Received: 22.03.2009 Accepted: 16.05.2009J Gastrointestin Liver DisSeptember 2009 Vol.18 No 3, 345-352Address for correspondence: A Koulaouzidis MD, MRCP(UK) Centre for Liver and Digestive Disorders RoyalInfirmaryofEdinburgh Edinburgh,EH164SA,UK Email:akoulaouzidis@hotmail.com

Soluble Transferrin Receptors and Iron Deficiency, a Step beyond Ferritin. A Systematic Review Anastasios Koulaouzidis1, Elmuhtady Said2, Russell Cottier3, Athar A Saeed4

1)CentreforLiverandDigestiveDisorders,RoyalInfirmaryofEdinburgh,MedicalDayCase&EndoscopyUnit,Edinburgh;2)BarnsleyGeneralHospital,GawberRoad,Barnsley;3)WarringtonGeneralHospital,LovelyLane,Warrington,Cheshire;4)QueenElizabethHospital,Gateshead,Tyne&Wear,UK

AbstractIrondeficiencyisacommondisorder;itcanalsobethe

firstindicatorofsignificantgastrointestinalpathology.Totalironstoresareevaluatedbyferritinmeasurement,butthisisoftendifficult,ascoexistentdiseasecanobscureferritinresults.Bonemarrow (BM) examinationwas previouslyfelttobesuperiortoallknownserologicalmarkersofironstatus,butithasanumberofdisadvantages.Thevalidityofmeasurementofsolubletransferrinreceptors(sTfR)asasurrogatemarkerofBMironstoreshasbeenthesubjectofvarious studies so far. Aim:TocriticallyreviewtheuseofsTfRasamarkerfortheevaluationofironstores.Methods: A systematiccomputerisedliteraturesearch,inordertoidentifystudiesthatcomparedsTfRmeasurementagainstBMironstain. Results:Twentyprospectivestudieswereidentified,ofwhichninefulfilledtheinclusioncriteria(sTfRmeasurementinanaemicadultsalongsideBMironstaining).Foratotalof979patients,adifferentsTfRreferencerangewasused,butlevelsof2.5mg/L(29.5nmol/L)wereconsistentlyusedas the threshold for iron-deficiency anaemia resulting ingood specificity and sensitivity.Conclusion:The use ofsTfR improves the clinical diagnosis of iron deficiencyanaemia,especially in thepresenceofcoexistingchronicdiseaseorgastrointestinalmalignancies.Thesafetyandcost-effectivenessofaferritin/sTfR-basedapproachtoexcludegastrointestinal cancer in the presenceof irondeficiencyhas to be provenwith a prospective,well standardised,multicentre trial or a meta-analysis.

KeywordsIron deficiency – bonemarrow– soluble transferrin

receptors–ferritin–endoscopy.

IntroductionIron deficiency (ID) is a commondisorder, affecting

almost 1.2 billion peopleworldwide [1]. In 1985, theWorldHealthOrganizationreportedthat15%to20%oftheworld’spopulationhadirondeficiencyanaemia(IDA)[2].Indevelopedcountriesmore than500millionpeopleareirondeficient[3-5].Irondeficiencyanemiaisaparticularlyimportantproblemformenandpostmenopausalwomen,asitcanbethefirstpresentingfeatureofanoccultgastrointestinalmalignancy[6-8].

It is important to remember that formost cliniciansID is associatedwith anaemia; the truth is that it is acontinuousprocessevolvinginthreestages.Thefirstphaseisthedepletionofstorageiron(stageI),wheretotalbodyironisdecreasedbuthaemoglobin(Hb)synthesisandredcellindicesremainunaffected.Boththeseindexeschangewhen the supplyof iron to bonemarrow (BM)becomesproblematic(irondeficienterythropoiesis–IDE,orstageII).InstageIIItheironsupplyisinsufficienttomaintainanormalHbconcentration,andeventuallyIDAdevelops[9].

Evaluation of iron deficiencyEvaluationof irondeficiencyisperformedbyvarious

methods.Redcellindices[meancorpuscularvolume(MCV)andmeancorpuscularhaemoglobin(MCHC)]mayconfirmamicrocytic, hypochromic picture,with rerum ferritinoften low.Unfortunatelyboth these testsare less reliablewithcoexistentillness.Anaemiaofchronicdisease(ACD)is a syndrome associatedwith chronic inflammatory andautoimmune conditions, malignancies, or infections (acute orchronic),andcausesastateof‘functional’ID.Recently,hepcidin(aliver-producedpeptide)wasfoundtobealinkbetweentheimmunesystemandthemovementofirontoandfromthestoragedepots.Hepcidinbindstoferroportin,a transmembrane iron exporter present onmacrophages,enterocytesandhepatocytes.Ithasbeendemonstratedthatinvitrohepcidininducestheinternalizationanddegradationofferroportin.Byregulatingthenumberofironexporters,hepcidinisinvolvedinthecontrolofironuptakeandreleasefrom enterocytes ormacrophages. It is conceivable that

346 Koulaouzidis et al

processesthataffecttheseregulatorymechanismscanresultindefectiveironmobilisationwithoutdeficiencyintheironstoresperse[10].

Thetwoentities(IDA&ACD)cancoexistandsothedetectionofIDinthepresenceofchronicdiseasecanbeanimportant diagnostic challenge.Efficientmanagement ofIDA requires, on most occasions, not only administration of iron therapy to replenish ironstores,butalso invasiveinvestigations (i.e. endoscopies) to find and treat an underlyingcause[11-12].Endoscopiesareknowntohavepotential life-threatening complications [13], whereasinappropriate administration of iron in ACD may aggravate theunderlyingdisease.

TheBritish Society ofGastroenterology guidelinesrecommend confirmation of ID before embarking onendoscopic tests [12].Measurements of peripheral bloodindices(i.e.MCV,MCHandferritin)canproveinadequatein certain patient groups because of known confounders(infection, inflammation or cancer).Haemoglobin (Hb)determination,themostwidespreadscreeningmethodforID,hasseriouslimitationswhenusedastheonlylaboratorymarkerforirondeficiencybecauseofitslowspecificityandsensitivity.ThisisbecauseanyindividualmustloosealargeportionoftotalbodyironforironstorestodecreaseandtheHbtofallbelownormallevels[14].

