Diagnosing Beta Thalassemia trait in a developing country

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Original research article/Praca oryginalna

Diagnosing Beta Thalassemia trait in a developing country

Shan-e- Rauf *, Ghassan Umair Shamshad, Fareeha Mushtaq, Saleem Ahmed Khan, Nadir Ali

ArmedForcesInstituteofPathology,Rawalpindi,Pakistan

Introduction

BetaThalassemia(BT)isoneofthemostprevalentinherited hemoglobindisorders intheworld[1], witha carrierrate of5% among thePakistani population[2]. It isestimated that with a 5% carrier rate, over5000 infants with Beta

ThalassemiaMajor(BTM)areborneveryyearinPakistan[3–5]. Screening forBeta Thalassemia trait(BTT) andidentiﬁcation of itscompoundheterozygoteswithvarianthemoglobinsis essentialfordiagnosisandgeneticcounselingthatholdsthe keyforpreventionandcontrolofBTM.

BTTissuspectedonﬁndinghypochromicandmicrocytic red cell indices with near normal hemoglobin levels and

*Correspondingauthorat:HouseNo.756-A,Street83,SectorI-8/4,Islamabad,Pakistan.Tel.:+923335631929;fax:+92515537821.

E-mailaddress:shan.e.rauf673@gmail.com(S.Rauf).

article info

Articlehistory:

Received:08.03.2016 Accepted:30.01.2017 Availableonline:09.02.2017

Keywords:

BetaThalassemiatrait

HPLC

CelluloseAcetateelectrophoresis

PolymeraseChainReaction

HbA2

abstract

Background:Beta Thalassemia trait (BTT) is diagnosed by detecting hemoglobin A2 (HbA2) >3.8% on either High Performance Liquid Chromatography (HPLC) or cellulose acetateelectrophoresis(CAE).HPLCisanaccurateandreproduciblebutcostlyalternative tomoreconventionalCAEwhichislaborintensivebuteasytointerpretandinexpensive.

Objective: TodeterminethesensitivityofCAE andHPLCkeepingPCR asgoldstandard for the diagnosis of BTT.Study Design: Cross sectional. Place and Duration of Study:

ArmedForcesInstituteofPathologyRawalpindi.May2014toJanuary2015.Patientand Methods:FivemlEDTAanti-coagulatedblood wascollectedfrom100PCRproven cases ofBTT.HbA2levels weremeasuredbyrunningsamplesdirectlyon HPLC.Butfor CAE, ﬁrstahemolysatewaspreparedwhichwasthenappliedtocelluloseacetatemembrane atanalkalinepH(7.9).AfterelutionofHbA2bandinTrisEDTAboratebuffer(pHof8.9), HbA2 concentration was calculated by measuring its absorbance in a photometer at awavelengthof416nm.Results: Meanageofthepatientswas28.88.1year.Themost commonmutationwasFr8–9(35%)followedbyIVS1-5(25%)mutation.MeanHbA2levels by CAE and HPLC were 4.970.42 and 5.540.59 respectively. All the patients had HbA2>4%onbothCAEandHPLC.Noneofourpatientshadfalsenegativeresulteither onCAEorHPLC.Conclusion:CAEhascomparablesensitivitywithHPLCfordetectionof BetaThalassemiaTrait.

ContentslistsavailableatScienceDirect

Acta Haematologica Polonica

journal homepage:www.elsevier.com/locate/achaem

http://dx.doi.org/10.1016/j.achaem.2017.01.001

zo.o.Allrightsreserved.

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slightly raised red blood cell count on routine complete bloodcount[1].LaboratoryconﬁrmationoftheBTTdepends primarily on detecting elevated levels of hemoglobin A2

(HbA2>3.8%) in the patient's blood sample [6]. The HbA2

levelscanbequantiﬁedbymanymethodsbutthetwomost commonlyusedareCellulose AcetateElectrophoresis(CAE) followed by elution and the automated method of High Performance LiquidChromatography (HPLC) [7]. CAE isthe conventionalandcommonlyusedtechniquefordiagnosisof BTT. It is reproducible, inexpensive and relatively easy to interpretasshowninFig.1.Additionallythisprocedurecan be performed in smaller and resource constrained laboratories.Theonlydrawbackisthatitislaborintensive[8].

