Rev. Bras. Hematol. Hemoter. vol.37 número5

revbrashematolhemoter.2015;37(5):316–319

w w w . r b h h . o r g

Revista

Brasileira

de

Hematologia

e

Hemoterapia

Brazilian

Journal

of

Hematology

and

Hemotherapy

Original

article

Frequency

of

Wr

a

antigen

and

anti-Wr

a

in

Brazilian

blood

donors

Janaína

Guilhem

Muniz

,

Carine

Prisco

Arnoni

,

Diana

Gazito

,

Rosangela

de

Medeiros

Person

_,

_Tatiana

_Aparecida

_de

_Paula

_Vendrame

_,

Flavia

Roche

Moreira

Latini

_,

_Lilian

_Castilho

a_{Associac¸ãoBeneficentedeColetadeSangue(Colsan),SãoPaulo,SP,Brazil}

b_{UniversidadeEstadualdeCampinas(Unicamp),Campinas,SP,Brazil}

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o

Articlehistory:

Received1June2015 Accepted3July2015 Availableonline29July2015

Keywords:

Anti-Wra Wra_antigen Diegobloodgroup Allelefrequency

a

b

s

t

r

a

c

t

Background:Wra_{isalow-incidenceantigen,whichisantitheticaltothehighprevalence}

redblood cellantigen,Wrb_.Anti-Wra_{isanaturallyoccurringantibodythatisfoundin} approximately1–2%ofblooddonors.Theaimofthisstudywastodeterminethefrequency ofWra_andanti-Wra_{inBrazilianblooddonors.}

Methods:Atotalof1662Brazilianblooddonorsweremolecularlyanalyzedusingthe

SNaP-shotmethodologytodeterminetheWR*A/BallelesandtopredictthefrequencyoftheWra antigen.Todetecttheanti-Wra_{,samplesfrom1049blooddonorswereanalyzedusinga} geltestwithWr(a+)redbloodcells.Theserumwastreatedwithdithiothreitol(DTT)to determinetheimmunoglobulinclasses.Immunoglobulin(Ig)-Gisotypeclassificationwas performedinageltestusingtheIgG1/IgG3card.Amonocytemonolayerassaywasemployed topredicttheclinicalsignificanceofIgGanti-Wra_.

Results:Ofthe1662donors,onlyonesamplehadtheDI*02.03alleleinheterozygous

predict-ingtheWr(a+b+)phenotype.Anti-Wra_{wasdetectedin34(3.24%)samples,64.7%infemales} and35.3%inmales.Regardingtheimmunoglobulinclass,eight(23.5%)casesofanti-Wra wereclassifiedasIgGand26(76.5%)asIgM.OftheeightcasesofIgGanti-Wra_,fourwere IgG1,twowereIgG3andthreeanti-Wra_{werenotIgG3orIgG1,andthusprobablyIgG2or} IgG4.TheresultsofthemonocytemonolayerassayshowedthatIgGanti-Wra_mightbeof clinicalsignificance.

Conclusion:Thisstudyshowsaverylowfrequency(0.06%)oftheWra_{antigeninBrazilian}

blooddonors.Additionally,itshowsthatthefrequencyofanti-Wra_{inthispopulationis} higherthanpreviouslyreported.

©2015Associac¸ãoBrasileiradeHematologia,HemoterapiaeTerapiaCelular.Published byElsevierEditoraLtda.Allrightsreserved.

∗ _{Correspondingauthorat:Colsan–Associac¸ãoBeneficentedeColetadeSangue,Av.Jandira1260,Indianópolis,04614-013SãoPaulo,SP,}

Brazil.

