w w w . r b h h . o r g
Revista
Brasileira
de
Hematologia
e
Hemoterapia
Brazilian
Journal
of
Hematology
and
Hemotherapy
Original
article
Evaluation
of
hemoglobin
performance
in
the
assessment
of
iron
stores
in
feto-maternal
pairs
in
a
high-risk
population:
receiver
operating
characteristic
curve
analysis
José
Carlos
Jaime-Pérez
∗,
Gisela
García-Arellano,
Nereida
Méndez-Ramírez,
Óscar
González-Llano,
David
Gómez-Almaguer
UniversidadAutónomadeNuevoLeón,Monterrey,Mexico
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received12November2014 Accepted15December2014 Availableonline17February2015
Keywords: Ferritins
Anemia,Iron-deficiency Diagnostictests,Routine
a
b
s
t
r
a
c
t
Objective:Byapplyingreceiveroperatingcharacteristiccurveanalysis,theobjectiveofthis studywastoseewhetherhemoglobinlevelsreflectbodyironstoresinagroupofpregnant womenattermwho,byusingserumferritinasthereferencetest,hadahighpre-test prob-abilityofhavingirondeficiencyanemia.Likewise,weevaluatedtheabilityofhemoglobin andmaternalserumferritinlevelstopredictirondeficiencyanemiainnewborns. Methods:Hemoglobinandserumferritinweremeasuredin187pregnantwomenatterm belongingtoagroupwithahighpre-testprobabilityofirondeficiencyanemiaandtheir newborns.WomenwithHb<11.0g/dLandnewbornswithcordHb<13.0g/dLwereclassified asanemic.Aserumferritin<12.0g/Linwomenandacordbloodserumferritin<35.0g/L
wereconsideredtoreflectemptyironstores.Receiveroperatingcharacteristiccurveanalysis wasappliedtoselectthecut-offpointsthatbetterreflectedironstores.
Results:TheHbcut-offpointselectedbyreceiveroperatingcharacteristiccurveanalysisin womenwas<11.5g/dL(sensitivity:60.82,specificity:53.33%,YoudenIndex:0.450).Mostof thenewbornshadnormalHbwhichprecludedthisanalysis.MaternalHb<11.0g/dLwas thecut-offpointthatbestreflectedirondeficiencyanemiainnewborns(sensitivity:55.88%, specificity:57.24%,YoudenIndex:0.217).Thebestcut-offpointofmaternalserumferritinto reflectemptyironstoresinnewbornswas<6.0g/L(sensitivity:76.47%,specificity:31.58%,
YoudenIndex:0.200).
Conclusion:Hemoglobinconcentrationperformedpoorlytodetectirondeficiencyanemiain womenattermwithhighriskforirondeficiencyandtheirnewborns.
©2015Associac¸ãoBrasileiradeHematologia,HemoterapiaeTerapiaCelular.Published byElsevierEditoraLtda.Allrightsreserved.
∗ Correspondingauthorat:AvenidaMaderoyGonzalitos,S/N,EdificioDr.RodrigoBarragán,2◦piso,ColoniaMitrasCentro,64460Monterrey,
NuevoLeón,Mexico.
E-mailaddress:carjaime@hotmail.com(J.C.Jaime-Pérez).
