Maternal anti-HLA class I antibodies are associated with reduced birth weight in thrombocytopenic neonates

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Paper I

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ContentslistsavailableatScienceDirect

Journal of Reproductive Immunology

j ourna l h o m e pa g e :w w w . e l s e v i e r . c o m / l o c a t e / j r e p r i m m

Maternal anti-HLA class I antibodies are associated with reduced birth weight in thrombocytopenic neonates

J. Dahl

^a

, A. Husebekk

^a

, G. Acharya

^c,e

, K. Flo

^c

, T.B. Stuge

^a

, B. Skogen

^b

, B. Straume

^d

, H. Tiller

^a,e,∗

aImmunologyResearchGroup,UiT,TheArcticUniversityofNorway,Tromsø,Norway

bNorwegianNationalUnitforPlateletImmunology,Tromsø,Norway

cWomen’sHealthandPerinatologyResearchGroup,UiT,TheArcticUniversityofNorway,Tromsø,Norway

dDepartmentofCommunityMedicine,UiT,TheArcticUniversityofNorway,Tromsø,Norway

eDepartmentofObstetrics&Gynecology,UniversityHospitalofNorthNorway,Tromsø,Norway

a r t i c l e i n f o

Articlehistory:

Received9June2015

Receivedinrevisedform16August2015 Accepted19October2015

Keywords:

Neonatalthrombocytopenia Birthweight

Antibodies Alloimmunization Placenta

a b s t r a c t

Inthiscomparativecross-sectionalstudy,possibleassociationsbetweenmaternalanti-HLAclassIanti- bodiesandbirthweightinneonatalthrombocytopeniaareexplored.Althoughcommonlydetectedin pregnanciesandgenerallyregardedasharmless,ithasbeensuggestedthatsuchantibodiesmightbe associatedwithfetalandneonatalalloimmunethrombocytopenia(FNAIT).AsalinkbetweenFNAITdue tohumanplateletantigen1a-specificantibodiesandreducedbirthweightinboyshaspreviouslybeen demonstrated,wewantedtoexplorewhethermaternalanti-HLAclassIantibodiesmightalsoaffect birthweight.Toexaminethis,suspectedcasesofFNAITreferredtotheNorwegianNationalUnitfor PlateletImmunologyduringtheperiod1998–2009wereidentified.Pregnancieswheretheonlyfinding wasmaternalanti-HLAclassIantibodieswereincluded.Anunselectedgroupofpregnantwomenpar- ticipatinginaprospectivestudyinvestigatingmaternal–fetalhemodynamicsattheUniversityHospital NorthNorwayduringtheyears2006–2010servedascontrols.Twenty-ninepercentofcontrolshadanti- HLAclassIantibodies.Thethrombocytopenicneonateshadasignificantlyloweradjustedbirthweight (linearregression,P=0.036)andsignificantlyhigheroddsofbeingsmallforgestationalage(OR=6.72, P<0.001)comparedwithcontrols.Increasinganti-HLAclassIantibodylevelsinthemotherweresignifi- cantlyassociatedwithlowerbirthweightandplacentalweightamongthrombocytopenicneonates,but notamongcontrols.Theseresultsindicatethatmaternalanti-HLAclassIantibodiesinthrombocytopenic neonatesareassociatedwithreducedfetalgrowth.Furtherstudiesareneededtotestifplacentalfunction isaffected.

1. Introduction

Howasemi-allogenicfetusmanagestosurvivepregnancyis stillquiteanenigma.Itis,however,clearthatthematernalimmune systemrecognizesandrespondstofetalantigens(ArckandHecher, 2013).

ThehumanleukocyteantigenclassI(HLAclassI)antigensare presentonallnucleatedcellsandplateletsinthehumanbody.The genesthatencodeHLAclassIantigensarethemostpolymorphic inthehumangenome.ExposuretoincompatibleHLAantigenscan activatethehostimmune systemandleadtotheproductionof

∗ Correspondenceto:UniversitetssykehusetNord-Norge,Kvinneklinikken,9038 Breivika,Norway.

