Description:

ChromogenixCoatest®SP4FactorVIIIisachromogenicassaykitfortheinvitrodiagnosticphotometricdeterminationoffactorVIIIactivityincitratedplasma.

KitComposition:

Reagents,Packaging,Storage,StABIlity,andMicroplate/TestTubePreparation

1. S-2765™15.4mg+I-2581,1vial.Chromogenicsubstrate(N-a-Z-D-Arg-Gly-Arg-pNA),15.4mg,syntheticthrombininhibitor,0.4mg,andmannitoladdedasabulkingagent.Reconstitutewith12.0mLofsterilewaterorNCCLStypeIIwater,toobtainaconcentrationof2.7mmol/L.Stabilityafterreconstitution:3monthsat2-8°C.
2. FactorIXa+factorX2.7IU,4vials.LyophilizedbovinefactorsIXaandXwithbovinealbuminaddedasastabilizingagent.Reconstitutewith3.0mLofsterilewaterorNCCLStypeIIwater.Stabilityafterreconstitution:12hoursat2-8°C.Thesolutioncanbestoredfrozeninaliquotsat-20°C(oratlowertemperature)for3months.Donotrefreeze.
3. CaCl26ml,1vial.Calciumchloridesolution,0.025mol/LReadytouse.Openedvialisstable3monthsat2-8°C.
4. Buffer,stocksolution20mL,1vial.20mLconcentratedTrisbuffercontainingNaClandBSA.Characteristicsoftenfolddilutedbuffer:Tris0.05mol/L,pH7.3,10mg/LCiprofloxacinand1.0%BSA.Dilute1:10(1+9)withsterilewaterorNCCLStypeIIwater.Prepareanewbufferworkingsolutioneachday.Onceopenedthebuffer.
5. Phospholipid2mL,1vial.Mixtureofhighlypurifiedphospholipidsand10mg/LCiprofloxacin.Readytouse.Openedvialisstablefor3monthsat2-8°C.Shakegentlybeforeuse.Whenkeptat2-8°Cthesealedreagentsarestableuntiltheexpirydateprintedonthelabel.Contaminationbymicroorganismsshouldbeavoidedoncethevialsareopened.

MeasurementPrinciple:

Inthepresenceofcalciumandphospholipids,factorXisactivatedtofactorXabyfactorIXa.ThisgenerationisgreatlystimulatedbyfactorVIII,whichmaybeconsideredasacofactorinthisreaction.ByusingoptimalamountsofCa²⁺andphospholipidsandanexcessoffactorsIXaandX,therateofactivationoffactorXissolelydependentontheamountoffactorVIII.FactorXahydrolysesthechromogenicsubstrateS-2765™thusliberatingthechromophoricgroup,pNA.Thecoloristhenreadphotometricallyat405nm.ThegeneratedfactorXaandthustheintensityofcolorisproportionaltothefactorVIIIactivityinthesample.HydrolysisofS-2765™bythrombinformedispreventedbytheadditionofthesyntheticthrombininhibitor,I-2581,togetherwiththesubstrate.

Background:

FactorVIIIisanon-enzymaticplasmaproteinthatisessentialfornormalbloodcoagulation. ThedeficiencyoffactorVIIIactivityinhumansisassociatedwithacongenitalbleeding disorder,calledhemophiliaA,whichaffectsabout1in5000males.HemophiliaApatients aretreatedwithfactorVIIIconcentrateformaintenanceofnormalhemostasisbut regrettablyprophylactictreatmentisnotingeneraluseworldwide.Duringlateryears recombinantfactorVIIIhasbeenapprovedfortherapeuticuse,whichminimizestherisk ofviraltransmission.ThereisnowalsogrowingevidencethatelevatedfactorVIIIactivity isariskfactorforthrombosis.Hence,factorVIIIlevelsareofimportancetomeasurenot onlyfordiagnosingandmonitoringhemophiliaAbutalsoforthrombophiliainvestigations. Withtheadventofchromogenicsubstratetechnologyaccurateandsensitivemethodsare availableforqualitycontrolandfortheclinicalcoagulationlaboratory.Itisthepurposeof thismonographtopresentanoverviewofbiochemicalandclinicaldataonfactorVIIIand toprovidecomprehensiveinformationonmethodologicalaspectsandontheuseofthe Coamatic®andCoatest®FactorVIIIkits.

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