http://dx.doi.org/10.1016/j.bjhh.2015.02.002
Introduction
Receiver operating characteristic (ROC) curve analysis is usedtoevaluatethediagnosticperformanceofatest.1 The ROCcurveprovidesaccurateinformationaboutthetrade-off betweenthe twomostimportantaspectsofdiagnostic per-formance:sensitivity(theprobability that atest resultwill bepositivewhenthediseaseispresent)andspecificity(the probabilitythatatestresultwillbenegativewhenthe dis-easeisnotpresent).1Inaddition,theareaundertheROCplot canbeexpressedasaquantitativeindex,A(z),that summa-rizesthediagnosticperformancedescribedbytheentireROC curve.ThevalueoftheA(z)indexvariesfrom0.5(noapparent accuracy,theROCcurvewillcoincidewiththediagonal)to1.0 (perfectaccuracy).2
Iron-deficiency (ID) anemia is the most common nutri-tionaldisorder inthe world.At least2billion people, that is,over30%oftheworld’spopulationareanemic,manydue toID.3 Theprevalence ofIDanemia isremarkably high in pregnantwomen,becauseoftheincreaseofironrequirements duringthesecondandthirdtrimesterswiththeexpansionof maternalbloodvolumeandfetalredcellmass.4Thisdisease particularlyaffectspregnantwomenandinfantsindeveloping countries.5Althoughthereisstrongevidencedemonstrating thattheacquisitionofironbythefetusislimitedbythe pres-enceofIDinthemother,6numerousstudieshaveshownthat thefetuscanaccumulateenoughirondespitemildto moder-atematernalID.7
The most commonly used test to screen for ID is the hemoglobin(Hb)concentration,whichreflectstheamountof functionalironinthebody.Althoughitisalateindicatorof ID,thelevelofHbisessentialforthediagnosisofIDanemia.8 TheCentersforDiseaseControlandPrevention(CDC)define gestationalanemiaduringthethirdtrimesterofpregnancyas aHblevel<11.0g/dLatsealevel9andalevelofserumferritin (SF)<12.0mg/Lisconsideredtobethegoldstandardforthe diagnosisofIDinpregnantwomen.9
Iron is transferred to the fetus through the placenta againstaconcentrationgradient,particularlyduringthelater stages of pregnancy, with the amount ofiron transported across the placenta increasing as pregnancy progresses.4 Several factors influence the concentration ofHb atbirth, including gestational age, gender, state of maternal iron reserves,andother factorsaffectingthematernal-fetaliron exchange.
Intermpregnancies,70–80%offetalironispresentinthe redcellsasHb.4 Anemiainnewbornsisestablishedwitha cordbloodHbconcentration<13.0g/dL.5Thecutoffcordblood serumferritin(CSF)indicatingirondeficiencyinthenewborn issetat<35.0mg/Latwhichpointtheironavailabletomeet normalbrainrequirements isrestricted.4 SF is,inaddition tothe directexaminationofstainableironinbone-marrow smears,themostspecificindicatorofbodyironreserves.10By usingSFasthereferencetestandapplyingROCcurveanalysis, theobjectiveofthisstudywastodeterminewhetherHblevels reflectthebodyironstoresinagroupofpregnantwomenat termwithahighpre-testprobabilityofhavingID.Likewise, theusefulnessofmaternalHbandSFlevelstopredictIDin newbornswasevaluated.
Methods
One hundred and eighty-seven pregnant women at term ofnormalpregnanciesand theirnewbornsattended atthe Hospital Universitario Dr.José E. Gonzálezofthe Universi-dadAutónomadeNuevoLeón,Monterrey,AC,Mexico,were enrolledinthisstudy.Informedconsentwasobtainedfrom allmothersandtheprinciplesoftheDeclarationofHelsinki werefollowed.Allofthewomenhadasinglepregnancy,were uninsuredandbelongedtothelow-incomeurbanpopulation ofMonterrey,Mexico,(altitude:524metersabovesea level). Womenwithcomorbidities,includingdiabetesandinfections, wereexcluded.
Samples of blood for Hb and SF measurement were obtainedfrom themotherand thenewborn’s cord.TheHb concentration was determined inan automated blood cell counter.Womenwithavaluelowerthan11.0g/dLwere clas-sified as anemic.7 Newborns withcordHb value<13.0g/dL were also classified as anemic.5 After being separated by centrifugation,serumwasstoredat−20◦CandtheSF
con-centrationwasmeasured induplicatewithintwoweeksof collection byanenzyme-linkedimmunosorbentassay (Fer-rizyme, Abbott Laboratories,North Chicago, IL) basedon a previously described modification of a method.11 A value lower than 12.0mg/L of SF is considered to reflect empty ironstores inpregnant womenatterm;12 innewborns the cutoff for CSF of severe ID is <35.0mg/L.4 No other red bloodcellindexorironsaturation dataareincludedinthis report.
ThestatisticalevaluationwasperformedusingROCcurve analysis1,2 employing MedCalcStatistical Software (version 14.8.1).Thediagnosticpropertiesofthetestswerecalculated basedonthepredictivevaluemodel.13,14
Results
Atotalof77(41.4%)pregnantwomenwereanemicatterm(Hb <11.0g/dL)and171(91.9%)wereID(SF<12.0mg/L).Themean Hb concentration in the study group was 11.11±1.57g/dL (range: 6.50–14.90g/dL), whereas the mean SF value was 5.78±7.20mg/L(range:0.0–50.0mg/L).