ThecationexchangeHPLCisarapid,accurate,reproduci- bleandlesslaborintensivealternativemethodfordetection ofmanyhemoglobinopathiesincludingBTT[9]. HPLCoffers the distinct advantage over conventional CAE as it can identify and quantify HbA2, fetal hemoglobin (HbF) and other hemoglobin variants moreaccurately. It isalso very usefulforpediatricgroupofpatients,asonly5mlofbloodis sufﬁcient for analysis. Its utility is more in diagnostic centerswhere thereis increased workload.Howevermajor drawbacks include cost considerations, heavy processing equipmentandexpertisetointerpretresults.Additionallyit hasbeenproventooverestimateHbA2percentageespecially in the presence of sickle hemoglobin (HbS) [10]; which is avariantofBetaglobinfoundinasigniﬁcantpercentagein differentethnicitiesofPakistanipopulation[11].

Polymerasechainreaction(PCR)isahighlysensitiveand speciﬁc method for diagnosis of BTT which can clearly identifythetypeofmutationaffectingBetaglobingene[12].

However, it requires expensive molecular equipment and highdegreeof technicalskill duetowhich itisnotcarried outroutinely during theworkupofBT patients.Itisthere- fore reserved for cases which present either withatypical redcellindicesand/orarenotdetectedonCAEorHPLC[13,14].

Previously nolocal studyhasdonecomparisonbetween CAEand HPLCbasedonHbA2levels inPCRconﬁrmed BTT patients. The aim of this study was to compare the sensitivity of CAE against HPLC to establish that CAE can still be used as an effective diagnostic tool not only in District Hospital laboratories (Level B) but also in Central/

Regional hospital laboratories of underresourced countries likePakistan[15].Pakistandespitehavingahighprevalence of BT has inadequate resources to cater for BT screening and diagnosis at mass level. Additionally fragmented and substandardtransfusionsysteminPakistanisinepttocater for the transfusion needs of BTM patients in the country [16]. Soinexpensive but effectivediagnostic modalitieslike CAEarerequiredtoscreenthemassesforBTTinPakistan.

Objective

To determinethe sensitivityof CAEandHPLCkeepingPCR asgoldstandardforthediagnosisofBTT.

Material and methods

Itwasacross-sectionalstudycarriedoutfrom May2014to January 2015 in the hematology department of Armed Forces Institute of Pathology (AFIP) Rawalpindi. A total of 100patientswereincludedinthisstudy.

Sampleselection

Allthosecases reportingforextendedfamily screeningand for prenatal diagnosis and found to bepositive for one of theBetaglobingenemutationsonPCRwereincludedinthis study. For controlgroup, PCR negative cases for BT mutations weretested. While all cases having silent mutations

Fig.1–ConventionalCelluloseAcetateElectrophoresistankwithbufferandelectricalsupplyandcelluloseacetatestrip showingnormalcontrol(NC),positivecontrol(PC)andPositiveforHbF(PF).ArrowsindicatingRaisedHbA2levelsonstrip.PC andPFonthestripshowingBTTandBTMrespectively

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like CAP+1 and history of recent blood transfusion were excluded.

After the approval of the study by institute's ethical committeeformedicalresearch,alltheproceduresfollowed were in accordance with the ethical standards of the responsiblecommitteeonhumanexperimentationandwith theHelsinkiDeclarationof1975,asrevisedin2000.Afteran informedconsent and recording of demographicdata,5ml of blood was collected in EDTA anticoagulant from all patients.Completebloodcounts(CBC)wereperformedusing SysmexKX21automatedhematologyanalyzer.