Despite that,whenhereditary anaemias are excluded,inthemajorityofcasesthepresenceofhypochromiaandmicrocytosisinthefaceoflowferritinmakesthediagnosisofIDfairlystraightforward[15].Thedifficultyisininterpretinglowmarkersinisolation,particularlywhentheHbisnormal[16].TheworkofJacobs,WaltersandBentleyestablishedserumferritinasanaccurateindicatorofstorageiron[17-21].NumerousstudiessincehavedemonstratedthesuperiorityofferritinoverothermeasurementsinidentifyingID,andthusferritinhasbeenthemostusefullaboratorymarkeroverthepast30years[15].

Intheirreviewof55studies,Guyattetal.reportedthatthemeanareaofthereceiver-operatorcharacteristic(ROC)curvesforferritinwas0.95±0.1,whilethatofMCVwasonly0.76[22].Aferritinlevelof≤12μg/Lisconsideredahighlyspecific indicatorof ID(98%specificity).Thesensitivityforthisisonly25%[23,24],andcanbeimprovedto92%ifacut-offferritinlevelof30μg/LisusedforIDdiagnosis,thusresultinginapositivepredictivevalue(PPV)of92%[24,25].Howeveritisknownthatferritinisanacutephasereactant and can be elevated irrespective of iron status in patientswithconcurrentchronicinflammatoryorinfectiousdiseases,recurrentacuteinfectionsandmalignancies[26].

The ‘gold standard’The examination of bonemarrow (BM) aspirate,

stainedwithPrussianblue for iron, is still considered asthebestmethodforevaluatingironstatusinpatientswithindeterminatelaboratoryfindings.Itsdrawbacksarethatitisinvasive,expensive,andthatresultsareoperatordependant.Barronetal.studiedacohortof108consecutiveunselected

patientsinwhomtheBMexaminationswerereportedasirondeficient,andfoundthatreportsofabsentBMhaemosiderinmaybeinaccurateinmorethan30%ofcases–evenwhenaccurate,thismaynotnecessarilysignifythepresenceofID[27].Measurementofserumferritinlevelsisstillrequiredtoconfirmaclinicaldiagnosis.Furthermore,upto35%ofBMaspiratemaybeinadequateforinterpretation[20].Inasmallretrospectivestudy,Gantietal.concludedthatthePPVofabsentBMironstoresforthediagnosisofIDAwasonly50%[28].AnotherrecentstudyshowedthatpatientswhohadstainableironintheirBMhadinfact‘functional’ID[29].

The invasivenatureofBMaspirationandbiopsyhasmadeittemptingtoidentifyaperipheralbloodtestwhichcouldactassurrogatemarkerofBMironstores.

Witteetal.examinedacohortof97consecutiveanaemicpatients anddevised a graph relating serum ferritinwitherythrocytesedimentationrate(ESR);theysuggestedthatitcouldallowaccurateconfirmationorexclusionofID[30].Twoyearslater,theyreportedthatatwo-dimensionallineargraphicrelationshipbetweenferritinandESRprovidedaPPVof97%,thusaidingtheconfirmationorexclusionofIDincommunityhospitalpractice[31].Othershaveproposedthataferritinlevelof70μg/LwasthenecessarysafetylimitforexclusionofIDA[32].Guyattetal.showedthat40%ofpatientswithferritin levelsbetween18μg/Land100μg/LhadIDonBMexamination[33].

There are twomain categories of laboratory tests foridentifying ID.Screeningmeasurements (Hb, transferrinsaturation,MCV,MCH,zincprotoporphyrinandreticulocytesHb) that detect iron deficient erythropoiesis (IDE) bydemonstratingeitherareducedsupplyofserumironorpoorhaemoglobinationofredcellsanddefinitivemeasurementsthatevaluatetissueironstatusbymeasuringproteinsderivedfrom either the iron store compartment inmacrophages(ferritin), or the iron utilization compartment in red cellprecursors[34].Ofnote,redcelldistributionwidth(RDW)usually precedes the othermarkers of IDE [35]. Solubletransferrinreceptors(sTfR)candetectstagesIIandIIIofID,andareconsideredasa‘dual’index.

Soluble transferrin receptorsThe first description of the presence of transferrin

receptors(TfR)on thesurfaceof reticulocyteswasmadein1963[36];theywerefurthercharacterisedascellsurfaceglycoproteinsin1981[37].TheoveralldynamicsofthesereceptorswasstudiedusingthemouseerythroleukemiaK562cellline[38].Wardetal.foundthatinthesecells,TfRmRNAsynthesiswasincreasedwheniron-deficientserum,oriron-chelatingagentswereaddedtotheculturemedium[39,40].Itwaslaterproposedthat,theup-regulationofreceptorsmustbemediated(atleastinpart)bythetransferrinlevels.

Thetransferrinreceptormediatescellularuptakeofironbybindingtheironcarrier-proteintransferrin(Tf).Followinginternalisation of the iron-transferrin-TfR complex, ironis released from its binding sites in the acidicmilieu of

Soluble transferrin receptors and iron deficiency 347

the endosomes (acidosomes), and theTf-TfRcomplex isreturnedbacktothecellsurface,whereapo-transferrinisreleasedagain[41].

The transferrin receptor iscomprisedof two identicalsubunits,eachcomposedof760amino-acidswithamolecularmassof95kDaltons.Eachpolypeptidesubunitcontainsatrans-membranesegmentwith21aminoacidresidues,anN-terminalcytoplasmicdomainwith61residues,andalargeC-terminalextracellulardomainwith671aminoacids.ThesolubleformofTfRinserumwasrecognisedsince1986[42].Kohgoetal.describeditsuseasasurrogatemarkerofBMerythropoiesisandredcellproduction[43].