E-mailaddresses:heidi.tiller@unn.no,heidi.tiller@gmail.com(H.Tiller).

alloantibodies.Itiswellknownthatanti-HLAclassIantibodiescan havesevereclinicalconsequences,suchastherejectionofallografts (Leeetal.,2002;Zhangetal.,2005)orthedestructionoftransfused platelets(Novotny,1999).

Maternalanti-HLA classIantibodiesarecommonly detected duringpregnancy(approximately30%ofpregnantwomen)(Morin- Papunenetal.,1984;Reganetal.,1991;Kingetal.,1996;Masson etal.,2013).Inthecontextofpregnancy,theseantibodiesaregen- erallyconsideredharmless.Reportshavedescribedanassociation betweenmaternalanti-HLAclassIantibodiesandrecurrentmiscar- riage(Sargentetal.,1988;Nielsenetal.,2010).Possibleassociations betweenmaternalanti-HLAclassIantibodiesandplacentalabrup- tion(Steinbornetal.,2004)andpreeclampsia(Buurmaetal.,2012) havealsobeensuggested.However,therearefewsystematicstud- iesonanti-HLA classI antibodiesand pregnancycomplications (Lashleyetal.,2013).

http://dx.doi.org/10.1016/j.jri.2015.10.003

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28 J.Dahletal./JournalofReproductiveImmunology113(2016)27–34

Fetalandneonatal alloimmunethrombocytopenia (FNAIT)is causedbymaternalalloantibodiestargetinghumanplateletanti- gens(HPAs)onfetalplatelets,mostcommonlyHPA-1a(Davoren etal.,2004;Skogenetal.,2010).FNAIToccursatafrequencyof about1.5per1000births(Dreyfusetal.,1997;Kjeldsen-Kraghetal., 2007).Intracranialhemorrhage(ICH)isthemostseverecomplica- tionandisreportedinaround10%ofpatientswithsevereFNAIT (Mueller-Eckhardtetal.,1989;Kamphuisetal.,2010).Numerous reportsdescribesuspectedcasesofFNAITwithmaternalanti-HLA classIantibodiesastheonlyﬁndingandapossibleexplanationof neonatalthrombocytopenia(Saitoetal.,2003;Moncharmontetal., 2004;Thudeetal.,2006;Gramatgesetal.,2009;Starcevicetal., 2010).Ithasthereforebeensuggestedthatmaternalanti-HLAclass IantibodiesmightcauseFNAIT.

We have previously demonstrated an association between maternalantibodiesagainstHPA-1aandreducedbirthweightin boys(Tilleret al.,2012).The aimof this study wasto explore whethertherearesimilarassociationsbetweenmaternalanti-HLA classIantibodiesandbirthweightinrelationtoneonatalthrom- bocytopenia.

2. Methods 2.1. Studypopulation

Thetwostudygroups(casesandcontrols)wereidentiﬁedand selectedfrom pregnant populations that were originally either clinical referrals to the Norwegian National Unit for Platelet Immunologyorparticipantsinadifferentstudy(Floetal.,2010, 2014).We performedasecondaryanalysisofthesedatausinga comparativecross-sectionalstudydesign.Selectionof thestudy populationispresentedasaﬂowchartinFig.1.

Allpregnancies referred totheNorwegian NationalUnit for Platelet Immunology in Tromsø, Norway, for suspected FNAIT duringtheperiod1998–2009wereidentified.Pregnancieswere includedas cases if maternal anti-HLA class I antibodies were detectedandneonatalthrombocytopeniawasconfirmed.Pregnan- cieswereexcludedifplatelet-specific(anti-HPA-)antibodieswere detectedorifotherreasonsforneonatalthrombocytopeniawere found.Informationregardingdemographiccharacteristics,obstet-

richistory,course,andoutcomeofpregnancywasobtainedfrom themedicalrecords.Allmaternalbloodsamplesweretakenpost- partum.