Molecularcharacterization

PCR for BT gene mutations was performed by multiplex amplificationrefractory mutationsystem(ARMS)in3sepa- ratereactionmixturesfor IVSI-5(G-C),Fr8–9(+G), Fr41–42 (-TTCT),IVSI-1(G-T),Del619bp,Cd5(-CT),Fr16(-C),IVSI-1 (G-T),Cd30(G-C),Cd30(G-A),IVSII-1(G-A),Cd15(G-A)and Cap+1(A-C).Thesegenotypescoveralmost98%ofBTgene mutations/deletionsinPakistanipopulation.DNAextraction wasdonebyusingPUREGENEgenomicDNApurificationkit (gentrasystems,USA).DNAamplificationwascarriedoutin a 20ml reaction mixture containing 5pM of the common primerand5pMeachofthetwoforwardprimers,0.5 units of Taq polymerase (Fermentas Life Sciences, Lithuania), mastermixturecontaining30mMofeachdNTP,10mMTris HCl (pH 8.3), 50mM KCl, 1.5mM MgCl2, 100mg/ml gelatin and0.1–0.3mgofgenomicDNA.

HbA2levelsmeasurement

SampleswererunonbothHPLCandCAEtomeasureHbA2

within24–48hof thecollection.Wholeblood sampleswere run directly on HPLC which automatically quantiﬁes the HbA2levelsbasedonchromatographicseparationofhemo- globin proteins following its passage through a column of polaraluminumcompoundsunder highpressure.However to measure HbA2 levels by CAE, ﬁrst a hemolysate was preparedwhichwasthen appliedoncellulose acetatemem- braneinanelectrophoretictankhavinganalkalinepHof7.9.

Anelectricalcurrent wasappliedwitha voltageof 200Vfor 30min.AfterwardsHbA2bandwaselutedinTrisEDTAborate buffer at pH 8.9 for 30min. Finally percentage of HbA2 was calculated in this elute by measuring its absorbance in aphotometerat awavelength of416nm.HbA2levels >3.8%

byCAEand/orHPLCwereconsideredasdiagnosticofBTT[17].

Data wascollected and analyzedonSPSS17. Mean and standarddeviationwerecalculatedforquantitativevariables like age, Total Red Blood Cells (TRBC), Hemoglobin (Hb), MeanCorpuscularVolume(MCV),MeanCorpuscularHemo- globin(MCH),Mean CorpuscularHemoglobinConcentration (MCHC), Red Cell Distribution Width (RDW) and HbA2. Frequenciesandpercentageswerecalculatedforqualitative variables like gender. Sensitivity of CAE and HPLC was calculatedbyusingtheformula“Sensitivity=TP/(TP+FN) 100⁰⁰. All PCR positive patients having HbA2>3.8% on CAE and/or HPLC were considered true positive, and those detectedpositiveonPCRbutnegative(HbA2<3.8%)onCAE and/orHPLCweretakenasfalsenegative.

Results

Atotalof 100patientswereincludedinthisstudy.Majority of thepatientswerebetween12and 44yearsof age.Mean age of the patients in this study was 28.88.1 years.

Distributionof patientsbygendershowed62patients(62%) weremaleand 38patients(38%)werefemale.Out ofthese 100 patients, 35(35%) patients had Fr 8–9 mutation which was the commonest mutation in these patients while 25 (25%) patientshadIVS1-5mutation.Restsofthe mutations withtheirfrequencyareshowninTableI.

AllofourpatientshadHbA2>4%onbothCAEandHPLC.

And none of our patients had false negative result. Mean HbA2levelsbyCAEwere4.970.42whileMeanHbA2levels asmeasuredonHPLCwere5.540.59.Allofthepatientsin this studyhadMCH< 27pg.Mean MCHvalueonCBCwas 18.91.4pg. Mean valuesof differentvariables are shown inTableII.