Humansuse80%oftheirbodyironforerythropoiesis,andalmostthesameproportionofTfRinthebodyisfoundin erythroid progenitor cells.Reticulocytes entering theperipheralbloodstreamcarryahighsurfaceconcentrationofthereceptor;asthecellsmaturethereceptorsareshedinto thecirculation[44].ReleaseofTfRhasbeenshownto be mediated by an integral membrane proteinase, and inhibited by amatrixmetalloproteinase and theTNF-Nproteaseinhibitor-2[41].

As the idea behindWitte’swork remained attractive,serumorsolubleTfR(sTfR)wasthenexttobescrutinisedwith regard to thevalidityof its use as amarker ofBMiron.WecriticallyreviewedtheexistingevidenceinordertoevaluateifsTfRmeasurementoranycalculatedvalues(suchasthesTfR/Log10ferritinratioorsTfR–Findex)couldstandasasurrogatemarkerofBMiron.

Review criteriaA comprehensive computerized literature search of

English-languagestudieswasperformedusingthe[Mesh]terms “receptors, transferrin” and ‘bonemarrow’’ in theMEDLINEdatabasewithlimitsinhumans,intheEnglishLanguage,andovertheageof19years.WecheckedMedline1966–May2007andwetrackedappropriatereferences.Atotalof31referenceswerefound.Twelvemorereferenceswere tracked from amanual review of related articles.Fromthetotal43articles,twentyrelevantreferences(sTfRmeasurementandBMironstaining)wereidentified;nineofwhichfulfilledtheinclusioncriteriaandwereincludedin our analysis.

Inclusion Criteria:Studieswereincludedonlyiftheysatisfiedthefollowingcriteria:

1. Examined anaemic adult patientswho had sTfRmeasurements documented.

2. Bonemarrow aspiration and iron stainwas thereference standard for detection of iron status.

Insomestudies,onlyasubgroupofpatientsfulfilledtheinclusioncriteria.Therefore,includedstudiesoughttohaveatleast50%ofpatientssubjectedtoBMexamination.

Exclusion criteria: letters, comments, and articles withoutoriginaldataandconferenceabstractswereexcluded.Wealsoexcluded2studiesbecauserecruitedpatientshadmalignantlymphomasandmyelodysplasticsyndromes[45,46].Haematologicalmalignanciesareknown to interfere

withthelevelofsTfRandqualifyasexclusioncriteriaformostoftheotherstudies.TwomorestudieswerenotincludedinourfinalanalysisastheycontainedpreliminaryresultsreportedlaterinalargerstudybyPunnonenetal[47-49].

Assessment ofmethodological quality: the followingcriteriawere used to assessmethodological quality [22](TableI).

Table I.QualityassessmentofthestudiesevaluatedStudy (year) Population Intervention Out-

comesTotalscore

Punnonen et al. 1997 3 2 1 6

Rimon et al. 2002 3 2 1 6

Means et al. 1999 2 2 2 6

Hanifetal.2005 2 2 1 5

Joosten et al. 2002 2 2 1 5

Junca et al. 1998 1 2 2 5

Lee et al. 2002 2 2 1 5

Suominen et al. 2000 1 2 1 4

Fitzsimonsetal.2002 1 2 1 4

Qualityassessment No.studies(%)

Population

Ideal 2 (22.2)

Best 4 (44.5)

Acceptable 3 (33.3)

Intervention

Ideal 9 (100)

Acceptable - (0)

Outcomemeasures

Ideal - (0)

Best 2 (22.2)

Acceptable 7 (77.8)

A. PopulationIdeal - prospective study, consecutive anaemic patients

(with explicit definition of anaemia), exclusion criteria;markedas3

Best - prospective study, not consecutive butwithexclusioncriteria;markedas2

Acceptable-anyotherprospectivestudy;markedas1B.InterventionIdeal-specifiedmethodoftesting(i.e.howlaboratory

testsweredone);markedas3Acceptable-anythingelse;markedas1C.Outcome–BonemarrowIdeal-BMexaminedby2ormoreblinded(toeachother

andthelabtests)readers;markedas3Best-BMexaminedby1personblindedtothelabtests,

or2readersnotblinded;markedas2Acceptable-anythingelse;markedas1Data Extraction:Datawas extracted independently

fromeachreport(byAKandES)usingapredefinedreviewform, anddisagreementwas resolvedby consensus.The

348 Koulaouzidis et al

extractorswereawareofthesiteoforiginofpublication,journal and year of publication.The following datawasrecordedfromeacharticle:authorandyearofpublication,sample size, number of anaemic patients, number of bone marrowexaminationsperformed,classificationofpatientsaccordingtobonemarrowironstatusandsensitivity,andspecificityof sTfRand/or sTfR–F index if thathadbeencalculated(TableII).

Studies breakdown and discussion

Punnonen et al (1997) studied 129 consecutive anaemic patients.AllunderwentBMaspirationtodefinethetypeofanaemiaanddetermineironstores[49].Allbloodsampleswereobtainedbeforeanybloodtransfusions,andpatientsonirontherapywereexcluded.Haematologicalmalignancies,haemolyticanaemias,B12orfolicaciddeficiencieswerealsoconsideredasexclusioncriteria[50-52].

Bonemarrowaspirationswereperformedfromsternaloriliaccrestsites.PatientswereassignedtothreegroupsaccordingtotheirBMiron.Forty-eightbelongedtothegroupofID(nostainableiron),64wereclassifiedashavingACD(stainable ironpresent),whilst 17withbothno stainableiron in theBMplus a concurrent inflammatory processor non-haematologicalmalignancy,were placed in thecombined(COMBI)anaemiagroup.MeasurementofsTfRwasperformedusingapolyclonalantibodyinasandwichEnzyme ImmunoAssay (EIA).The referencedistributionlevelswere0.85to3.5mg/L.Thecalculatedareasunderthereceiveroperatorcharacteristics(AUCROC)forsTfRwere0.98forthewholepatientpopulation.Thesamevaluewas

obtainedforthesubpopulationofuncomplicatedIDAandACDpatients,aswellastheonecontainingtheACDandCOMBIanaemias.The logarithmic transformationof theferritinvalues,andcalculationofthesTfR–FIndexprovidedan outstanding indicator of iron depletion (AUCROC:1.00).