Of 82 mothers who fulﬁlled the inclusion criteria, 62 con- sented to participate. Therewas one twin pregnancy. Thirteen neonates were further excluded from analysis: eight for other possiblereasonsfor neonatalthrombocytopenia(twowithcon- genitalcytomegalovirusinfections,onewithJacobsen’ssyndrome, onewithmaternalimmunethrombocytopenicpurpura,onewith neonatalhemochromatosis,onewithNoonan’ssyndrome,onewith Down’ssyndrome,onecaseofneonataldeath18daysafterbirth, wheretheautopsyshowedunderdevelopedbonemarrow),andﬁve caseswherematernalserawereunavailableforantibodyanalysis.

Thus,datafrom50casesoveraperiodof11yearswereincluded forfurtheranalysis.

An unselected population of pregnant women originally includedinaprospectivestudyinvestigatingmaternal-fetalhemo- dynamicsattheUniversityHospitalofNorthernNorwayduring theperiod2006–2010servedascontrols(Floetal.,2010,2014).

Maternalbloodsamplesweretakenat22–24weeksofgestation.

Additionalmaternalbloodsamplesacquiredwithinthreedaysof deliverywereavailableforsevencontrols.Allsamplesweretested forthepresenceofmaternalanti-HLAclassIantibodiesandcate- gorizedaseitheranti-HLAclassIantibody-negativeor-positive.Of 250pregnanciesinthecontrolgroup,72(29%)testedpositivefor maternalanti-HLAclassIantibodies.Plateletcountswereobtained from45randomlyselectedneonatesinthecontrolgroup,noneof whichwasthrombocytopenic.

All pregnancies were dated based on ultrasonography per- formed in the second trimester. Preeclampsia was diagnosed accordingtocurrentISSHPcriteria(Tranquillietal.,2014).

2.2. Deﬁnitions

Smallforgestationalage(SGA)wasdeﬁnedasbirthweightless thanthe10thpercentile forgestational agebasedonsingleton percentilecurves(Skjaervenetal.,2000).

Infantsbornbefore37+0 gestationalweeksweredeﬁnedas premature.

Fig.1.Casesconsistedofpregnancieswherethemotherwasanti-HLAclassIantibody-positiveandtheneonatehadsuspectedFNAIT,whilecontrolsconsistedofnormal pregnancies.

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Fig.2.Maternalantibodylevelversuspredictedvaluesforthebirthweightinpregnancieswithmaternalanti-HLAclassIantibodiesandthrombocytopenicneonates(cases), andinnormalpregnancieswithmaternalanti-HLAclassIantibodies(anti-HLAclassIantibody-positivecontrols).*Bothregressionlinesadjustedformaternalage,parity, preeclampsia,gestationalageatdelivery,andsexofthefetususingmultiplelinearregression.

Table1

Unadjustedmaternalandneonatalcharacteristicsforpregnancieswithmaternalanti-HLAclassIantibodiesandthrombocytopenicneonates(cases)versusnormalpregnancies(controls).

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Table2 Comparisonofbirthweight,riskofSGAa,placentalweight,andPW/BWbbetweenpregnancieswithmaternalanti-HLAclassIantibodiesandthrombocytopenicneonates(cases)versusnormalpregnancies(controls). Effectestimatesofoutcomevariable AdjustingvariableEffectestimate(B)of birthweightingrams (95%CI) PEffectestimate(OR)of SGA(95%CI)PEffectestimate(B)of placentalweightin grams(95%CI)