E-mailaddress:janagmuniz@yahoo.com.br(J.G.Muniz).

http://dx.doi.org/10.1016/j.bjhh.2015.07.002

revbrashematolhemoter.2015;37(5):316–319

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Introduction

TheDiegobloodgroupsystemiscarriedonband3,a multi-passmembraneglycoprotein,whichisencodedbytheSLC4A1

gene.TheDiegosystemiscomposedof22antigens:threepairs ofantitheticalantigens,Dia_andDib_,Wra_andWrb_,Wuand DISK,and16verylowfrequencyantigens.1_Wra_andWrb anti-gensarerelatedtoaSNPinexon16(1972G>A)thatencodesa LysineinWra_{oraglutamicacidinWr}b_{ataminoacidposition} 658.2

TheWra_{antigen,firstdescribedbyHolmanin1953,hasan} incidenceofaround1in1000inCaucasianpopulations,butit isnotreportedinotherethnicgroups.3_{AlthoughtheWr}a anti-genhasaverylowincidence,anti-Wra_{isarelativelycommon} antibodysinceitisoftenanaturallyoccurringantibody.4_The describedincidenceofanti-Wra_{intheseraofnormaldonors} variesindifferentstudies;ithasbeenestimatedat1of100 inhealthyvolunteerblooddonors.5_{Theimmunoglobulin(Ig)} classofanti-Wra_{canbeIgM,IgGorIgMplusIgG.Alloanti-Wr}a israrelyinvolvedinhemolytictransfusionreactions,however therearesomecasesreportinghemolyticdiseaseofthefetus andnewborn(HDFN)causedbyanti-Wra_.1

Antibodiesagainstlow-incidenceantigens,including anti-Wra_{,aredifficulttoidentify,becausethescreeningandpanel} cellsrarelyexpresstheseantigens.6,7 _{Hence,littleisknown} aboutthe frequencyofanti-Wra _{inmany populations. The} knowledgeofthemolecularbasisoftheDiegobloodgroup systemandthedevelopmentofmolecularassaystoidentify theDIalleleshasallowedthefrequencyoftheseallelestobe assessedindifferentpopulations.Theaimofthisstudywas todeterminethefrequencyoftheWra_{antigenandanti-Wr}a inaBrazilianpopulationofblooddonors.

Methods

Atotalof1662bloodsampleswereobtainedfromhealthy vol-unteerBrazilianblooddonorsattheAssociac¸ãoBeneficente

deColetadeSangue(Colsan),SãoPaulo,Brazil.The popula-tionstudiedwasfromSoutheastofBrazilanditiscomposed ofahighlyadmixedpopulation.

Molecularanalysis

DNAwasextractedusingtheQIAmpDNAMiniKit(Qiagen® Inc. Valencia, CA, USA) according to the manufacturer’s instructions. To determine the WR*Aand WR*B alleles and predictthefrequencyoftheWra_{antigen,genotypingwas} per-formedusingapreviousdescribedSNaPshot®_{protocol(Latini} etal.8_{).Fragmentanalyseswereperformedina3500xLGenetic} Analyzer(AppliedBiosystem,FosterCity,CA,USA)asshown inFigure1.

Antibodyscreening

Inordertoinvestigatetheoccurrenceofanti-Wra_,serum sam-plesfrom1049blooddonors(638maleand411femaledonors) wereinitiallycross-matchedwithaWr(a+)redbloodcell(RBC) from our collection in a gel test by an automated system (WADiana® _{8XT, Grifols,Barcelona,Spain). Thepresenceof} anti-Wra _{inpositivecross-matcheswasconfirmedwithtwo} sources ofWr(a+) RBCs from commercial panels(BioRad®_, LagoaSanta,Brazil).

Immunoglobulinclasses

To determine the Ig classes (IgG or IgM), the serum was treatedwithdithiothreitol (DTT,Sigma-Aldrich, Brazil).The IgG isotypeclassificationwasperformedinageltestusing theIgG1/IgG3card(BioRad®_{,LagoaSanta,Brazil).}

Monocytemonolayerassay

To predict the clinical significanceof anti-Wra_{, the} mono-cyte monolayer assay (MMA)was performed as previously described9_{intwosampleswithanti-Wr}a_{classifiedasIgG1and} onesampleclassifiedasIgG3.Usinganopticalmicroscopy,600

1200

DI*4

DI*3

DI*2 _YT*1

CO*1 KEL*7

KEL*4 DO*2

DO*5

DO*4

Vel+ 1000

800

600

400

200

0

318

Figure2–Monocytemonolayerassayslides(1000×).(A)Negativecontroland(B)Monocytemonolayerassaywithanti-Wra.

monocyteswerecountedtodeterminethepercentageof reac-tivemonocytes(RBCadheredandphagocytized).MMAresults <4%wereconsiderednegativewhileresults≥4%were

consid-eredpositive.