TheefficiencycurvesfordistinctionbetweenIDAandACDsuggestcut-offlimitsforsTfRandforsTfR–Findexof2.7and1.5mg/Lrespectively.Suchacut-offlevelforsTfR(2.7mg/L)hadsensitivity,specificity,PPV,negativepredictive value (NPV) and efficiencyof 0.94 each.Theequivalent values for the sTfR–F indexwere 0.98, 1.00,1.00, 0.98, 0.99.

Ayearlater(1998),Juncàetal.reportedontheirstudyof37anaemicpatients(10ofthemhadhypoferritinaemiadue to chronic blood losses andwere used as controlsof the sTfR technique) with concurrent infectious orinflammatorypathology[53].Thosefromthegroup(n=27)withhyperferritinaemiaunderwentBMaspiration.TwelvewerefoundtohavelowBMironandfifteen(n=15)normalorincreasedBMironcontent.TheBMsmearswereevaluatedbytwopathologistsandmarrowirongradewasrecordedas0 (absent), + (reduced), ++ (normal) or +++ (increased).

They simplified the results into absent or low vs.normal or highBM iron. sTfRwasmeasuredwith animmunoenzymometricassayand the referencerangewas3.1– 4.5mg/L.Applyingmultivariate logistic regressionanalysis theyfoundthatsTfRwas thefirstvariable tobeexcludedfromthemodelasnon-significant,as itdidnotimprove its predictive value at any cut-off level. At a level of4.5mg/Lthesensitivitywasonly50%andthespecificity

Table II.ReviewresultsIDA ACD Combi

anaemiasTfR sTfR-FIndex

Name of study

IDA on BM

sTfRmean

ACD-total

sTfRmean

no sTfRmean

sTfRcut-off level

sens% spec% PPV% NPV% Indexlevel

sens% spec%

Punnonen 1997

48 6.2±3.5

64 1.8±0.6

17 5.1±2.0

2.7 94 94 94 94 1.5 98 100

Junca1998 n/a n/a 15 3.39±1.9

12 5.63±3.3

2.8 84 47 n/a n/a n/a n/a n/a

Means 1999 24 52.7±39.9*

121 23.7±12.3*

n/a n/a 71 74 n/a n/a n/a n/a n/a

Suominen 2000

19 2.92±0.76

11 1.81±0.48

n/a n/a 2.3 n/a n/a n/a n/a 13.5 n/a n/a

Joosten 2002

27 34±11 38 25.5±10

7 n/a 28 68 61 n/a n/a n/a n/a n/a

Rimon 2002

49 n/a 14 n/a n/a n/a n/a n/a n/a n/a n/a 1.5 88 93

Lee 2002 31 n/a 48 n/a 41 n/a 1.8 97 88 n/a n/a 1.36 100 98

Fitzsimons2002

6 n/a 12 23.9* 15 40.2* 28.1* 93 92 n/a n/a n/a n/a n/a

Hanif2005 86 9.68±2.48

90 2.96±1.28

n/a n/a n/a 83.3 100 100 87 n/a n/a n/a

IDA:irondeficiencyanaemia,sTfR:solubleTransferrinreceptor,ACD:anaemiaofchronicdisease,Combianaemia:ACD+IDA,no:number,sens:sensitivity,spec:specificity,PPV:positivepredictivevalue,NPV:negativepredictivevalue,*sTfRlevelsinnmo/L(sTfRlevelsfortherestexpressedinmg/L)

Soluble transferrin receptors and iron deficiency 349

74%.ThisstudyinvolvedasmallnumberofpatientsandthesTfR–Findexwasnotestimated.

Ayearlater(1999),Meansetal.studiedthepredictivevalueofsTfRfortheBMironresultsinaheterogeneousgroup of patients [54]. Thiswas a large, three-centrestudythatincluded145anaemicpatientsundergoingBMexamination for diagnostic purposes (noBMaspirationwasperformedsolelyforstudypurposes).Theinvestigatorsexaminedthestainedmarrowsmearsinablindedmanner,i.e.withoutanyinformationaboutthepatientsandreportedtheBMironcontentinasemi-quantitativescale.Subjectsreceiving iron or erythropoietin therapywere excluded.Of the 216 individuals recruited, 71were excluded forvariousreasons[15wereexcludedlaterduetoconcomitanttreatmentwithiron,somehadBMbiopsyforcancerstagingorhaematologicalmalignancies(leukaemia/myeloma),11hadB12deficiency or other haemolytic states,while 45patientshadunsatisfactoryoruninterruptibleBMsamples].The remaining 145 entered the final analysis, includingsomewithmalignanciesandliverdisease(n=87).SolubleTfRwasmeasuredbyenzyme-linkedimmunosorbentassay(ELISA)andthereferencerangewas8.8–28.1nmol/Lforwhiteand10.6–29.9nmol/Lforcolouredsubjects.MeansTfRconcentrationforpatientswithstainableironontheBMaspiratewas23.7±12.3nmol/L(mean±SD),whilethevalueforthosewithabsentBMironwere52.7±39.9nmol/L.Meansetal.testedasequentialalgorithm(predictorofBMiron)forbothferritinaloneandferritin/sTfRincombination.Theferritinalgorithmcorrectlyidentified42%ofthepatientswithabsentBMiron(sensitivity)and70%ofthosewithBMstainableforiron(specificity).