PEffectestimate(B)of PW/BW(95%CI)P Studygroup(0=controls,1=cases)−167(−323,−11)0.0366.72(3.22,14.03)<0.001−33.6(−86.7,19.5)0.2140.015(0.003,0.028)0.012 Maternalage(years)1.6(−10.7,13.9)0.8011.00(0.93,1.08)0.9911.1(−2.6,4.9)0.5570.000(−0.001,0.001)0.803 Parity(0=nulliparous,1=multiparous)129(5254)0.0420.62(0.29,1.32)0.21626.0(−12.4,64.5)0.1830.001(−0.007,0.010)0.750 Preeclampsia(0=no,1=yes)−102(−328,124)0.3735.01(1.82,13.82)0.00253.6(−19.6,126.7)0.1510.039(0.022,0.056)<0.001 Gestationalageatdelivery(weeks)213(188,237)<0.00131.6(24.0,39.2)<0.001−0.007(−0.008,−0.005)<0.001 Sex(0=boy,1=girl)−36(−150,77)0.5290.74(0.38,1.47)0.3965.2(−30.0,40.5)0.770−0.002(−0.010,0.006)0.701 aSmallforgestationalage. bPlacentalweight/birthweightratio.

Thrombocytopeniawasdeﬁnedasaplateletcount<150×10⁹/L.

Severe thrombocytopenia was deﬁned as a platelet count

<50×10⁹/L.

Aplacentalweight/birthweightratio(PW/BW-ratio)wascalcu- latedandincludedintheanalyses,asthisratiohasbeenconsidered tobeabetterpredictoroflong-termfetalhealththanbirthweight orplacentalweightalone(Hemachandraetal.,2006;Shehataetal., 2011).

2.3. Laboratoryanalysis

Screeningforanti-HLAclassIantibodieswasdoneusinganin- housemonoclonalantibodyimmobilizationoftheplateletantigen (MAIPA)technique(Killieetal.,2010),orwithFlowPRA1Screen- ingTest(OneLambda,CanogaPark,CA,USA).InMAIPA,samples weretestedagainstpaternalplateletswhenavailable.Ifpaternal plateletswerenotavailable,thestudysamplewastestedagainst random donor platelets from at least four donors. Allsamples thattestednegativeforantibodiesinMAIPApanelswithrandom donorplateletsweresubsequentlyalsotestedusingtheFlowPRA 1ScreeningTest(OneLambda)touncoverfalsenegatives.

Aftertheprimaryidentiﬁcationofanti-HLAclassIantibody- positive cases,all sampleswerere-tested using theFlowPRA1 ScreeningTesttoobtaincomparablevalues.Aratioofthemedian ﬂuorescenceintensity(MFI)foreachsampletotheMFIofthenega- tivecontrolfortheassaywascalculatedandusedwhencomparing anti-HLAclassIantibodylevels.

Alllaboratoryworkandanalyseswereperformedbyexperi- encedbioengineers attheNorwegian NationalUnitfor Platelet ImmunologyinTromsø,Norway.

2.4. Statistics

AlldatawereanalyzedusingSPSSsoftware(version21.0;SPSS, Chicago,IL,USA).

The normality of data distribution was tested using the Kolmogorov–Smirnov test. An independent samples t-test was usedtocomparemeansforcontinuousvariables.Fisher’sexacttest wasusedtocomparefrequenciesforcategoricalvariables.APvalue of<0.05wasconsideredsigniﬁcant,and95%conﬁdenceintervals (CI)werereportedwhereappropriate.

Regression models were applied to further evaluate differ- encesinbirthweight,frequencyofSGA,placentalweightandthe PW/BWratiobetweenstudygroups.Inallregressionmodelswe adjustedformaternalage,parity(nulli-ormultiparity),preeclampsia(yes/no),sexofthefetus, andgestationalageatthetimeof delivery.Whenlookingatassociationsamongmaternalantibody levelandbirthweight,SGA,placentalweight,orPW/BWwithin eachstudygroup,maternalantibodylevelwasincludedasaninde- pendentvariable.Wedidnotadjustforgestationalageatdelivery whenSGAwasdeﬁnedasthedependentvariable.

Missingdataweretreatedbypairwisedeletionwhencompar- ingallunadjusteddataandbylist-wisedeletionwhenperforming regressionanalyses.

Data onsmoking habitswere available for70% of cases. All regressionanalyseswereperformedbothwithandwithoutadjust- ingforsmokinghabits.Datapresentedintheresultsdidnottake intoaccountsmokinghabitsunlessotherwisestated.