As all these100BTT cases weredetected bybothHPLC and CAE thus there was not even a single false negative case on either HPLC or CAE. The sensitivity of both the methods for diagnosis of BTT was calculatedby using the followingformula:

Sensitivityofhemoglobinelectrophoresis=HPLC

¼TP=ðTPþFNÞ100¼100=ð100þ0Þ100¼100%

Discussion

Thalassemia is the commonest inherited disorder world- wideandinourcountryBTisthemostcommonsinglegene disorder [3].PreventionofBTMbirthsisofvitalimportance as the average income in Pakistan is $1115 per year [18]

whereastheaverageannualcostformanagementofaBTM patient is> 10times theincome.It isthereforeimportant to diagnoseBT accurately and timely.Thelaboratory diagnosis of BT can be done by a step-wise approach starting with a detailed clinical and family history; CBC including redcellindices,ReticulocytecountandRedbloodcell(RBC) morphology; protein based analytic methods like alkaline and acid hemoglobin electrophoresis, HPLC and reserving PCR for difﬁcult cases and genetic counseling [13]. An increaseinthe HbA2levels intherange of4–6% isspeciﬁc for BTT after the third month of life. And high HbA2

concentrationsarearesultofBTinalmostallinstances[19].

TableI–Betaglobingenemutationanalysis Typeof

mutation

Totalno.of cases

Frequency (percentage)

Fr8–9mutation 100 35(35%)

IVS1-5mutation 100 25(25%)

Fr41–42(12%) 100 12(12%)

Del619(10%) 100 10(10%)

Cd15(6%) 100 6(6%)

IVS1-1(4%) 100 4(4%)

Cd5(3%) 100 3(3%)

Fr16(3%) 100 3(3%)

Cd30(2%) 100 2(2%)

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Keeping in mind economicconstraints of the country and high prevalence of the disease, cost effective, reliable and appropriatediagnostic tools holdkey toeradicateor tackle thisdisease.

HPLC has emerged as an alternative method overcon- ventional CAE for diagnosis of BTT in most of the health caresetupsand largediagnostic centersworldwideaswell as in this country. It can also accurately identify and quantitate abnormal hemoglobins in mass screening pro- grams.However it isexpensiveandcosts PakistaniRupees 1000 ($10) per test compared to conventional CAE which costsonlyRupees100–150($1–1.5)pertest.

We conducted this study to evaluate and compare sensitivityof CAEagainstHPLC inPCRpositive patientsto ascertainitsutilityasadiagnostictoolinoursetup.Outof our100PCRpositiveBTTcases,allwerediagnosedbyCAE.

Asallof ourcases werecarriers andsilentmutationswere excluded,noneofourcasesshowedaborderlineincreasein HbA2(3.5–3.8%).Fivehundred carriersweretestedonHPLC inavalidationstudyinIndia. Out of these500cases,only 7caseshadborderlineincreaseinHbA2whichonmolecular testingshowed presence of silentmutations [20]. Although 2003 survey of College of American Pathologist hasshown superiority of HPLC over conventional gel electrophoresis but ourresultsshow that electrophoresis is comparable to HPLC[21].

Manystudies haveshownthe precisionandaccuracy of CAE in diagnosis of BTT. And traditionally it has been consideredasmethodofchoicefordetectionandquantita- tion ofdifferenthemoglobinvariants[6,12]. Tyagiet al.in IndiashowedsimilarresultslikeoursinwhichallBTTcases weredetectedbothonelectrophoresisandHPLC[22].Khosa et al. did a comparative analysis of CAE and HPLC for quantitative determination of HbA2 levels in 40 BTT cases andshowed 100%sensitivityfor boththemethods.And in this study,CAE wasrecommended tobemoresuitable for diagnosisofBTTinpoorcountrieslikePakistan[23].Despite beingslowandlaborintensivemethod,most(296of387)of the laboratories participating in the College of American Pathologists Hemoglobinopathies Survey program reported results for HbF, HbA2 and Hemoglobin identiﬁcation using conventional CAE methods. While some laboratories use acombination of CAEand HPLCtoidentify and quantitate hemoglobinvariants.