The authors found that sTfRwas the only laboratorytestused (amongst serum iron,Tf,Tf saturation, ferritin,MCV)whichshowedmeanvaluesinthenormalrangeforpatientswithdetectableBMironandmeanvaluesoutsidethenormalrangeinthosewithoutdetectableBMiron.Despitethis,thesTfRassayinthisstudyfailedtoidentify29%ofthe patientswith absentBM iron.The authors explainedthis tobe inherent to thestudydesignasonly7%of therecruitedsubjectsunderwentBMexaminationprimarilyfortheevaluationofanaemia.

Suominen et al. (2000) studied a group of 30 anaemic patientswithrheumatoidarthritis(RA)andperformedBMexaminationinallofthem[55].TheinvestigatorsdeterminedbothsTfRandsTfR–FindexandaccordingtotheirprotocoltheysupplementedpatientswithdiminishedorexhaustedBMstoreswithiron.InonemorestudyfromTurku,Finland,itwasreportedthatansTfRlevelof2.3mg/LandansTfR–Findex of 1.35was themost efficient in discriminatingbetweenabsentandrepleteBMironstores.

In2002,fourmorestudiesonthatsubjectwerepublished.Joosten et al. prospectively studied elderly hospitalisedpatients(meanage83years)inordertocheckthevalidityofsTfRindiscriminatingIDAfromACD[56].Afterexcludingcasesofhaematologicalmalignancies,haemolyticanaemia,B12 or folate deficiency,moderate renal failure, recentsurgeryortherapywithiron/transfusion,theyrecruited83

anaemicpatientswhoallunderwentBMaspiration.Thirty-fourhadnostainableBMironandwereclassifiedasIDA;38hadironrepleteironstoresfromeitheracuteinfectionsor chronic inflammatory conditions, andwere classifiedasACD;11were excluded from further analysis as theyhad normalBM iron and no evidence of chronic/acuteinfection,inflammationormalignancy.Theirresultsshowedthatatalevelof2.8mg/LforthedetectionofID,thesTfRmeasurementhadasensitivityof68%andspecificityof61%.The authors concluded that sTfRmeasurement providedresultssimilartothatofferritinintheirstudypopulation,partly because of the broader spectrum of coexistingunderlying diseases.

Rimon et al. also focused on elderly inpatients and studied a population of 49 anaemic and 14 non-anaemic individuals(n=63)[57],allofwhomhadBMexaminations.Aswellasmeasuringtheusualhaematologicalparametersand sTfRvalues, they also calculated the sTfR–F index.Thiswasawell-conducted,prospectivestudywithstringentinclusionandexclusioncriteriaandagoodstudysize.Themeanageofthecohortwas83years.UsingasTfR–Findexlevelof1.5theycameupwithaspecificityof92.9%andsensitivityof87.8%forthedetectionofIDA(PPVof97.7%andNPVof68.4%).

Fitzsimonsetal.(2002)examinedBMfrom29anaemicpatientswith rheumatoid arthritis (RA), 6 patientswithsimpleIDA,andhealthyvolunteers.Theyemployed125I-Tfand59Fe-TfinordertoassesstheinvitroerythroblastsurfaceTfR expression, and the uptake of high purityerythroblastfractionspreparedfromtheBMsamples.SerumTfRvaluesweremeasuredonlyfortheRAanaemiagroup,whichwas subdivided as eitherRA-ACD (marrow ironpresent)orRA-IDA(marrowironabsent),onthebasisofvisiblereticuloendothelialBMironstores.TheyconcludedthatsTfRlevelsinRA-ACDremainwithinthenormalrange.RAerythroblasts,howeverarestillabletorespondtoanyadditionalworseningof theironsupplycausedbyabsentreticuloendothelial iron stores.This additional responsecausesthehighlysignificantincreaseinserumsTfRvaluesseenbetweenRA-IDAandRA-ACD[58].

Laterthatyear,Leeetal.reportedtheirstudyoftheuseofsTfRandsTfR–Findexinanaemicpatientswithnon-haematologicalmalignanciesandchronicinflammation[59].Theauthorsrecruited120anaemicadults(meanage54years)whounderwentBMexaminationforanaemiaand81non-anaemiccontrols.Thegroupwasdividedinto4subgroupsaccordingtoBMresults:IDA(n=31),ACD(n=48),COMBIwithchronicinflammatorydisease(n=15)andCOMBIwithmalignancy(n=26).ExclusioncriteriaweresimilartothoseofpreviousstudiesandsTfRwasmeasuredwiththeuseofanimmunoturbidimetricmethod.AtansTfR–FIndexlevelof1.36,thesensitivityfordetectionofIDAwas100%andthespecificity98%,whilethesameparametersforasTfRlevelof1.8mg/Lwere97%and88%,respectively.

Hanif et al. (2005) conducted a studywherein 176patientswere subjected intoBMexamination [60].TheauthorsreportedexcellentPPVforthetest(sTfR)inboth

350 Koulaouzidis et al

IDAandACD,but theNPVfor the latter statewasonly74.1%.ThisstudyhadnoclearsTfRlevelusedfordefiningIDA,andthereportedPPVandNPVindifferentsubgroupsof IDA and ACD appear confusing.

Thestudiesweexaminedareheterogeneousas to thepopulationofrecruitedpatients(TableIII)andtheexclusioncriteriaused(TableIV).Mostof them,withfournotableexceptions [49, 54, 59, 60],were limitedby the numberofBMexaminationsperformed,andwererestrictedtoamaximumof3centres[54].Furthermorereferencerangesfor themeasurement of sTfR are blighted as there areseveralcommercialassaysavailable,andthetechniqueisnot standardized [61, 62].This leads to confusion in thetransferability of results fromone study to another, bothnationallyandinternationally;howeverthisproblemisnotuniquewithin theremitofdiagnostic testing.TheremustbeincreasedpressuretoestablishinternationalguidelinesforthecalibrationofsTfR,asfirsthighlightedbySkiknein1998[62].