2.5. Ethics

ThestudywasapprovedbytheRegionalCommitteeforMedical ResearchEthics,NorthNorway(Ref.no.REKNORD2013/1863:date ofapproval15.05.2014).Informedwrittenconsentwasobtained fromallwomenincludedinthisproject.

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Fig.3.Maternalantibodylevelversuspredictedvaluesfortheplacentalweightinpregnancieswithmaternalanti-HLAclassIantibodiesandthrombocytopenicneonates (cases),andinnormalpregnancieswithmaternalanti-HLAclassIantibodies(anti-HLAclassIantibody-positivecontrols).*Bothregressionlinesadjustedformaternalage, parity,preeclampsia,gestationalageatdelivery,andsexofthefetususingmultiplelinearregression.

3. Results

Childreninthecasegrouphadasignificantlylowerbirthweight (3005gversus3485g,P=0.003)andahigherfrequencyofbeing SGA(48%versus12%,P<0.001)comparedwithcontrols.Almost 20%ofinfants(9/48)inthecasegroupwerebornpremature,com- paredwith5%amongcontrols(P=0.004).Therewasnosignificant differenceinplacentalweightbetweencasesandcontrols.Fiveof theneonates(10%)inthecasegrouphadICH.Clinicalcharacteris- ticsdidnotdiffersignificantlybetweenthetwocontrolgroups, except for thefrequency of nulliparous mothers. Maternal and neonatalcharacteristicsarepresentedinTable1.

Mean adjusted birth weight among cases was signiﬁcantly lowercomparedwithcontrols(adjustedweightdifference167g, P=0.036,Table2).TheriskofbeingSGAwassigniﬁcantlyhigher amongcasesthanamongcontrols(OR=6.72,P<0.001,Table2).

Incontrast,therewasnosigniﬁcantdifferenceinadjustedbirth weightorfrequencyof SGAbetweenanti-HLAclassIantibody- positiveand-negativecontrols(datanotshown).Therewereno signiﬁcantadjusteddifferencesbetweenboysandgirlsforbirth weight,riskofSGAorplacentalweightinanyofourstudygroups (datanotshown).

Meanmaternalanti-HLAclassIantibodylevelwassignificantly higher among cases compared with anti-HLA class I antibody- positive controls (unadjusted, P=0.001, Table 1). Further, the meanmaternalanti-HLAclassIantibodylevelwassignificantly higherincaseswheretheinfantwasSGAatdeliverycompared withcaseswheretheinfantwasnotSGA (unadjusted,P=0.037, data not shown). An increasing level of anti-HLA class I anti- bodieswassignificantlyassociatedwithdecreasingbirthweight

amongcases(ˇ=−4.9,P=0.004,Fig.2).Althoughnon-significant, thetrendamonganti-HLAclassIantibody-positivecontrolswas opposite, withthelevel ofmaternal anti-HLAclassI antibodies increasingtogetherwithincreasingbirthweight(ˇ=1.7,P=0.140, Fig. 2). There was a significant association between maternal anti-HLA class I antibody level and frequency of SGA among cases when smoking was includedas an independentvariable (OR=1.026,P=0.021,datanotshown).Thisassociationwasnotsig- nificantwhensmokingwasnotincludedintheregressionmodel (OR=1.011,P=0.058,datanotshown).

Anincreasinglevelofanti-HLAclassIantibodieswasfurthersig- nificantlyassociatedwithdecreasingplacentalweightamongcases (ˇ=−1.7,P=0.008).Thisassociationhadanoppositetrendamong anti-HLAclassIantibody-positivecontrols,althoughitwasnon- significant(ˇ=0.748,P=0.061)(Fig.3).Thecaseshadasignificantly higherPW/BWratiothancontrols(meanadjusteddifference0.015, P=0.012).Therewasnosignificantassociationbetweenmaternal anti-HLAclassIantibodylevelandPW/BWratioforanyofthestudy groups(datanotshown).

Thefrequencyofpreeclampsiaamongcaseswas13%.Fiveout ofsixinfantswhosemotherhadpreeclampsiawereSGAatbirth.