Figure1showsthegraphgeneratedbyROCanalysis.An Hbconcentration<11.5g/dLwasfoundtobethebestcutoff point toreflectemptyironstores basedonaferritin value <12.0mg/L.ThesensitivityoftheHblevelatthisvaluewas 60.82%withaspecificityof53.33%.ThediagnosticpowerofHb, expressedastheareaundertheROCcurve(AUC),was0.656, witha95% confidenceintervalof0.583–0.7244.TheYouden indexofHbconcentrationtoreflectcorporalironstoresinthe studygroup,asevaluatedbySF,was0.450.Thecorresponding valuesfordifferentROC-derivedHbcutoffvaluesareshown inTable1.
Table1–Performanceevaluationfordifferentreceiveroperatingcharacteristiccurve-derivedhemoglobincutoffvaluesin pregnantwomenatterm.
Maternalhemoglobincutoffvalue(g/dL)
<10.8 <11.0 <11.3 <11.5a <11.7 <12.0 <14.9
Sensitivity(%) 42.7 47.4 55.6 60.8 67.8 73.1 100.0
Specificity(%) 100.0 80.0 66.7 53.3 46.7 33.3 0
Positivepredictivevalue(%) 100.0 96.4 95.0 93.7 93.5 92.6 91.9
Negativepredictivevalue(%) 13.3 11.8 11.6 10.7 11.3 9.8
Positivelikelihoodratio 2.4 1.7 1.3 1.3 1.1 1.0
Negativelikelihoodratio 0.6 0.7 0.7 0.7 0.7 0.8
a OptimaloperatingpointchosenbyROCanalysis.
100
80
60
40
20
0
0 20 40 60 80 100
100-specificity
Sensitivity
AUC: 0.656
Figure1–Receiveroperatingcharacteristic(ROC)curve analysisofhemoglobin(Hb)toassessironstoresin pregnantwomenatterm(areaundertheROCcurve [AUC])=0.656.
ROCanalysistoassessitsefficiencyinrespecttoneonateiron stores.
AROCcurveanalysiswascarried outtoassess the per-formance ofmaternalHb in theassessment ofironstores innewbornsbasedonCSF(<35.0mg/L).Theresultinggraph shows an AUC of 0.591 with a 95% confidence interval of 0.517–0.662(Figure 2).Theoptimalcutoffpointofmaternal HbthatbestreflectedIDinnewbornswas<11.0g/dL,atthis pointthesensitivitywas55.88%withaspecificityof57.24%. TheYoudenindexforthistestwas0.200.Again,itis impor-tanttoconsiderthatonly3.2%ofneonateswereanemic.The correspondingvaluesobtainedbyROCanalysisfordifferent cutoffpointsofmaternalHbareshowninTable2.
ROCcurveanalysiswasappliedtoassesstheperformance ofmaternalSFinreflectingironstoresinnewbornsbasedon avalueofCSF<35.0mg/L;agraphwithanAUCof0.600anda 95%confidenceintervalof0.525–0.671wasobtained(Figure3). ThebestcutoffpointofmaternalSFchosentoreflectempty ironstoresinthenewbornwas<6.0mg/L,withasensitivityof 76.47%andaspecificityof31.58%.Thelackofefficiencywas confirmedbyaYoudenIndexof0.217.Differentcutoffpoints wereusedandthecorrespondinganalysisisshowninTable3.
100
80
60
40
20
0
0 20 40 60 80 100
100-specificity
Sensitivity
AUC: 0.591
Figure2–Receiveroperatingcharacteristic(ROC)curve analysisofmaternalhemoglobin(Hb)toassessironstores innewborns(areaundertheROCcurve[AUC])=0.591.
100
80
60
40
20
0
0 20 40 60 80 100
100-specificity
Sensitivity
AUC: 0.600
Table2–Performanceevaluationfordifferentreceiveroperatingcharacteristiccurve-derivedmaternalhemoglobincutoff valuesforirondeficiencyinnewborns.