Currently, there is noNationalQualityAssessmentScheme so it still remains impractical to compare acrossdifferent laboratory analyses,which is necessary if sTfRis tobeutilizedonawiderbasis.ThesTfRassay isalsoclaimedtobecost-prohibitiveincomparisontoestablished‘traditional’laboratoryindicesofironassessment,butwiththe continued proliferation of high-throughput analyzersandutilisatingmicro-samplingtechniques,costshavefallensignificantly (unpublished authors’ data).Of note is the

Table III.Populations,characteristicsStudy (year)

No Mean age (SD)

Anaemia cause

sTfRreferencevalues

Punnonen et al. 1997

129 n/a n/a 0.85-3.05‡

Junca et al. 1998

37 hypo-F:65 IDA, GI loss

3.1-4.5‡

hyper-F:65.7 ACD

Means et al. 1999

216 IDA: 43(12) IDA various

White:8.8-28.1*

ACD: 51(18) Black:10.6-29.9*

Suominen et al. 2000

30 range: 26 - 79

RA n/a

Joosten et al. 2002

83 83 various to 28.1*

Rimon et al. 2002

63 83(2.8) various 0.85-3.05‡

Lee et al. 2002

201 54 various n/a

Fitzsimonset al. 2002

44 IDA: 59.8(19.3)

IDA

RA

8.8-28.1*

ACD: 63.1

Hanif2etal. 005

176 n/a various 1.3-3.33‡

IDA:IronDeficiencyAnaemia;ACD:AnaemiaofChronicDisease;RA:RheumatoidArthritis;n/a:notapplicable;hypo-F:hypoferritinaemic;hyper-F:hyperferritinaemic;GIloss:gastrointestinalbloodloss;sTfRreferencevalues:*nmol/L, ‡ mg/L

Table IV.ExclusioncriteriaperstudyStudy (year)

Exclusioncriteria

Typeofexclusioncriteria

Punnonen et al. 1997

yes Oralirontherapy,haematologicalmalignancies,haemolyticanaemia,B12/folatedeficiency

Junca et al. 1998

no n/a

Means et al. 1999

yes Ironand/orerythropoietintherapy,haemolysis&B12deficiency

Suominen et al. 2000

n/a n/a

Joosten et al. 2002

yes Haematologicalmalignancies,haemolysi,B12/folatedeficiency,creatinine>200μg/L,irontherapy,transfusionorsurgery

Rimon et al. 2002

yes Notconsented,irontherapy,B12/folatedeficiency,acuteGIbleed,malignancies,renal or liver failure

Lee et al. 2002

yes Haematologicalmalignancies,haemolysis,B12/folatedeficiency,irontherapy,bonemarrowhypercellularity

Fitzsimonset al. 2002

no n/a

Hanifetal.2005

yes Thallasaemia,sideroblasticanaemia

immunoturbidimetricmethod,whichguaranteesresultsin11 minutes.

STfRmeasurementsappeartohavehighsensitivity,butsufferfromlowspecificitywhenitcomestodifferentiatingirondeficiencyfromothercausesofincreasederythropoiesis(e.g. haemolytic states such as ineffective erythropoiesisfromB12/folatedeficiencyandthalassaemia).Thiscanbepartiallycorrectedbyincorporatingferritininthecalculatedindex.

OnthebasisofthisreviewweconcludethatifsTfRorthecalculatedsTfR–FindexistobeusedonabroaderscalefortheassessmentofBMironstores,aprospective,multicenterstudy is required.Sucha studywouldneed toutilize theexisting ‘gold standard’, i.e.BM iron stain, as a cross-referenceandapplyuniformexclusionandinclusioncriteria.AsTfRlevelof2.5mg/L(29.5nmol/L)appearsareasonablethresholdforidentificationofiron-deficiencyanaemia(withgoodspecificityandsensitivity)(Fig.1)[63].

With the integration of Clinical Chemistry andHaematologydepartments,theassessmentofironstatusisbeginningtoenjoyamoreunifiedapproach.TheuseofsTfRassTfR–Findexcanimprovethediagnosisofirondeficiencyanaemia,especially in thepresenceofcoexistingchronicdisease,butfurtherresearchisrequired[64,65].

ConclusionThe use of sTfR improves the clinical diagnosis of

iron deficiency anaemia, especially in the presence ofcoexistingchronicdiseaseorgastrointestinalmalignancies.Thesafetyandcost-effectivenessofaferritin/sTfR-basedapproachtoexcludegastrointestinalcancerinthepresence

Soluble transferrin receptors and iron deficiency 351

ofirondeficiencyhastobeprovenwithaprospective,wellstandardised, multicentre trial or a meta-analysis.

AcknowledgementsWethankMageedAbdalla,MScforhisinvaluablehelp

withFigure1,AKaragiannidis,MDforhishelpwiththetables,andShivaramBhat,MBBCh&SarahDouglas,BScfortheirhelpwithproofreading.

References 1. GrosboisB,DecauxO,CadorB,CazaletsC,JegoP.Humaniron

deficiency.BullAcadNatlMed2005;189:1649-1663. 2. DeMaeyerE,Adiels-TegmanM.Theprevalenceofanaemiainthe

world.WorldHealthStatQ1985;38:302-316. 3. CookJD.Iron-deficiencyanaemia.BaillieresClinHaematol1994;

7: 787-804. 4. LookerAC,DallmanPR,CarrollMD,GunterEW, JohnsonCL.

PrevalenceofirondeficiencyintheUnitedStates.JAMA1997;277:973-976.

5. HercbergS,PreziosiP,GalanP.IrondeficiencyinEurope.PublicHealthNutr2001;4:537-545.

6. RockeyDC,Cello JP.Evaluation of the gastrointestinal tract inpatientswith iron-deficiency anemia.NEngl JMed1993; 329:1691-1695.

7. McIntyre AS, Long RG. Prospective survey of investigations in outpatients referredwith irondeficiency anaemia.Gut 1993; 34:1102-1107.

8. IoannouGN,RockeyDC,BrysonCL,WeissNS.Irondeficiencyandgastrointestinalmalignancy:apopulation-basedcohortstudy.AmJMed2002;113:276-280.