Becauseoftheknownassociationbetweenpreeclampsiaandfetal growthrestriction, werepeatedtheanalysesafterexcluding all preeclampticpregnancies;lowerbirthweight,andtheriskofhav- inganSGAinfantwerestillsigniﬁcantlyandsimilarlyassociated withmaternalanti-HLAclassIantibodiesamongcases(datanot shown).

ToevaluatetheuseofMFIfromtheFlowPRAIScreeningTestas aquantitativemeasure,wecomparedtheseresultswiththoseof theMAIPAassays.Wefoundastrongdegreeofcorrelationbetween

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theopticaldensityvalueobtainedfromMAIPAandtheMFIfrom FlowPRA(Spearman’scorrelationcoefﬁcient=0.680,P<0.001).

Asthesamplingtimevariedbetweenthecaseandthecontrol group,weevaluatedwhethersamplingtimecouldhaveinﬂuenced theobserveddifferenceinanti-HLAclassIantibodylevels.Samples takenbothantenatallyandpostpartumwereavailablefromseven participantsintheanti-HLAclassIantibody-positivecontrolgroup.

Samplestakenatweeks22–24hadanMFIthatwasonaverage 66%higherthantheMFIofthepostpartumsamples(mean1.66, 95%CI0.94–2.38).Althoughnotstatisticallysigniﬁcant,thetrend indicatesthattheanti-HLAclass1antibodyleveltendstofallafter delivery.Ifthistrendisrepresentative,thedifferenceinantibody levelbetweencasesandcontrolswouldhavebeenevenlargerif sampleshadbeentakenatthesametimeinbothstudygroups.

Neonatalplateletcountswereavailablefor46ofthe50neonates in the case group.The mean platelet countin this group was 29×10⁹/L(SD19×10⁹/L).Themeanplateletcountintheumbilical cordbloodof45randomlyselectedcontrols(antibodypositiveand antibodynegative)was290×10⁹/L(n=45,SD62×10⁹/L).Noneof thecontrolshadthrombocytopenia.

4. Discussion 4.1. Mainﬁndings

Thrombocytopenicneonates born to motherswith anti-HLA classIantibodieshadasigniﬁcantlylowerbirthweightcompared withcontrols.Anincreasinglevelofmaternalanti-HLAclassIanti- bodieswaslinearlyandinverselyassociatedwithbirthweightand placentaweightamongthethrombocytopenicneonates.Therewas nodifferenceinbirthweightbetweentheantibody-positiveand antibody-negativecontrols.

4.2. Strengthsandlimitations

Consideringtherarityoftheconditionwedescribe,thesizeof ourstudypopulationislargeandwithfewmissingdata(TableS1).

Thecaseswereselectedfromnation-widereferralsofpregnancies whereFNAITwassuspected.AsFNAITinvestigationsareunder- takenonlyattheNorwegianNationalUnitforPlateletImmunology, thecasegroupisconsideredrepresentativeofNorwegianFNAIT pregnanciesin general.Mostof theneonatesinthecase group hadseverethrombocytopenia.Aselectionbiastowardmoresevere casesispossibleowingtotheretrospectivestudydesign.Toassess theexternalvalidityofthecontrolgroup,theclinicalvariableswere comparedwithequivalentdatafromtheMedicalBirthRegistryof Norway(MBRN)for2010(Norwegian,2012).Exceptforalowerfre- quencyofsmokers(6.4%versus18.5%intheMBRN),allmaternal andneonatalcharacteristicsweresimilartodatafromtheMBRN (TableS2).Thecontrolsincludedinthisstudymaythereforebe consideredrepresentativeofanormalbackgroundpopulationof Norwegianpregnancies.Theparticipantsinbothgroupswereorigi- nallyidentiﬁedprospectivelyandduringoverlappingtimeperiods.

Wethereforeconsiderthetwogroupscomparable.Themajority ofourstudyparticipantswereWhiteEuropean.Ourﬁndingsmay thereforenotberepresentativeofotherethnicities.

