Description:
TheTechnochrom®FXIIIkitisareagentkitforthedeterminationofbloodcoagulationFactorXIII(FXIII)activityforuseinresearchtodetectinheritedoracquiredFXIIIdeficiencies,abnormalFXIIIwithdecreasedactivityandelevatedFXIIIlevel.InheritedFXIIIdeficiencyisarare,butseverebleedingdiathesiswithoccasionalwoundhealingimpairmentandinwomenwithhABItualabortion.AcquiredFXIIIdeficiencyduetoananti-FXIIIautoantibodyisalsoaveryseverehaemorrhagicdiathesis.ConsumptionofFXIIIinvariousdiseases(malignantdiseases,ChrondiseaseHenochSchoenleinpurpura,majorsurgery,etc…)usuallyresultsinmoderatedecreaseofFXIIIlevel.TheassaycanalsobeusedformonitoringFXIIIreplacementtherapy.WhenFactorXIII(FXIII)isactivateditistransformedintoanactivetransglutaminase(activeFXIII[FXIIIa]).ThedeterminationofFXIIIactivityisbasedonthemeasurementofammoniareleasedduringthetransglutaminasereaction.FXIIIpresentintheplasmasampleisactivatedbythrombinandCa2+.Fibrinformedfromfibrinogenbythrombinacceleratesthisreaction.Thepolymerizationoffibrinispreventedbyatetrapeptide.TheformedFXIIIathencross-linkstheaminesubstrateglycineethylester(GEE)totheglutamineresidueofspecificpeptidesubstratePI(1-12),andammoniaisreleased.IntheindicatorreactiontheamountofreleasedammoniaismonitoredinaglutamatedehydrogenasecatalyzedNADPH-dependentreaction.TheconsumptionofNADPHismeasuredspectrophotometricallybythedecreaseofabsorbanceat340nm.WithinatimewindowthedecreaseofabsorbanceisdirectlyproportionaltotheFXIIIactivity.
Advantages:
- Easyandfast(10minutes),one-stepreaction
- LimitedinterferencesduetouseofNADPHinsteadofNADH
- Ammoniaproducingreactionsaresubtractedfromtheresultsbyusingablank
- Sensitivity:0.6%
- Referencerange:69-143%
- Linearity:upto300%
- Canbeperformedwithautoanalyzersandspectrophotometersat340nm
- CalibrationwithCoagulationReferenceplasma,whichwascalibratedagainsttheWHOFXIIIstandard
KitComposition:
- 3x3FXIIIActivatorReagent
- 3x3FXIIIDetectionReagent
- 3x3FXIIINADPHSolution
- 3x1FXIIIInhibitorReagent
- 1x6StabilizerSolution
AssayPrinciple:
Background:
FactorXIII
FactorXIII,alsoknownasfibrinstabilizingfactor,isaheterodimer(FXIII-A2B2)composedoftwocatalyticA-subunitsandtwocarrierB-subunits.WiththrombincleavageoftheA-subunitfollowedbydissociationoftheB-subunitinthepresenceofcalcium,theactivesiteisexposedintheA-subunit.ActivatedFXIII(FXIIIa)isatransglutaminasethatlinksorligatesthefibrousscaffoldconsistingofstaggeredfibrinunits.Transglutaminaseenzymesmaybetheearliestclottingenzymesinevolution,but,invertebrateblood,conversionofFXIIItoFXIIIaistightlyregulated.FXIIIaalsocrosslinksinhibitorsoffibrinolysistofibrin,suchasalpha2-antiplasmin.FXIIIastabilizesthefibrinclot,andisessentialforaproperlyfunctioningfibrinmatrix.
FactorXIIIDeficiency
WhilestudieselucidatingtheroleofFXIIIinclottingbeganinthe1940s,itwasn’tuntil1966thatthefirstfamilywithcongenitalFXIIIdeficiencywasidentified.AsFXIIIisessentialforstabilizingthefibrinmatrix,thelossofitsactivitywouldresultinhemorrhage.FXIIIdeficiencyisaveryrarediseaseandmostcasesareduetomutationscausinglossofthecatalyticAsubunits,butthereareafewcasesthatresultindeficiencyduetoalackoftheBcarriersubunit.Congenitaldeficiencyisanautosomalrecessivedisorderwithanestimatedincidenceofaround1in2million.Althoughclottingmightbenormalandthusmanyofthecommonlyusedlaboratoryclottingtestsremainnormal(ie,prothrombintime[PT]andactivatedpartialthromboplastintime[aPTT]),ahemorrhagicconditionoccursbecauseofthelackofcross-linkingduringcoagulation.Theseverityofbleedingcanrangefromverymildtolife-threatening,andotherconditionscanresultaswell,suchashabitualabortioninwomen.CasesofacquiredFXIIIdeficiencyalsoexist,andalthoughalsorare,inhibitorsblockFXIIIactivitybyvariousmechanisms.Earlydiagnosisiscriticalastheonsetcanbesuddenandsevere.ThesepatientsrequirereplacementoftheFXIII.WhilereportsvaryontheFXIIIlevelsthatwillcausesymptoms,itisclearthatverylowFXIIIlevelsareespeciallyharmful.ThereisongoingworkonusingrecombinantFXIII,andatleast1productisapprovedforuseintheUnitedStates.ItshouldbenotedthatFXIIIhasawiderangeofproteintargets,suggestingadditionalimportantrolesinhealthanddisease.
FactorXIIIAssays
AssaysforFXIIIacoincidewiththeongoingdiscoverythatFXIIIplaysinthecoagulationsystem. Urea-basedassaysplayedaroleinunderstandingtheneedforthiscomponentinclotstabilization,asaformedclotwasdegradedincertainconcentrationsofureawithoutit.Theurea-solubilityassaysubsequentlywasusedtofindthefirstfamilywithFXIIIdeficiency.Asthepropertiesoftheproteinbecameclearer,assaysbasedonitsmechanismofassaywereemployed.Itwasrecognizedthatsmallamines,suchasglycineethylester,playedaroleinfibrinstabilization,andthatFXIIIawasspecifictowardsaminesubstrates.AmineincorporationthusbecameapowerfultoolinstudyingFXIIIaquantitation.WhenFXIIIaattachesglycineethylestertoaspecificpeptidesubstrate,ammoniaisreleased.Variousmethodshavebeenavailablefordeterminingammoniacontentandthishasbeenutilizedformanyyearsindifferentassays.Inthesecolorimetricassays,releasedammoniaismonitoredinanNAD(P)Hdependentglutamatedehydrogenase(GlDH)reaction.Thedecreaseinabsorbancemeasuredat340nmovertimeisproportionaltoFXIIIaactivity. NADHandNADPHabsorbat340nmwhereasNADandNADPdonot.TheuseofNADHorNADPHasacofactorinthereactionvariesdependingontheassay.SpontaneousbreakdownofNAD(P)Hcanoccurbutthiscanbecompensatedbyusingareagentblank(usingiodoacetamide).Inaddition,anycompoundspresentinthesamplethatreactwithNAD(P)Hundertheconditionsofthisassaycangivediscrepantresultsfordeterminationoftheammoniaconcentration.ThissidereactioncanbeasaresultofthepresenceofotherenzymesthatutilizeNADH;however,theuseofNADPHasacofactorcaneliminatesomeoftheseinterferences,asinthecaseoflactatedehydrogenase(LDH)whichcannotutilizeNADPH.WithuseofablankandinanNADPH-dependentreaction,thesensitivitycanbeaslowas0.6%.