9. MetzgerothG,AdelbergerV,Dorn-BeinekeA, et al. Solubletransferrinreceptorandzincprotoporphyrin--competitorsorefficientpartners?EurJHaematol2005;75:309-317.

10. KemnaEH,TjalsmaH,WillemsHL, SwinkelsDW.Hepcidin:fromdiscoverytodifferentialdiagnosis.Haematologica2008;93:90-97.

11. Logan EC,Yates JM, Stewart RM, FieldingK, KendrickD.Investigation and management of iron deficiency anaemia in general practice: a cluster randomized controlled trial of a simple managementprompt.PostgradMedJ2002;78:533-537.

12. GoddardAF, JamesMW,McIntyreAS, et al.Guidelines for theManagement of IronDeficiencyAnaemia.BSG2005;ClinicalPractice –Guidelines.www.bsg.org.uk/pdf_word_docs/iron_def.

pdf(accessed28thFebruary2008) 13. GreenJ.GuidelinesonComplicationsofGastrointestinalEndoscopy.

BSG2006;ClinicalPractice –Guidelines.www.bsg.org.uk/pdf_word_docs/complications.pdf(accessed28thFebruary2008)

14. CookJD.Diagnosisandmanagementofiron-deficiencyanaemia.BestPractResClinHaematol2005;18:319-332.

15. BrugnaraC. Iron deficiency and erythropoiesis: newdiagnosticapproaches.ClinChem2003;49:1573-1578.

16. WrightCM,KellyJ,TrailA,ParkinsonKN,SummerfieldG.Thediagnosisofborderlineirondeficiency:resultsofatherapeutictrial.ArchDisChild2004;89:1028-1031.

17. JacobsA,WorwoodM.Theclinicaluseofserumferritinestimation.BrJHaematol1975;31:1-3.

18. WaltersGO,MillerFM,WorwoodM.Serumferritinconcentrationand iron stores in normal subjects. JClinPathol 1973; 26: 770-772.

19. BentleyDP,WilliamsP.Serumferritinconcentrationasanindexofstorageironinrheumatoidarthritis.JClinPathol1974;27:786-788.

20. KrauseJR,StolcV.Serumferritinandbonemarrowironstores.I.Correlationwithabsenceofironinbiopsyspecimens.AmJClinPathol1979;72:817-820.

21. MazzaJ,BarrRM,McDonaldJW,ValbergLS.Usefulnessoftheserumferritinconcentrationinthedetectionofirondeficiencyinageneralhospital.CanMedAssocJ1978;119:884-886.

22. GuyattGH,OxmanAD,AliM,WillanA,McIlroyW,PattersonC.Laboratorydiagnosisofiron-deficiencyanemia:anoverview.JGenInternMed1992;7:145-153.

23. AliMA,LuxtonAW,WalkerWH.Serumferritinconcentrationandbonemarrowironstores:aprospectivestudy.CanMedAssocJ1978;118: 945-946.

24. MastAE,BlinderMA,GronowskiAM,ChumleyC,ScottMG.Clinicalutilityofthesolubletransferringreceptorandcomparisonwithserumferritininseveralpopulations.ClinChem1998;44:45-51.

25. WeissG,GoodnoughLT.Anemiaofchronicdisease.NEnglJMed2005;352:1011-1023.

26. LipschitzDA,CookJD,FinchCA.Aclinicalevaluationofserumferritinasanindexofironstores.NEnglJMed1974;290:1213-1216.

27. BarronBA,HoyerJD,TefferiA.Abonemarrowreportofabsentstainableironisnotdiagnosticofirondeficiency.AnnHematol2001;80: 166-169.

28. GantiAK,MoazzamN,LaroiaS,TendulkarK,PottiA,MehdiSA.Predictivevalueofabsentbonemarrowironstoresintheclinicaldiagnosisofirondeficiencyanemia.InVivo2003;17:389-392.

29. ErvastiM,Kotisaari S,Romppanen J, PunnonenK. In patientswhohave stainable iron in the bonemarrowan elevated plasmatransferrinreceptorvaluemayreflectfunctionalirondeficiency.ClinLabHaematol2004;26:205-209.

30. WitteDL,KraemerDF,JohnsonGF,DickFR,HamiltonH.Predictionofbonemarrowironfindingsfromtestsperformedonperipheralblood.AmJClinPathol1986;85:202-206.

31. WitteDL,AngstadtDS,DavisSH,SchrantzRD.Predictingbonemarrowironstoresinanemicpatientsinacommunityhospitalusingferritinanderythrocytesedimentationrate.AmJClinPathol1988;90: 85-87.

32. CoenenJL,vanDieijen-VisserMP,vanPeltJ,etal.Measurementsofserumferritinusedtopredictconcentrationsofironinbonemarrowinanemiaofchronicdisease.ClinChem1991;37:560-563.

33. GuyattGH,PattersonC,AliM,etal.Diagnosisofiron-deficiencyanemiaintheelderly.AmJMed1990;88:205-209.

Fig 1.sTfRcut-offlevelandmeanwithitsconfidenceintervals.

352 Koulaouzidis et al

34. CookJD,FlowersCH,SkikneBS.Thequantitativeassessmentofbodyiron.Blood2003;101:3359-3364.

35. OskiFA.Irondeficiencyininfancyandchildhood.NEnglJMed1993;329:190-193.

36. JandlJH,KatzJH.Theplasma-to-cellcycleoftransferrin.JClinInvest1963;42:314-326.

37. TrowbridgeIS,OmaryMB.Humancellsurfaceglycoproteinrelatedtocellproliferationisthereceptorfortransferrin.ProcNatlAcadSciUSA1981;78:3039-3043.

38. vanRenswoudeJ,BridgesKR,HarfordJB,KlausnerRD.Receptor-mediatedendocytosisof transferrinandtheuptakeoffeinK562cells: identificationofanon-lysosomalacidiccompartment.ProcNatlAcadSciUSA1982;79:6186-6190.

