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Research ArticleDiagnosis of Severe Fetal Anemia Based on Perinatal Outcomes:A Comparative Analysis of the Current Reference Values

Zilma Silveira Nogueira Reis,1,2 Gabriel Costa Osanan,1 Tiago Lanfernini Ricardo Coelho,3

Cezar Alencar De Lima Rezende,1 Henrique Vitor Leite,1 and Antônio Carlos Vieira Cabral1

! Department of Gynecology and Obstetrics, Federal University of Minas Gerais, Brazil"Obstetrics and Gynaecology Department, Universidade Federal de Minas Gerais (UFMG), Avenida Professor Alfredo Balena,!#$, Funcionarios, Belo Horizonte, %$.!%$.!$$Minas Gerais, Brazil

% Federal University of Minas Gerais, Brazil

Correspondence should be addressed to Zilma Silveira Nogueira Reis; Zilma@medicina.ufmg.br

Received ' July !"#$; Revised !! September !"#$; Accepted !! September !"#$

Academic Editor: Bruno Annibale

Copyright © !"#$ Zilma Silveira Nogueira Reis et al. (is is an open access article distributed under the Creative CommonsAttribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.

Objectives. To compare current criteria for severe fetal anemia diagnosis.Methodology. A cohort study analyzed #"% alloimmunizedfetuses that underwent cordocentesis due to risk of anemia. Concordance among the diagnostic criteria for severe fetal anemia,hemoglobin de)cit >* g/dL, hemoglobin de)cit !% g/dL, and hemoglobin concentration <".%%MoM, was analyzed using Cohen’sKappa index. Perinatal mortality, fetal hydrops, and fetal acidosis were used to discuss discordances. Results. (ere was fairconcordance among the three criteria analyzed: ".&" (Kappa index, IC +%%: ".'* to ".+$) when comparing hemoglobin de)cit>*." g/dL andhemoglobin concentration<".%%MoMcriteria, ".'$ (Kappa index, IC +%%: ".,* to ".'+) when comparing hemoglobinde)cit !%." g/dL and hemoglobin de)cit >*." g/dL reference, and ".** (Kappa index, IC +%%: ".', to ".+") when comparinghemoglobin de)cit!%." g/dL and hemoglobin concentration <".%%MoM standards. Eighteen cases were classi)ed di-erentlydepending on the criteria used.(e cut-o- point of hemoglobin de)cit !% g/dL was the best criterion to discriminate fetuses withpoor perinatal outcome in our study. Conclusions. Relevant discordances in classi)cation of severe fetal anemia were pointed out.Some criteria may underestimate the real gravity of fetal anemia.

1. IntroductionMaternal alloimmunization still a-ects a large number ofpregnancies, particularly in developing countries [#, !].(esepregnancies need speci)c follow-up at tertiary referral centersto carry out proper monitoring, in the view of a high risk ofperinatal morbidity and mortality [$, ,]. When severe fetalanemia is suspected by a noninvasive method, cordocentesisis necessary to assess fetal hemoglobin concentration andthen to determine the need of an intrauterine transfusion(IUT) [%, ']. In this context, perinatal outcome also willdepend on timely diagnosis and treatment of fetal anemia.For severely anemic fetuses the transfusion therapy is a life-saving procedure [*–+]. However, IUT carries risks for bothmother and fetuses. In this way, it is important to determinewhich fetus is anemic and so it will need an IUT [*–+].

In this high-risk context, assessment of the degree of fetalanemia is an essential strategy for managing these pregnan-cies [#"]. (ere are three main references for diagnosis andclassi)cation of fetal anemia. (e )rst one was proposed byNicolaides et al., published in #++& [##]. (ese criteria usefetal hemoglobin deviation or de)cit (mean hemoglobin forgestational age minus measured hemoglobin) as parameterfor determining the severity of anemia.(e authors consideras severely anemic fetuses those at high risk of hydropsthat would generally occur when the hemoglobin de)cit was>* g/dL.

(e second criterion was proposed by Bahado-Singhet al., published in #++& [#!].(ese authors also use the con-cept of hemoglobin de)cit as a parameter to classify the sever-ity of anemia. (ey de)ne severe anemia as a hemoglobin

! Anemia

de)cit !% g/dL, since the risk of fetal hydrops is o.en belowthis below this value [#$].