Our study shows a CV of 8.39% for HbA2 at mean concentration of 4.97% for electrophoresis and for HPLC it was 10.72% at a mean HbA2 concentration of 5.54%. BTT diagnosis requires HbA2>3.8% and at this concentration,

CVisbetter for electrophoresisthan HPLC.Howeverit was 5.02 per cent0.72 for HPLC and 7.011.56% for electro- phoresisinastudybyTangvarasittichaietal.[7].Themean and SD valuesof HbA2in ourstudywere4.970.42% and 5.540.59% for electrophoresis and HPLC respectively. In our study we noted that all of our BTT cases had MCH<27pg which is usually recommended as an initial screening testfor BTT [24–27]. So MCH can be usedas an initial screening test followed by HbA2 quantitation for diagnosis of BTT. Based on the results of our study and resource constraints of our country, we recommend that MCH<27pg and HbA2>3.8% by CAE are the reliable and appropriatemethodforscreeninganddiagnosisofBTT.

The equipment and technology has improved over the years for the diagnosis of hemoglobindisorders. HPLChas advantagesinmassscreeningprogramsandhighworkload.

Theconventional CAE istimehonored cheapmethodology and has sensitivity comparable to HPLC as found in this study. Considering the economic restraints in developing countrieslikePakistan,itisrecommendedthatconventional CAE can be done at all levels of hospital care with good accuracyfordiagnosisofBTT.

Authors’ contributions/Wkład autorów

SeR – study design, data collection and interpretation, statistical analysis, manuscript preparation, literature search.SAK–datacollection,statisticalanalysis.GUS–data interpretation.FM–manuscriptpreparation.NA–literature search.AS–Studyconcept/design.

Conﬂict of interest/Konﬂikt interesu

Nonedeclared.

Financial support/Finansowanie

Nonedeclared.

Ethics/Etyka

The work described in this articlehas been carried out in accordance with The Code of Ethics of the World Medical Association(DeclarationofHelsinki)forexperimentsinvolving TableII–Meanvaluesofdifferentvariables

Variable Number Minimum Maximum MeanSD Pvalue

Age(years) 100 12 44 28.88.1 –

Hemoglobin(g/dl) 100 8.1 14.5 11.41.6 –

TRBC(10⁹/l) 100 4.87 8.16 6.06 0.87 –

MCV(ﬂ) 100 55.9 80.1 64.6 4.0 –

MCH(pg) 100 13.7 23.6 18.91.4 –

MCHC 100 24.5 33.3 29.31.4 –

RDW 100 33.1 45.9 37.82.4 –

HbA2onHPLC(%) 100 4.4 6.68 5.540.59 <0.0001

HbA2onhemoglobinelectrophoresis(%) 100 4.0 5.6 4.970.42 <0.0001

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humans; EU Directive 2010/63/EU for animal experiments;

UniformRequirementsformanuscriptssubmittedtoBiome- dicaljournals.

references/pi smiennictwo

[1] OuZ,LiQ,LiuW,SunX.ElevatedHbA2asamarkerforb Thalassemiatraitinpregnantwomen.TohokuJExpMed 2011;223(3):223–226.

[2] AhmedS,AnwarM.Haematologicalandgeneticfeaturesof deltabeta-ThalassemiainPakistan.JCollPhysiciansSurg Pak2006;16(1):19–22.

[3] KhattakMF,SaleemM.Prevalenceofheterozygousbeta ThalassemiaintheNorthernareasofPakistan.JPakMed Assoc1992;42:32–34.

[4] SharmaNP,GuptaSC,AtalRR,MelhotraTN,AgarwalAK, KapoorKK.AbnormalhemoglobinsinthePakistaniarmed forcespersonnel.IndJMedRes1976;64:883–890.

[5] AhmedS,SaleemM,ModellB,PetrouM.Screening extendedfamiliesforgenetichemoglobindisordersin Pakistan.NEngJMed2002;347:1162–1168.

[6] KotilaTR,AdeyemoAA,MewoyekaOO,ShokunbWA.B ThalassemiatraitinwesternNigeria.AfrHealthSci2009;9 (1):46–48.

[7] TangvarasittichaiS,TangvarasittichaiO,JermnimN.

Comparisonoffastproteinliquidchromatography(FPLC) withHPLC,electrophoresisandmicrocolumn

chromatographytechniquesforthediagnosisofb Thalassemia.IndianJMedRes2009;129(3):242–248.

