbFHbA0HbEHbA2HbDHbSHbC

16(0.16)72(0.72)50(0.5)44(0.44)46(0.46)36(0.36)50(0.5)42(0.42)

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HbE(0.44%), HbD(0.46%), HbS(0.5%), HbD(0.3%)

Rama Univ. J. Med Sci 2015;1(1):6-9- Nitu et al (2015)

of quantifying HbF and HbA2 along with detectingother variants in a single screening test[12]. Cationexchange HPLC is emerging as one of the bestmethods for screening and detection of varioushemoglobinopathies with rapid and reproducibleand precise results [13]. Beta thalassemia traitformed the largest sub group of abnormalhemoglobin (8.9%). The characteristichematological findings in a typical case of betathalassemia trait include microcytosis with raisedRBC counts.

Hemoglobin rate is quite reduced than normal. Themutations common in an Indian setting includeIVS1-5(G-C), 619 bp deletion, IVS 1-1(GT),CD8/9(+G), CD41/42(-CTTT), CD15 (G-A),CD30 (G-C) [14]. HbA2 levels >7% are usually seenwith being the major concern in this study,quantification of HbA2 and HbF along with othervariant levels by HPLC was of prime importance inour laboratory where facilities for genetic studiesare available. In our study HbF frequency washigher (0.72) in the cases of beta-thalassemia.Presence of HbF variant may be useful fordetection of homozygous β-thalassemia variants.HbE and HbA2 traits were found in 44 (0.44) and46 (0.46) cases respectively.Detection of other variants becomes important dueto complex interactions in cases with doubleheterozygous and homozygous states, which maylead to severe hematological abnormalities.Findings must be supplemented by hemogramfindings, family/ sibling studies, hemoglobinelectrophoresis, other confirmatory techniques andmolecular studies based on HPLC findings and ona case-to-case basis.

Conclusion:The hallmark of classical beta-thalassemia is thepresence of an elevation of HbA2 and HbF, wherethe recommended method of measurement is doneby automated HPLC. HPLC forms a rapid, accurateand reproducible tool for early detection andmanagement of hemoglobinopathies andthalassemia. This is especially important in view ofhigh incidence of beta-thalassemia. Early detectionof traits will prevent occurrence of thlassemiamajor in offspring. Detection of other variantsbecome important due to complex interaction incases with heterozygous and homozygous stateswhich may lead to severe hematologicalabnormalities. Due to high prevalence of Hbdisorders, premarital screening routinely done forprevention of high risk marriages. The presentstudy conducted using HPLC reflects themagnitude of hemoglobinopathies and thalassemia

in a hospital based small population which may bein fact the tip of an iceberg, but this type of studycan definitely help to increase awareness amongpatients suffering from these disorders.

Acknowlegment: The authors are grateful to Dr.Yashwant Rao, Department of Pediatric, G.SV.MMedical College Kanpur and to Dr. S.K.SinghDepartment of Pediatrics, Ursula HorsmanMemorial Hospital, Kanpur for providing bloodsamples of beta thalassemia patients and theirfamily.

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