And )nally, the third criterion was described by Mari etal., published in !""".(is author uses a di-erent parameterfor diagnosis and classi)cation of fetal anemia, the multiplesof median (MoM) of hemoglobin levels (calculated by divid-ing the measured hemoglobin value by the expected value forgestational age). Mari et al. de)ne, as severely anemic fetuses,the presence of fetal hemoglobin concentration of less than".%%MoM at a blood cord sample [#"].(is cut-o- point wasalso based on the risk of fetal hydrops.

In this manner the purpose of this study was to make acomparative analysis of these three criteria used to diagnoseand classify fetal anemia, based on perinatal outcome offetuses followed and treated at a university referral center forRh alloimmunization in Brazil.

2. Patients and Methods

A cohort study analyzed #%# fetuses that underwent cordo-centesis due to suspected anemia. Fetal blood was sampledfrom January #+++ to December !""+, at the Fetal MedicineCenter at the Clinical Hospital of the Federal University ofMinas Gerais, a tertiary referral center for alloimmunizedpregnancies in Brazil. Clinical records were retrospectivelyaccessed.(e study was approved by UFMG Research EthicsCommittee.

Eligibility criteria for inclusion in the study were fetusessubmitted to cordocentesis due to the risk of anemia, accessto information in medical records such as fetal hemoglobinlevels before )rst cordocentesis; and reliable gestational age,con)rmed by the latest reliable menstruation date and by anultrasound examination performed at !" weeks of gestation.

During this ##-year period, $!+ pregnancies were referredto our FetalMedicine Center due to rhesus alloimmunization(Figure #). From this total, #%# (,%.+%) needed cordocentesisto con)rm fetal anemia and to evaluate the need of IUT. Fromthese #%# cases, ,' were excluded from this analysis due tounclear or insu/cient clinical records (," cases had unre-liable gestational age at )rst cordocentesis and six medicalrecords had no information about hemoglobin values at the)rst IUT).

Cordocentesis was indicated, between #+ and $, weeksof gestation, based on previous obstetric history, indirectCoombs test titer, presence of fetal hydrops, or alteredcardiofemoral index (CFI) and peak systolic velocity middlecerebral artery (MCA-PSV) [#$, #,]. Fetal hemoglobin con-centration was measured using the HemoCue, a photometrictechnique on drops of blood taken immediately before thetransfusion. In addition, the blood was sent for con)rmationof the hemoglobin concentration measured using conven-tional techniques and for determination of fetal gasometry.(e hemoglobin de)cit was calculated based on the di-er-ence between the hemoglobin expected for the gestationalage and that found in the puncture. IUT was performedwhen the fetal hemoglobin concentration de)cit was !% g/dL[#!].

Finally we analyzed concordance among the criteriaproposed by Nicolaides et al. [##], Bahado-Singh et al. [#!],

Total number of pregnant women referred from 1999 to 2009 (n = 329)

Pregnant women not subjected to

cordocentesis (n = 178)

Pregnant women subjected to

cordocentesis (n = 151)

Pregnant women excluded for lack of

data (n = 46)Pregnant women

included in the study (n = 105)

Anemic fetuses (n = 91) !(n = 14)

Nonanemic fetuses

F01234 #: Flow diagram of enrolled fetuses at risk of fetal anemiafrom Rh.

and Mari et al. [#"] (Table $), by means of diagnosis of severeanemia and prediction of perinatal outcome.

For the statistical descriptive analysis, variables werepresented using central tendency and dispersion measures,according to the nature of their frequency distribution andabsolute and relative frequencies.

In order to provide better understanding of the concor-dances and discordances among the diagnostic criteria ofanemia, the fetal hemoglobin levels were plotted on a graphsuperimposed on the reference criteria curves.(e Interrateragreement among the three criteria (for diagnosis of severeanemia) was obtained by Cohen’s Kappa index, with respec-tive con)dence intervals of +%%. For this purpose referencesfor fetal severe anemia diagnosis were compared.

Perinatal outcome parameters such as pH value and hyd-rops [#%] at )rst cordocentesis, perinatal mortality, and %thminute APGAR index were compared to the criteria. A.ercomparing the current standards, the cases were distrib-uted into three categories of agreement: concordant severeanaemia cases, concordant nonsevere anaemia cases, anddiscordant severe anaemia cases.

To detect di-erences in perinatal outcome among thegroups the nonparametrical Kruskal-Wallis and Pearson’sChi-square test were utilized. Statistical signi)cance wasde)ned as " < 0.05. Statistical analysis calculations were per-formed using MINITAB Release #,.#!." #+*!–!"", MinitabInc. so.ware.

Anemia $

T5674 #: Characteristics of alloimmunized pregnancies included in the study.