Maternalhemoglobincutoffvalue(g/dL)
<9.0 <10.0 <11.0a <12.0 <13.0
Sensitivity(%) 20.6 35.3 55.9 76.5 88.2
Specificity(%) 93.4 82.2 57.2 28.3 9.2
Positivepredictivevalue(%) 41.2 30.8 22.6 19.3 17.9
Negativepredictivevalue(%) 84.0 85.0 85.3 84.3 77.8
Positivelikelihoodratio 3.1 2.0 1.3 1.1 1.0
Negativelikelihoodratio 0.9 0.8 0.8 0.8 1.3
a OptimaloperatingpointchosenbyROCanalysis.
Table3–Performanceevaluationfordifferentreceiveroperatingcharacteristiccurve-derivedmaternalserumferritin cutoffvaluesforirondeficiencyinnewborns.
Maternalserumferritincutoffvalue(mg/L)
<6.0a <8.0 <9.2 <10.0 <11.1 <12.5 <13.9
Sensitivity(%) 76.5 88.2 91.2 94.1 100.0 100.0 100.0
Specificity(%) 31.6 19.1 17.1 13.2 12.5 9.2 8.6
Positivepredictivevalue(%) 20.0 19.6 19.7 19.5 20.4 19.8 19.7
Negativepredictivevalue(%) 85.7 87.9 89.7 90.9 100.0 100.0 100.0
Positivelikelihoodratio 1.1 1.1 1.1 1.1 1.1 1.1 1.1
Negativelikelihoodratio 0.8 0.6 0.5 0.5 0 0 0
a OptimaloperatingpointchosenbyROCanalysis.
Discussion
Although its diagnostic properties are not ideal, SF was selectedasthereferencetestinthisstudytoevaluatebody ironstoresinpregnantwomenattermandtheirnewborns. Thisisthesecondmostaccurateassayaftertheexamination ofstainableironinthebonemarrow, andtheonlymethod thatcanprovideasemi-quantitativeindicationofthelevels ofsuchstores;itsdiagnosticefficiencyis91%andspecificity is100%iniron-repleteindividuals.12
Eventhoughmeasurementssuchaspositiveornegative predictivevaluesarehighlydependentontheprevalenceof adiseaseinthestudiedpopulation,thesensitivityand speci-ficityofatestareindependentofprevalence,thusallowingthe drawingofvalidconclusionsoftestperformancewhenusing ROCcurveanalysis,suchasinthepresentstudy.
Hblevelswerestudiedinagroupofwomenattermwitha highpre-testprobabilityofhavingemptyironstoresinorder todescribethebehaviorofHblevelsunderdemanding condi-tionsandtoassesswhetherSFshouldberoutinelyevaluated toassessironstatusinthisdisadvantagedgroup.
Highprevalences of anemia and ID were found in this low-income,uninsuredurbanpopulationofpregnantwomen in Mexico. The Hb level selected by ROC curve analysis (11.5g/dL) washigher than the 11.0g/dL recommended for womenduringthethirdtrimesterofpregnancy.9Atthislevel, thesensitivityofhemoglobintodetectemptyironstores com-paredtoSFwaslow(60.82%),but remarkablysimilartothe sensitivityof61%offerritinitself(<12.0mg/L)whencompared tostainableironinbonemarrowsmearsfromwomenof repro-ductiveage.Atthisvaluehowever,SFspecificityreached100% forthecorrectdiagnosisofIDinwomenofreproductiveage.10 MaternalHbspecificitytodetectemptyironstoresinpregnant
womeninthe presentstudy was53.33%, leadingtoapoor overallefficiencyindiagnosingIDasexpressedbytheYouden indexof0.450.
ofSFand Hbwasconductedduringearlypregnancy,when theincidenceofgestationalanemia islowest,representing bothendsofthegestationalIDspectrum.Thecurrentstudy consistedofuninsured women attermbelonging toalow incomepopulation and athigh risk ofbeing iron-depleted attendedinapublichospital.Thiscouldhelptoexplainthe significantcontrastbetweenpreviousstudiesandthecurrent findingsregardingtheusefulnessofHbtoassessironstores.