Toremovepossibleconfounders,weexcludedallcaseswhere otherpossiblecausesofthrombocytopeniacouldbefoundinthe patient’s medical records,but undisclosed causescouldstill be present.

ThefrequencyofICHamongcaseswas10%,whichissimilar towhat hasbeen previously reportedfor children withsevere FNAIT(Mueller-Eckhardtetal.,1989;Kamphuisetal.,2010).The frequencyofmaternalanti-HLAclassIantibodiesamongcontrols was29%,whichisalsoinaccordancewithpreviousreports(Morin-

Papunenetal.,1984;Reganetal.,1991;Kingetal.,1996;Masson etal.,2013).Consideringthatneonatalinfectionsareacommon causeofneonatalthrombocytopenia,thenumberofneonatesin thecasegroupexcludedbecauseofneonatalinfectionswaslow.

However,newbornsdiagnosedwithinfectiontypicallywouldnot bereferredforFNAITinvestigation,thusexplainingthelownumber ofinfectionsinourstudypopulation.

WeusedﬂuorescenceintensitiesobtainedfromtheFlowPRA IScreeningTestasameasureofanti-HLAclassIantibodylevel.

ThepanelofantigenspresentintheFlowPRAIScreeningTestcon- tainsallofthemostcommonandseveralrareHLAclassIantigens describedinourpopulation.However,someoftheantigensare representedat differentfrequencies,meaning thatcertain anti- bodyspeciﬁcitieshavemorebindingsitesthanothers.TheFlowPRA I Screening Test is not validated as a quantitative test by the manufacturer,althoughMFIiscommonlyconsideredasemi-quan- titativemeasureofantibodylevel(Akalinetal.,2008;Lefaucheur etal.,2010).Theantibodylevelresultsinthisstudymustbeinter- preted withcaution. The essence of our data is primarily that antibodylevelseemstomatter,butfutureassaysdesignedtomea- sureanti-HLAclassIantibodylevelarewarranted.

4.3. Interpretation

Theassociationbetweenmaternalanti-HLAclassIantibodies and birth weightin relationtoneonatal thrombocytopenia has notbeendescribedbefore.However,asimilarassociationbetween maternalantibodiesagainstHPA-1aandreducedbirthweightin boyshasbeenreported(Tilleretal.,2012),andneonatalthrom- bocytopeniaingeneraliscommonlydetectedingrowth-retarded newborns(Beineretal.,2003;EngineerandKumar,2010).Thelack ofsignificantassociationsbetweenmaternalanti-HLAclassIanti- bodiesandbirthweightorplacentalweightamongcontrolscould indicatethatanti-HLAclassIantibodiesalonedonotinfluencefeto- placentalgrowth.Itis possiblethat thelowerbirth weightwas mediatedthroughfetalthrombocytopenia,ratherthanthroughthe anti-HLAclass1antibodies.Theobservationthatantibodylevels weremuchhigheramongcasesthanamongcontrolsmakesitdif- ficulttodrawanyconclusionsonthismatter.Prospectivestudies areneededtoclarifywhethertherecouldbeathresholdlevelof anti-HLAclassIantibodiesabovewhichfetoplacentalgrowthmay beaffected.

Alternatively,boththematernalanti-HLAclass1antibodiesand theneonatal thrombocytopenia mayhave beenepiphenomena, withanundisclosedthirdfactoractuallycausingthelowerbirth weight.Apreviousstudyreportedthatthebreadthandstrengthof anti-HLAantibodiesincreasedwithinflammationintransplanted patients(Lockeetal.,2009),anditwasalsorecentlyshownthat antibody-mediatedplateletdestructionbyhumanphagocytesis enhanced bytheinflammatory markerC-reactiveprotein (CRP) (Kapuretal.,2015).Boththeobservedthrombocytopeniaandthe highanti-HLAclassIantibodylevelsamongcasesmaythereforebe secondarytoanincreasedstateofinflammationcausedbyother factors.Evenso,theirpresencemaystillhaveaffectedfetoplacental growth.