Gestational and perinatal characteristics # Values Variation or %Maternal age, years (mean ± SD) #"% 29.7 ± 5.3 #+ to ,$Parity (median, range) #"% , # to ##Anti-D antibodies (alone), #(%) ++ %, %,.%%Anti-D + anti-C antibodies, #(%) ++ $! $!.%%GA, weeks, at )rst cordocentesis (mean ± SD) #"% 27.9 ± 3.9 #+ to $,Fetal hemoglobin (g/dL), at )rst cordocentesis (mean ± SD) #"% 9.4 ± 3.4 !.! to #'.,Fetal severe anemia (>*." g/dL hemoglobin de)cit)!, #(%) #"% !, !!.+%Fetal severe anemia (!%." g/dL hemoglobin de)cit)!!, #(%) #"% ,# $+.#%Fetal severe anemia (hemoglobin < ".%%MoM!!!, #(%) #"% $" !&.'%Fetal pH, at )rst cordocentesis (median, range) #"# *.$% *."*–*.,+Fetal hydrops at )rst cordocentesis, #(%) #"% !' !,.&%Perinatal mortality, #(%) #"% !" #+.#%Apgar % (median, range) &+ + ! to #"!Nicolaides et al. [##], !!Bahado-Singh et al. [#!], and !!!Mari et al. [#"]; SD: standard deviation, GA: gestational age.

3. Results

Most of the pregnant women (',.&%, # = 68) arrived atthis university center during third trimester of gestation.(ehydrops incidence at )rst cordocentesis was !,.&% (# = 26).In these cases, &".'% (# = 122) of pregnant women showedan indirect Coombs test titre !# : ',. (e characterizationof the pregnancies evaluated in the study was presented inTable #. (e lack of a speci)c Rh immunoprophylaxis, atbirth and at miscarriage, was the major cause of maternalsensitization (&+.+%, # = 95).(e most prevalent erythrocyteantibody found in this study was the anti-D (&*%, # =86/99) (Table #). (e perinatal mortality rate found was#+.#% (# = 20). Severe fetal anemia frequency ranged from!!.+% to $+.#%, depending on the diagnosis criterion adopted(Table #).

(e fetal hemoglobin concentration values, according tothe respective gestational age, were plotted on a graph withthe reference standards (the three criteria) for fetal anemia(Figure !). Eighteen fetuses could be classi)ed as nonseverelyor severely anemic, depending on the criterion employed(hollow triangles).

Concordance on the diagnosis of severe anaemia was".&" (Kappa index, IC +%%: ".'*–".+$) when comparing thecriteria of Nicolaides et al. (hemoglobin de)cits >*." g/dL)and Mari et al. (hemoglobin concentration <".%%MoM) [##,#!], even though seven diagnoses were discordant.

Moderate concordance (".'$, Kappa index, IC +%%: ".,*–".'+) was observed when comparing Bahado-Singh et al.(hemoglobin de)cit !%." g/dL) [#!] and Nicolaides et al.(hemoglobin de)cit >*." g/dL) [##] criteria for severe anemia.However, #& diagnoses were discordant.

Finally, fair concordance was observed: ".** (Kappaindex, IC +%%: ".',–".+") comparing Bahado-Singh et al.(hemoglobin de)cit !%." g/dL) and Mari et al. (hemogl-obin concentration<".%%MoM) criteria. All the $" cases con-sidered severely anemic by Bahado-Singh et al. (hemoglobinde)cit !%." g/dL) [#!] criteria were also classi)ed as sev-erely anemic by Mari et al. (hemoglobin concentration

36343230282624222018

181716151413121110

987654321

Gestational age (weeks)

Foet

al ha

emog

lobi

n (g

/dL)

Discordant

Concordant in lack of severe anaemia

Concordant in severe anaemia

F01234 !: Fetal hemoglobin concentration values assessed at )rstcordocentesis, plotted under the severe anemia diagnosis threshold,according to the anemia di-erent criteria. Solid triangles: concor-dant cases. Hollow triangles: discordant cases.

<".%%MoM) [#"]. Once more, ## cases showed discordantclassi)cation.

Perinatal results were presented in Table !. (e out-come was characterized by high mortality (!"%) and highincidence of hydrops ($$.$%). Weight at birth ranged from$+" to $#&" g (median !#&" g). Only the hemoglobin de)cit!%." g/dL criterion for severe anemia [#!] could identify allcases with poor perinatal outcome.