Inthenewborngroup,thecomparisonofHbandCSFusing ROCcurveanalysistoassessIDwasprecludedbecausealmost 97%ofnewbornshadnormalHb.Intheanalysisconductedto determinetheusefulnessofmaternalHbtoassessironstores innewborns,thecutoffpointrecommendedforwomeninthe thirdtrimesterofpregnancy(11.0g/dL),7lackedsensitivityand specificity(55.88%and57.24%respectively).TheAUC(0.591) demonstratedlowdiagnosticefficacyofHbasanindicatorof storedironinneonatesofwomenwithahighprevalenceofID. Despitethe high prevalenceofmaternalanemia andID withoutanemia,only3.2%ofneonateswereanemic,whereas 18.3%hadID(CSF<35.0mg/L).Althoughithasbeenshown that maternal iron stores strongly influence (although not exclusively)ironstoresinnewborns,6 from ourresultsand those ofothers5,19 it can beconcludedthat the fetus uses availableironinapreferentialwaytosupporterythropoiesis, despiteaninadequatematernalironsupplyandemptyiron stores.Thisisconsistentwithdataobtainedfromastudy car-ried out in China5 inwhich, asin the current population, anemiaand ID were common inpregnant womenatterm whilethepercentagesofnewbornswithbothwereverylow.
WhenanalyzingtheefficiencyofmaternalSFtoassessiron stores innewborns, the resultsdid notshow that the test performedwellbecausetheoptimalcut-offpointof mater-nalSFidentifiedusing ROCanalysis(6.0mg/L)isunderthe goldstandardforthediagnosisofirondeficiencyinpregnant women(SF<12.0mg/L).Theseresultsarenotconsistentwith otherstudies,whichconcludethatironstoresinneonatesare associatedwithmaternalonesonlyifmaternalironstatusis atrisk.5OurresultsmaysupportthetheoryofKilbrideetal.19 whosuggestedthatthehematologiceffectsofempty mater-nalironstoresaremoreevidentlaterinchildhoodthanduring thenewbornperiod.Theanalysiscarriedoutinour popula-tionfoundnocorrelationbetweenHbandmaternalSFwith ironstoresinnewborns.OthermarkersofIDininfantshave beenstudied,suchasreticulocytehemoglobincontent(CHr), whichisconsidered anearlymarkerofIDduetotheshort lifespanofreticulocytes(about24h),andunlikeothermarkers ofID,including SF,it hastheadvantageofnotbeing influ-encedbychronicinflammationprocessesandacuteinfection. Thistesthasbeenshowntobeasensitiveeffectivemarkerto evaluateironstoresinnewbornsatterm,20aswellinpreterm newborns.21
Itisimportanttonoticehowever,thatincontrasttothe simplicityandlowday-to-dayvariationofHbconcentration measurement,theSFassayhasarelativelyhigherinherent methodological variability, witha 5%generalcoefficient of variation,whichcanincreaseto15%forthesameindividual overaperiodofweeks.22Incomparison,serumiron determi-nationofthesamesubjecthasaday-to-dayoverallcoefficient ofvariationof30%,asaconsequenceofitshighphysiological variability.22
Insummary,thelowperformanceindicesforHb inthis study,particularlyalackofsensitivityandlowdiagnostic effi-ciency,confirmeditspoorvalueinreflectingbodyironstores whentheprobabilityofirondepletionishigh.23Thepresent reportalsoillustratesthe usefulnessofROC curveanalysis appliedtoacommonclinicallaboratorytestandhowitcan helpindefininganappropriaterangeofvaluesandaspecific cut-offpointforaparticularpopulation.
Conclusions
There wasapoorcorrelation betweenmaternalHb andSF withironstoresinneonates,despitethehighprevalenceof IDandanemiainwomenatterm.Duetofinancialandlogistic restrictionsindevelopingcountries,itisimportanttovalidate inexpensive,sensitivemarkerstoguideironprophylaxisor therapyduringpregnancy.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgment
TheauthorsgratefullyacknowledgeDr.NeriÁlvarezVillalobos forhisstatisticalanalysis.
r
e
f
e
r
e
n
c
e
s
1.DwyerAJ.InpursuitofapieceoftheROC.Radiology. 1997;201(3):621–5.
2.HanleyJA,McNeilBJ.Themeaninganduseoftheareaunder areceiveroperatingcharacteristic(ROC)curve.Radiology. 1982;143(1):29–36.