[8] OuCN,RognerudCL.Diagnosisofhemoglobinopathies:

electrophoresisvs.HPLC.ClinChimActa2001;313(1–2):187–194.

[9] SachdevR,DamAR,TyagiG.DetectionofHbvariantsand hemoglobinopathiesinIndianpopulationusingHPL:report of2600cases.IndianJPatholMicrobiol2010;53(1):57–62.

[10] HeadCE,ConroyM,JarvisM,PhelanL,BainBJ.Some observationsonthemeasurementofhemoglobinA2andS percentagesbyhighperformanceliquidchromatographyin thepresenceandabsenceofaThalassemia.JClinPathol 2004;57(3):276–328.

[11] GhaniR,ManjiMA,AhmedN.Hemoglobinopathiesamong ﬁvemajorethnicgroupsinKarachi,Pakistan.Southeast AsianJTropMedPubHealth2002;33(4):855–861.

[12] ClarkeGM,HigginsTN.Laboratoryinvestigationof hemoglobinopathiesandThalassemias:reviewandupdate.

ClinChem2000;46(8):1284–1290.

[13] RaoS,KarR,GuptaSK,ChopraA,SaxenaR.Spectrumof hemoglobinopathiesdiagnosedbycationexchange-HPLC andmodulatingeffectsofnutritionaldeﬁciencyanemias fromnorthIndia.IndianJMedRes2010;132:513–519.

[14] WeatherallDJ,CleggJB.TheThalassemiasyndromes, 3rd ed.,Oxford:BlackwellScientiﬁcPublications;1981.

[15] LewisSM,BainBJ,BatesI.DacieandLewispractical haematology,10thed.,Philadelphia:ChurchillLivingstone;

2006.

[16] HassanAZ,UsmanW.Bloodsafetysystemreformsin Pakistan.BloodTransfus2014;12(4):452–457.

[17] Thelaboratorydiagnosisofhemoglobinopathies.BrJ Haematol1998;101:783–792.

gdp-per-capita[cited27.10.16].

[19] TanGB,AwTC,DunstanRA,LeeSH.Evaluationofhigh performanceliquidchromatographyforroutineestimation ofhemoglobinA2andF.JClinPathol1993;46:852–856.

[20] ColahRB,SurveR,SawantP,D'SouzaE,ItaliaK, PhanasgaonkarS,etal.HPLCstudiesin

hemoglobinopathies.IndianJPaediatr2007;74(7):657–662.

[21] HG-CHemoglobinopathyParticipantSummary.2003 CollegeofAmericanPathologists.

[22] TyagiS,SaxenaR,ChoudryVP.HPLC–hownecessaryisit forHemoglobinopathydiagnosisinIndia?IndianJPathol Microbiol2003;46:390–393.

[23] KhosaSM,UsmanM,MoinudinM,MehmoodHO,QamarK.

Comparativeanalysisofcelluloseacetatehemoglobin electrophoresisandhighperformanceliquid chromatographyforquantitativedeterminationof hemoglobinA2.BloodRes2015;50(1):46–50.

[24] BhukhanvalaD,SeliyaV,ShahA,GupteS.Studyofparents ofb-Thalassemiamajorchildrentodeterminecutoffvalues ofhematologicalparametersfordiagnosisofb-

Thalassemiatraitandassessmentofanemiainthem.

IndianJMedSci2013;67(5–6):117–122.

[25] AliN,MoizB,Bin-AzharW,ZaidiN,MemonR.Carrier detectionforbeta-ThalassemiatraitingeneralPakistani population:awayforward.Hematology2012;17(4):237–240.

[26] YousafzaiYM,KhanS,RaziqF.Beta-Thalassemiatrait:

haematologicalparameters.JAyubMedCollAbbottabad 2010;22(4):84–86.

[27] PranpanusS,SirichotiyakulS,SrisupunditK,TongsongT.

Sensitivityandspeciﬁcityofmeancorpuscularhemoglobin (MCH):foralpha-Thalassemia-1traitandbeta-Thalassemia trait.JMedAssocThai2009;92(6):739–743.