As mentioned before, when comparing the current crite-ria for the diagnosis of severe anemia (two at each time), thecases could be distributed into three categories: the concor-dant severe anemia cases, the concordant nonsevere anemiacases, and the discordant severe anemia cases (Table !). (eAPGAR score and the pH values at cord blood sample (at )rstcordocentesis) were similar among these groups (Table !).However, the presence of hydrops and perinatal death werestatistically higher in the group of concordant severe anemia

, Anemia

T5674 !: Perinatal outcome, according to concordance in classifying anemia severity using the several criteria analyzed.

$ Concordantnonsevere anemia

casesDiscordant severeanemia cases

Concordant severeanemia cases "

hb ! %." g/dL de)cit [#!] versus hb < ".%%MoM [#"] (# = 64) (# = 11) (# = 30)Fetal pH8 #"# *.$+ (*.#"–*.,+) *.$$ (*.#+–*.,$) *.$% (*."*–*.,%) "."*'!Fetal hydrops+ #"% & (#!.%%) ! (!"%) #' (%#.'%) <".""#!!Perinatal death #"% % (*.&%) $ ($"%) #! ($&.*%) ".""#!!Apgar % &+ + (!–#") + (+-+) + (!–#") ".'"$!hb > *." g/dL de)cit [##] versus hb ! %." g/dL de)cit [#!] (# = 64) (# = 18) (# = 23)Fetal pH8 #"# *.$+ (*.#"–*.,+) *.$, (*.#+–*.,') *.$, (*."*–*.,%) "."'+!Fetal hydrops+ #"% & (#!.%%) ' ($$.$%) #! (%!.!%) ".""#!!Perinatal death #"% % (*.&%) % (!*.&%) #" (,$.%%) ".""#!!Apgar % &+ + (!–#") + (+-+) + (!–#") ".$#&!hb > *." g/dL de)cit [##] versus hb < ".%%MoM [#"] (# = 74) (# = 8) (# = 23)

Fetal pH8 #"# *.$& (*.#"–*.,+) *.$* (*.$"–*.,$) *.$, (*."*–*.,%) ".#*!!Fetal hydrops+ #"% #" (#$.%%) , (%"%) #! (%!.!%) <".""#!!Perinatal death #"% & (#".&%) ! (!%%) #" (,$.%%) ".""!!!Apgar % &+ + (!–#") + (+-+) + (!–#") ".%$+!Note: ##st cordocentesis, ! Kruskal-Wallis test, !!Chi-square test, hb: foetal hemoglobin value at )rst cordocentesis.

T5674 $: Criteria for diagnosis and classi)cation of fetal anemia.

Mild anemia Moderateanemia Severe anemia

Nicolaides et al. [##] Hemoglobin de)cit <! g/dL Hemoglobin de)cit !! g/dL to * g/dL Hemoglobin de)cit >* g/dLBahado-Singh et al. [#!] Hemoglobin de)cit !! g/dL to

less than % g/dL — Hemoglobin de)cit !% g/dLMari et al. [#"] Hemoglobin concentration from

".&, to ".'%MoMHemoglobin concentration from less

than ".'% to ".%%MoMHemoglobin concentration

less than ".%%MoM

cases than in the group of concordant nonsevere anemia(Table !).

In this study, the standard that uses a cut-o- point ofhemoglobin de)cit !% g/dL [#!] to de)ne severe fetal anemiawas the only criterion available to classify as severely anemicfetuses, all the cases that had poor perinatal outcome.

4. Discussion

(e absence of previous studies that compare current cri-teria for the diagnoses of severe fetal anemia coexists withan uncritical clinical use of them. (is study provides aretrospective analysis that compares these three standardsto the perinatal outcome of pregnancies complicated bymaternal alloimmunization at our service. We also highlightthe potential discrepancies among these three criteria whenclassifying fetuses as severely anemic. It is important toemphasize that this study was not addressed to evaluaterecommendations to indicate cordocentesis or to comparethe noninvasive methods to predict fetal anemia or evento analyze current protocols to manage Rh-alloimmunizedpregnancies [!, #,].

By pointing out concordances and discordances in clas-sifying the severe anaemia, this study may help to de)ne

protocol standards for the management of pregnancies com-plicated by maternal alloimmunization. It is important tonote that such a comparative approach was possible, justbecause it took place at a referral center for the care ofalloimmunized pregnancies. However, as all retrospectivestudies, some peculiar limitations were present in this anal-ysis. An important one referred to the changes (worldwide)in the protocol for handling these pregnancies, especiallywhen the MCA-PSV [#"] substituted the amniocentesis inthe prediction of fetal anemia. (is fact reduced drasticallythe utilization of invasive procedures for diagnosing fetalanemia. (e MCA-PSV measurement by ultrasound turnedsafer prediction of fetal anemia in these pregnancies and thenreduced the risk (including fetal death) related to unnecessarycordocentesis. Furthermore, the experience of our servicewith another noninvasive method, the CFI [&, +, #'], addeda new and important parameter to be used in associationwith theMCA-PSV to predict important fetal anemia. Finallythe present study faced another limitation related to theincompleteness of data (due to information not inserted intothe records) or even the loss of cases, leading to exclusions.