39. WardJH,KushnerJP,RayFA,KaplanJ.Transferrinreceptorfunctioninhereditaryhemochromatosis. JLabClinMed1984;103:246-254.

40. KohgoY,NiitsuY,NishisatoT,etal.Externalizationoftransferrinreceptorinestablishedhumancelllines.CellBiolIntRep1987;11:871-879.

41. KaupM,DasslerK,WeiseC,FuchsH.Sheddingofthetransferrinreceptor is mediated constitutively by an integral membrane metalloprotease sensitive to tumornecrosis factor alphaproteaseinhibitor-2.JBiolChem2002;277:38494-38502.

42. KohgoY,NishisatoT,KondoH,TsushimaN,NiitsuY,UrushizakiI.Circulatingtransferrinreceptorinhumanserum.BrJHaematol1986;64:277-281.

43. KohgoY,NiitsuY,KondoH,etal.Serumtransferrinreceptorasanewindexoferythropoiesis.Blood1987;70:1955-1958.

44. ShihYJ,BaynesRD,HudsonBG,CookJD.Characterizationandquantitationofthecirculatingformsofserumtransferrinreceptorusingdomain-specificantibodies.Blood1993;81:234-238.

45. BjernerJ,AmlieLM,RustenLS,JakobsenE.Serumlevelsofsolubletransferrinreceptorcorrelatewithseverityofdiseasebutnotwithironstoresinpatientswithmalignantlymphomas.TumourBiol2002;23: 146-153.

46. MatsudaA,MisumiM,ShimadaT,etal.Solubletransferrinreceptoranditsratiotoerythroblastsinbonemarrowmaybeanewdiagnostictool to distinguish between aplastic and refractory anemia.ActaHaematol2004;111:138-142.

47. PunnonenK, IrjalaK,RajamäkiA. Iron-deficiency anemia isassociatedwithhighconcentrationoftransferrinreceptorinserum.ClinChem1994;40:774-776.

48. SuominenP, PunnonenK,RajamakiA, IrjalaK.Evaluation ofnewimmunoenzymometricassayformeasuringsolubletransferrinreceptor to detect irondeficiency in anemic patients.ClinChem1997;43:1641-1646.

49. PunnonenK,IrjalaK,RajamakiA.Serumtransferrinreceptoranditsratiotoserumferritininthediagnosisofirondeficiency.Blood1997;89:1052-1057.

50. HuebersHA, BeguinY, Pootrakul P, EinspahrD, Finch CA.Intact transferrin receptors inhumanplasmaand their relation toerythropoiesis.Blood1990;75:102-107.

51. CarmelR,SkikneBS.Serumtransferrinreceptorinthemegaloblasticanemiaof cobalamindeficiency.Eur JHaematol 1992; 49: 246-250.

52. BeguinY,LampertzS,DeGrooteD,IgotD,MalaiseM,FilletG.SolubleCD23andother receptors (CD4,CD8,CD25,CD71) inserumof patientswith chronic lymphocytic leukemia.Leukemia1993;7:2019-2025.

53. JuncàJ,Fernández-AvilésF,OriolA,etal.Theusefulnessoftheserumtransferrinreceptorindetectingirondeficiencyintheanemiaofchronicdisorders.Haematologica1998;83:676-680.

54. MeansRT Jr,Allen J, SearsDA, Schuster SJ. Serum solubletransferrinreceptorandthepredictionofmarrowaspirateironresultsinaheterogeneousgroupofpatients.ClinLabHaematol1999;21:161-167.

55. SuominenP,MottonenT,RajamakiA, IrjalaK.Singlevaluesofserumtransferrinreceptorandtransferrinreceptorferritinindexcanbeusedtodetecttrueandfunctionalirondeficiencyinrheumatoidarthritis patientswith anemia.ArthritisRheum2000; 43: 1016-1020.

56. JoostenE,VanLoonR,BillenJ,BlanckaertN,FabriR,PelemansW.Serumtransferrinreceptorintheevaluationoftheironstatusinelderlyhospitalizedpatientswithanemia.AmJHematol2002;69:1-6.

57. RimonE,LevyS,SapirA,etal.Diagnosisofirondeficiencyanemiaintheelderlybytransferrinreceptor-ferritinindex.ArchInternMed2002;162:445-449.

58. FitzsimonsEJ,HoustonT,MunroR,SturrockRD,SpeekenbrinkAB,Brock JH.Erythroblast ironmetabolism and serum solubletransferrin receptor values in the anemiaof rheumatoid arthritis.ArthritisRheum2002;47:166-171.

59. LeeEJ,OhEJ, ParkYJ,LeeHK,KimBK.Soluble transferrinreceptor (sTfR), ferritin, and sTfR/log ferritin index in anemicpatientswithnonhematologicmalignancyandchronicinflammation.ClinChem2002;48:1118-1121.

60. HanifE,AyyubM,AnwarM,AliW,BashirM.Evaluationofserumtransferrin receptor concentration in diagnosing and differentiating irondeficiencyanaemiafromanaemiaofchronicdisorders.JPakMedAssoc2005;55:13-16.

61. Kuiper-KramerEP,VanRaanJ,VanEijkHG.Anewassayforsolubletransferrinreceptorsinserum:timeforstandardisation.EurJClinChemClinBiochem1997;35:793.

62. SkikneBS.Circulatingtransferrinreceptorassay—comingofage.ClinChem1998;44:7-9.

63. KoulaouzidisA, SaeedAA,AbdallahM, SaidEM.Transferrinreceptorlevelassurrogateperipheralbloodmarkerofirondeficiencystates.ScandJGastroenterol2009;44:126-127.

64. KoulaouzidisA,Bhat S. Investigating iron status inmicrocyticanaemia:zincprotoporphyrinandsolubletransferrinreceptorhavearole.BMJ2006;333:972.

65. KoulaouzidisA,CottierR,BhatS,SaidE,LinakerBD,SaeedAA.A ferritin level >50microg/L is frequently consistentwith irondeficiency.EurJInternMed2009;20:168-170.