Results

Wra_antigen

Ofthe1662genotypedbloodsamples,onlyonesample pre-sentedtheWR*Aalleleinheterozygous.Itwasgenotypedas

WR*A/WR*BpredictingtheWr(a+b+)phenotype.

Anti-Wra

Anti-Wra _{was detected in 34 samples from 1049 screened} blooddonorsrepresentingafrequencyof3.24%.Regardingthe Igclasses,8/34(23.5%)wereIgGand26/34(76.5%)wereIgM.Of theeightIgGanti-Wra_{,fourwereclassifiedasIgG1andone} wasisotypedasIgG3.Threesampleswere notclassified as IgG1orIgG3;theseareprobablyIgG2orIgG4,Igclassesthat arenotinvolvedinseveretransfusionreactions.Asshownin

Table1,ahigherfrequencyofanti-Wra_{wasobservedinfemale} donors(p=0.0036,Fisher’sexacttest).

Table1–Frequencyofanti-Wra_{andimmunoglobulin(Ig)}

class.

Igclass Isotype Donor Total

Female Male

IgG IgG1 3 1 4

IgG3 0 1 1

IgG2orIgG4 3 0 3

IgM – 16 10 26

Total 22 12 34

MMAresults(Figure2)showthatWra_{antibodiesclassified} asIgGcanpotentiallybeclinicallysignificant,asIgG1 antibod-iespresented7–7.5%ofreactivemonocytesand12.7%ofIgG3 hadreactivemonocytes.

Discussion

Thisstudyshowsnovelinformationregardingthepresenceof anti-Wra_{inaBrazilianpopulationofblooddonors.Although} thefrequencyoftheWra _{antigen(1:1662)islowerthanthat} previouslyreportedinEuropeans(1:1000),3_{theoccurrenceof} anti-Wra _{washigher(1:31)whencomparedtootherstudies} whereitrangedfrom1in80to1in200.Thefrequencyof anti-Wra_{foundinthisstudyissimilartothatfoundinSpain(1:37),} howeverthepresenceoftheantigeninSpanishpopulationis around2-times(1:785)6_{thefrequencyfoundinBrazilians.}

Themechanismsinvolvedinanti-Wra_{productionarestill} unclear. Some authors believe that, besidesthe alloimmu-nization in response to antigenexposure, certain proteins

that can cross-react with the Wra _{antigen are formed}

whentheimmunesystembecomesmoreactive.7_Situations described to beinvolved inanti-Wra _{alloimmunization are} also related to immune system activation, including

preg-nancy, autoimmune hemolytic anemia and patients with

otherRBCantibodies.7_{Therefore,ourhypothesisisthatthe} differenceinanti-Wra_{distributionbetweengenderscouldbe} associatedto pregnancy, asanti-Wra _{was foundin5.2%of}

womenand1.8%ofmen.

revbrashematolhemoter.2015;37(5):316–319

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canbeofclinical significance.Due tothelow incidenceof theWraantigenand the lowriskofhemolytictransfusion reaction,theuseofscreeningpanelscontainingWr(a+)RBCs isnotrequired. TherebyWra _{incompatibletransfusion can} occur,but fewcasesofhemolytictransfusionreactionwere described,beenestimatedtobe1in500,000.11

Insummary,theWra_{antigenhasaverylowfrequencyin} Brazilianblooddonorsandanti-Wra_{hasahigherfrequency} thanreportedinotherpopulations.Consideringthelow fre-quencyoftheantigenandthefewcasesofmildHDFNrelated toanti-Wra_{,clinicalimpactisdiscussableaswellthe} require-mentofRBCreagenttoidentifythem.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

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