Most reports on neonatal thrombocytopenia and low birth weight are based onthe assumption that thrombocytopenia is secondarytoreduced fetalgrowthand/orplacentalfailure.It is noteworthythatthepossibilitythatthecause–effectmaybethe otherwayaround hasnotbeen considered.Therefore, itis not possibletoassess or ruleout therole of maternalalloantibod- iesinassociationwithneonatalthrombocytopeniainfetalgrowth restrictionbasedonpreviousstudies.Theobservedassociationin thisstudybetweenincreasinganti-HLAclassIantibodyleveland decreasingbirthweightandplacental weightisinteresting and supportsthehypothesisthatmaternalanti-HLAclassIantibod-

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iesmightaffectfetalgrowth,eitherdirectlyorindirectlyviafetal thrombocytopenia.Futureresearchincludingplacentalstudiesis warrantedtotestthesehypotheses.

Anti-HLAclassIantibodiesaredirectedagainstantigenswitha highdegreeofpolymorphism.Thedistributionofantibodyspeci- ﬁcitiesinthisstudymaythereforehavedifferedbetweencasesand controls,thuscontributingtothedifferencesobserved.Additional analysisofantibodyspeciﬁcitieswouldclarifythis.

Thefrequencyofpreeclampsiaamongcaseswasunexpectedly highat13%.Afterremovalofallpreeclampticpregnanciesfrom ouranalysesthedescribedresultsdidnotchange,supportingthe theorythatotherfactorsinthecasegroup(i.e.,anti-HLAclass1 antibodiesor fetal thrombocytopenia)were mainlyresponsible forthelower birthweight.Thehighfrequencyof preeclampsia amongHLAclassI-immunizedpregnanciesinrelationtoneona- talthrombocytopeniaisstillinteresting.Anincreasedfrequencyof preeclampsiaamongmothershavingpreterminfantswiththrom- bocytopenia was previously reported (Beiner et al., 2003), and apossibleconnectionbetweenthematernalproductionofanti- bodies duringpregnancy and thedevelopmentof preeclampsia hasbeensuggested(Buurmaetal.,2012).Anexacerbatedmater- nalinflammatoryresponseisfoundinpreeclampticpregnancies (RedmanandSargent,2004),andinflammationhasfurtherbeen shown toincreasethebreadthand strength ofanti-HLA classI antibodies(Lockeetal.,2009).Therewasnoassociationbetween anti-HLAclassIantibodiesandpreeclampsiaamongcontrols,but thedifferentselection strategies ofthe studygroups mayhave influencedthis.Thepossiblelinkbetweenmaternalanti-HLAclass Iantibodiesandpreeclampsiashouldbefurtherstudied.

AsHLAantibodiesarecommonandneonatalthrombocytopenia occursrelativelyfrequently,itisdifﬁculttodrawadeﬁniteconclu- sionbasedonthisobservationalstudy.However,thehypotheses generatedfromourstudydeservetobefurtherevaluated.

5. Conclusions

Maternalanti-HLAclassIantibodiesareassociatedwithreduced birthweightin neonateswithsuspectedFNAIT.Furtherstudies areneededtodisclosewhethermaternalanti-HLAclassIantibod- iesoccurringduringpregnancycanaffectfetalgrowth,oriftheir presenceissimplyanepiphenomenon.

Conﬂictofinterest

Wehavenoconﬂictsofinterest.

Acknowledgements

TheauthorswishtothankKristineTollefsenatUniversityHos- pitalNorthNorway andEirinListauBertelsenatUiT, theArctic UniversityofNorway,for helpwithlaboratoryanalysis.Weare alsogratefultoÅseVårtunatUniversityHospitalNorthNorwayfor assistancewithprovidingplasmasamplesfromthecontrolgroup.

FundingwasobtainedfromtheNorthernNorwayRegionalHealth Authority.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,athttp://dx.doi.org/10.1016/j.jri.2015.10.003.

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