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formedbyanelectrochemiluminescencemethod(RocheDiagnostics®)Wehave tried todefine thecriticalplasmalevel whichcausesbiotininterferenceforeachimmunoassay.2MATERIAL ANDMETHODS21PreparationofsamplesWefollowedtheproceduredescribedbyLietal(9)withslightmodificationsBriefly,biotin(Sigma-Aldrich) wasdissolvedinaphosphate-buffered saline(PBS) solution freeofcalciumandmagnesiumtoprepareastocksolutionwith100 µg/mLThissolutionwas seriallydiluted withPBS to yield working solutions withdifferentbiotinconcentrations(10 µg/mL7500500025001250625and 3125ng/mL) with which we spiked the samples Weusedcommercialqualitycontrolsas samplescontainingatleast twodifferentanalyteconcentrations Samples were spiked with thedifferent workingbiotinsolutionsresultinginthefollowingfinalbiotinconcentrationsinthesamples3125625125250 500750and1000ng/mLAsreferenceweusedthesamecontrolsamplesspikedwithanequivalent volumeofPBSEach serumbiotindilution was testedin triplicate Atleast twolevelsofanalyteconcentration wereassayedtoassess theinfluenceofanalyteconcentrationon theinterferenceTable1showstheanalyteconcentrationineachpool22ImmunoassaysAnalytesweremeasuredonthecobase801(TSHfreethyroxinfreeT3FSHLHprolactinestradiolprogesteronetestosteronealpha-fetoproteincarcinoembrionicantigenCA15-3CA125prostate specific antigen freeprostate specific antigen vitaminB12 folate 25-hydroxyvitaminD)and cobas e411(parathyrine cortisolC-terminalcollagentypeIpeptidesCA19-9antigenHE4antithyroglobulin antibodies and antithyroperoxidase antibodies)Bothcompetitiveandsandwichimmunoassaysareelectrochemiluminescence-basedBriefly,ananalyteanalogueinthecompetitivedesignordetectionantibodiesin theimmunometricdesignislabeled witharuthenium chelate Streptavidin-coveredmagneticmicroparticlesbindtothebiotinylatedanalyte(orcaptureantibody)andattachtothesurfaceofanelectrodeduetomagneticforceFinally,anelectriccurrentwhichinducesachemiluminescentreactioninrutheniumisappliedbeingtheintensityofchemiluminescencerelatedtoanalyteconcentration23StatisticsStatisticalcomparisonbetweentheresultsobtainedbyanalyzingthebiotin-spikedsamplesandthereferencePBS-spikedsampleswasperformedusingMicrosoftExcelTherelativebiaswiththeinterferent-freespecimenwascalculatedusingtheequation100x(Ci-C0) /C0 whereCiis the concentration obtained when analyzing1INTRODUCTIONImmunoassaysare widelyused tomeasureahuge varietyofanalytesincludinghormonestumorandcardiacbiomarkers vitaminsordrugs Thehighly specificinteractionbetween streptavidinandbiotinhasbeenexploitedforthedevelopmentofrobustandsensitiveimmunoassaysbymanymanufacturers(1)BiotinisawatersolublevitaminalsoknownasvitaminB7 whichis foundin thenormaldiet(eggsporkcereals)and thatservesasacofactorforcarboxylationreactionsinvolvedincellularmetabolism(23)TheAdequateIntake(AI)levelinadultshasbeenestimatedin 30 µg/day(4)Biotindeficiencyisnotacommondisorder,butsupplementationisprescribedfrequentlyforindividualswithintestinalmalabsorptionpregnantwomenorpatientswithmetabolicdisorders(4-6)whichreceivedosesrangingfrom10to40mgperday(5-7)Morerecently,veryhighdosesofbiotin(300mgperday)havebeenproposedinsomeclinicalprotocolsaspartof the treatmentofmultiplesclerosisandotherdemyelinatingdisorders(78)Inaddition self-medication with supraphysiologicaldosesofbiotinhasemergedasawaytoreducehairlossorfortifyhairandnails(up 20mgperday)Beingnotconsideredasdrugbypatientsmanydonotmention that theyareusingit when theirdoctorasksthemabouttheirmedications(7)Exogenousbiotinhasbeenrecognizedasaninterferencefactorin streptavidin-biotinbasedimmunoassaysbecausebiotininthe samplecompetes withbiotinylatedreagents for thebinding siteson streptavidin(79) The firstcasein whichbiotininterference wasreportedwasamisdiagnosisofGrave´sdiseaseduetofalselylowTSHandhighfT4results(10)Asiswellknownbiotincancausefalselylowresultsinthesandwich(immunometric)assaysusedtomeasurelargemoleculesand falseincreasesin thecompetitiveassays(711)usedformeasuringsmallmoleculesThereforebiotininterferencecancauseelevatedfreeT3andfreeT4concentrationsandlowTSHlevelsaswellasspuriouslyhighcortisolemiaswithbluntedACTHconcentrationsSimilarly,elevatedconcentrationsof25OHvitaminDassociatedwithsuppressedPTHlevelshavebeendescribed(7) These factscanconfuse thecliniciansleading tomisdiagnosesandeventuallyinappropriate treatments(10-12) Typicaldietaryintakeofbiotinhasbeenreported tobeinsufficient toaffectthe streptavidin-biotin-basedimmunoassaysHowever, supplementscontaining10mgormoreofbiotinarecauseofspuriousresultsinseveralimmunoassays(13)Oraladministrationofbiotindosesof10mgresultedinpeakplasmaconcentrationsrangingfrom53ng/mL to141(14) whereas the thresholdmost frequentlydescribedascriticalis>50ng/mL(15)Thepurposeofourstudyistodeterminetheinfluenceofbiotininseveralendocrine tumormarker,and vitaminassaysper240ANALESRANFwwwanalesranfcomInterferenciasporbiotinaenvariosinmunoensayosdeelectroquimioluminiscenciaJoséLuisMartín-CalderónAnRealAcadFarmvol87nº3(2021)·pp239-246