Description:

TheAR-chipwasspecificallydesignedforthequantitativeanalysisofthewhitethrombusformationprocessmediatedbytheactivationofbothplateletsandcoagulationsystemunderflowconditions.Developmentofwhitethrombiconsistingofactivatedplateletsandfibrincanbeanalyzedandvisualizedbymeasuringflowpressurewaveformsandvideo-microscopicinspection,respectively.

Imageanalysisofwhitethrombiformation

fibrinrichplateletthrombusformation

fibrinrichplateletthrombusformation(GPIbantagoNIST)

Characteristics:

CharacteristicsoftheAR-chipassay

• Usesatissuethromboplastin-andcollagen-coatedmicrochip
• Requiresonlyasmallamountofwholeblood(450μl)
• Simplesettingofoptionalwallshearrate(200-800s-1）
• Dual-monitoringsystem(byflowpressurewaveformandreal-timevideoimaging)
• Easyoperationcontrolledbythecomputerinterlockingsystem

MeasurementPrinciple:

ArecalicifiedbloodsamplecontainingCTI(corntrypsininhibitor)isplacedinareservoirthatisconnectedtoahigh-precisionmicropump.Themicropumpinfusesmineraloilintothereservoir,whichimpelsthesamplebloodthroughtheinletportandintotheflowpaththatleadstothetissuethromboplastin-andcollagen-coatedanalyticalpath.

Theprocessofwhitethrombusformation(WTF)intheflowchamberiscontinuouslymonitoredbyavideo-microscope(10x)positionedundertheflowchamberandbyapressuresensorthatrecordspressurechangesintheflowpath.Theflowrateofthemicropumpcanbeeasilyadjustedasneededbasedonthedesiredexperimentalconditions(range:4-40μl/min).

WhenbloodflowsthroughtheanalyticalpathoftheAR-chip,theextrinsiccoagulationpathwayandplateletsaresimultaneouslyactivatedonthetissuethromboplastin-andcollagen-coatedsurfaces.Theactivatedcoagulationsystemandplateletsthenamplifytheirrespsonsesresultinginthrombusgrowth,whichoccludesthecapillaryandincreasestheflowpressure.

Parameters:

Flowpressureanalysis

WhenbloodflowsthroughtheanalyticalpathofAR-chip,theplateletsandcoagulationsystemaresimultaneouslyactivatedonthecollagen-andtissuethromboplastin-coatedsurface.Smallwhitethrombithatconsistofactivatedplateletsandfibrinfibersgraduallyincreaseinsizeandeventuallyoccludethecapillary,resultinginanincreaseofflowpressure.Thus,flowpressurepatternsreflectthewhitethrombusformationprocess.

Definitionsoftheparametersusedforthequantificationofthewhitethrombusformationprocess

Threespecificparameters,T₁₀,T₈₀,andAUC₃₀,areusedtoquantifywhitethrombusformationinsidetheAR-chip.

T₁₀(=timetoreach10kPa)isdefinedastheonsetofwhitethrombusformationandrepresentstheduration(sec)fortheflowpressuretoincreaseto10kPafrombaseline.

T₈₀(=timetoreach80kPa)isdefinedasthecompleteocclusionofcapillaryduetothrombusformationandrepresentstheduration(min)fortheflowpressuretoincreaseto80kPafrombaseline.

AUC₃₀(=areaundertheflowpressurecurvefor30min)isusedtoquantifythrombogenicitywhenT₈₀cannotbeobtainedduetoreducedthrombusformationorfragilethrombi.

Antithromboticagents

AswhitethrombithatdevelopinAR-chipsconsistofactivatedplateletsandfibrinfiberscontainingabundantthrombin,theAR-chipassayiscapableofanalyzingsingleandintegratedefficacies,basedoncommonparameters,ofvariousantithromboticagents,includinganticoagulant,antiplatelet,andthrombolyticagents,undervariableflowconditions.

WhitethrombiformedinanAR-chip
(red,fibrin;green,activatedplatelets)

Imageanalysisofwhitethrombusformation

WhitethrombusformationintheAR-chipcanbeautomaticallymonitoredandrecordedusingabuilt-invideomicroscopeatthree-minutesintervals.

Imagesofwhitethrombiformedinthecapillarycanbeautomaticallyprocessedfromtherecordedvideo,andtheareacoveredwithwhitethrombicanbeidentifiedandcalculatedusingtheZIAprogram.Thus,inadditiontoflowpressureanalysis,thewhitethrombusformationprocesscanbeopticallyanalyzedandquantifiedinAR-chipmeasurements.

AssayProtocol:

PlaceanAR-chipontheassaystageandletstandfor1-3mintoallowstABIlizationofthetemperature.

Connectthereservoirtothenozzle.

Mixacitratedwhole-bloodsamplewiththeCa/CTIreagent,pipet450μlofthemixedsampleintothereservoir,coverwithalid,andthenremovethecap.

ConnectthereservoirtotheAR-chip.

Pushthestartbutton.