3.WHO.Micronutrientdeficiencies.Irondeficiencyanaemia. Availablefrom:http://www.who.int/nutrition/topics/ida/en/
4.SiddappaAM,RaoR,LongJD,WidnessJA,GeorgieffMK.The assessmentofnewbornironstoresatbirth:areviewofthe literatureandstandardsforferritinconcentrations. Neonatology.2007;92(2):73–82.
5.ShaoJ,LouJ,RaoR,GeorgieffMK,KacirotiN,FeltBT,etal. Maternalserumferritinconcentrationispositivelyassociated withnewbornironstoresinwomenwithlowferritinstatusin latepregnancy.JNutr.2012;142(11):2004–9.
6.Jaime-PérezJC,Herrera-GarzaJL,Gómez-AlmaguerD. Sub-optimalfetalironacquisitionunderaniron-deficient maternalenvironment.ArchMedRes.2005;36(5):598–602.
7.O’BrienKO,ZavaletaN,AbramsSA,CaulfieldLE.Maternal ironstatusinfluencesirontransfertothefetusduringthe thirdtrimesterofpregnancy.AmJClinNutr.
2003;77(4):924–30.
8.CentersforDiseaseControlandPrevention(CDC).
Recommendationstopreventandcontrolirondeficiencyin theUnitedStates.MorbMortalWklyRep.1998;47(RR-3):1–29.
9.CentersforDiseaseControlandPrevention(CDC).CDC criteriaforanemiainchildrenandchildbearing-agedwomen. MorbMortalWklyRep.1989;38(22):400–4.
determinationsinapopulationsampleofwomen.BrJ Haematol.1993;85(4):787–98.
11.LinpisarnS,KrickaLL,KennedyJH,WhiteheadTP.Sensitive sandwichenzymeimmunoassayforserumferritinon microtitreplates.AnnClinBiochem.1981;18Pt1:48–53.
12.WorwoodM.Ferritininhumantissuesandserum.Clin Haematol.1982;11(2):275–307.
13.GalenRS,GambinoSR.Beyondnormality.Thepredictive valueandefficiencyofmedicaldiagnosis.NewYork:Wiley; 1975.
14.SanfordJ.Understandinglaboratorytestresults.MedClin NorthAm.1987;71(4):639–52.
15.TiwariM,KotwalJ,KotwalA,MishraP,DuttaV,ChopraS. Correlationofhaemoglobinandredcellindiceswithserum ferritininIndianwomeninsecondandthirdtrimesterof pregnancy.MedJArmedForcesIndia.2013;69(1):31–6.
16.AsbergAE,MikkelsenG,AuneMW,AsbergA.Emptyiron storesinchildrenandyoungadults-thediagnosticaccuracy ofMCV,MCH,andMCHC.IntJLabHematol.2013;14:1–7.
17.CasanovaBF,SammelMD,MaconesGA.Developmentofa clinicalpredictionruleforirondeficiencyanemiain
pregnancy.AmJObstetGynecol.2005;193(2): 460–6.
18.HassanR,AbdullahWZ,NikHussainNH.Anemiaandiron statusofMalaywomenattendingandantenatalclinicin KubangKerian,Kelantan,Malaysia.SoutheastAsianJTrop MedPublicHealth.2005;36(5):1304–7.
19.KilbrideJ,BakerTG,ParapiaLA,KhourySA,ShuqaidefSW, JerwoodD.Anemiaduringpregnancyasriskfactorfor iron-deficiencyanemiaininfancy:acase–controlstudyin Jordan.IntJEpidemiol.1999;28(3):461–8.
20.TorsvikIK,MarkestadT,UelandPM,NilsenRM,MidttunO, BjorkeMonsenAL.Evaluatingironstatusandtheriskof anemiainyounginfantsusingerythrocyteparameters. PediatrRes.2013;73(2):214–20.
21.LorenzL,PeterA,PoetsCF,FranzAR.Areviewofcordblood concentrationsofironstatusparameterstodefinereference rangesforpreterminfants.Neonatology.2013;104(3):194–202.
22.WorwoodM.Thelaboratoryassessmentofironstatus–an update.ClinChimActa.1997;259(1-2):3–23.