(e )rst study to propose a classi)cation for fetal anemiain a group of alloimmunized pregnancies was performed byNicolaides et al. [##]. To achieve it, the authors determined

Anemia %

a normal reference range of fetal hemoglobin, based on !#"samples of umbilical cord blood from conceptuses withoutanemia, undergoing prenatal diagnosis. Subsequently, theycompared the normal range of hemoglobin concentrationto the levels found in umbilical cord from alloimmunizedfetuses, in a way that they could evaluate severity of anemia.(us the authors de)ned severe anemia as the presence ofhemoglobin de)cit >* g/dL, since hydropic fetuses in theirstudy had a hemoglobin concentration * to #" g/dL below thenormal mean for gestational age. (is criterion is probablythe most traditional reference to de)ne and classify fetalanemia nowadays. However, in our study, some fetuses withmarkers of poor perinatal outcome were not classi)ed asseverely anemic by this reference.

(e second criterion studied was the one created byBahado-Singh et al. [#!]. (ese authors, based on the refer-ences established by Nicolaides et al. [##], decided to reducethe cut-o- for the diagnosis of severe anemia to a hemoglobinde)cit !% g/dL, in the attempt to increase sensitivity to detectseverely anemic cases, and so to ensure that the anemicfetuses would be treated before development of hydrops.Moreover, he classi)es severity of anemia in only two groups:mild and severe anemic fetuses. In the present study, allconceptuses that showed hydrops or died during the perinatalperiod were classi)ed as severely anemic by this criterion.

(e third and last criterion evaluated was de)ned byMariet al. [#"]. (is author, as Nicolaides et al. [##], determinedthe normal range of hemoglobin concentration from hisown population of fetuses [#", #,] and classi)ed fetal anemiainto mild, moderate, and severe anemia. Di-erently fromNicolaides et al. [##], Mari et al. [#"] found that hemoglobinconcentration increases exponentially with advancing ges-tation and so they decided to classify severity of anemiaby means of MoM (multiples of median) for haemoglobinconcentration (in order to adjust for the e-ect of gesta-tional age on the measurement). With this approach, theauthors demonstrated that fetuses with severe anemia hada hemoglobin value <".%%MoM for a given gestational age[#", #,]. In our analyses, unfortunately this de)nition of severefetal anemia also did not include all fetuses with signi)cantmarkers of poor perinatal outcome.

Evaluating the di-erent criteria of severe anemia diagno-sis, we found that there are important divergences capableof modifying perinatal outcome, especially regarding theoccurrence of fetal hydrops and perinatal mortality. On com-paring Bahado-Singh et al. [#!] and Mari et al. [#"] criteria,it was possible to observe ## divergent cases. Out of these,three cases ($"%) were classi)ed as having a severe form ofanemia by Bahado-Singh et al. [#!] but not by Mari et al.[#"], progressed to perinatal death. (e same discordancehappened with the other two cases (!"%) that were hydropicat the )rst cordocentesis and were not considered severelyanemic by Mari standards [#"]. Also on comparing Bahado-Singh et al. [#!] and Nicolaides et al. [##] standards, #& fetuseswere divergent in severe anemia classi)cation were observed.At this time, )ve cases (!*.&%) were classi)ed as havinga severe form of anemia by Bahado-Singh et al. [#!] butnot by Nicolaides et al. [##], progressed to perinatal death.Moreover other six ($$%) that were hydropic would not

considered severely anemic by Nicolaides et al. [##] cut-o-point.

Among the three recommendations, we believe thatde)ning severe anemia in the presence of hemoglobin de)cit!% g/dL [#!] could o-er more safety intervention by allowingearlier and more timely treatment for these conceptuses,despite its risk. It is necessary for other prospective clinicaltrials to con)rm our )ndings. In any event, we hope that thisstudy may contribute to a better management of pregnanciescomplicated by maternal alloimmunization.

Conflict of Interests

(ere is no con9ict of interests with any trademark or sof-tware.

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' Anemia

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