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HUP3011HumanPathology1 PracticalWorkshopNotes

UnderstandingDiseaseProcesses
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PlateletFunctionTesting:
LightTransmissionAggregometry
I.PrinciplesofLightTransmission[Born]Aggregometry
Plateletaggregationtestingmeasurestheabilityofvariousagoniststoplateletstoinduceinvitro
activation and platelettoplatelet activation. Classically Born aggregometry uses platelet rich
plasma[PRP]butwholebloodaggregometrycanbealsoused.

IntheBornaggregometer,PRPisstirredinacuvetteat37Candthecuvettesitsbetweenalight
courseandaphotocell.Whenanagonistisaddedtheplateletsaggregateandabsorblesslightand
sothetransmissionincreasesandthisisdetectedbythephotocell.

YoucanalsoseetheprinciplesofBornaggregometryasananimationonthissite:
[http://www.plateletresearch.org/3/aggregometry.htm]



HUP3011HumanPathology1 PracticalWorkshopNotes
UnderstandingDiseaseProcesses
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II.LightTransmissionAggregometry:Variability
Variable Explanation
PreAnalytical
Variables
Drugswhichcaninterferewithplateletfunctionincludeaspirinandantiinflammatorydrugs,specific
antiplateletdrugsincludingclopidogrelandimidazole.However,therearenumerousotherdrugs
whoseprimaryroleisnottoinhibitplateletfunctionbutneverthelesscandosoe.g.antibiotics,anti
depressants,betablockersetc
Foodstuffs
Ahighfatdietcanleadtothepresenceofchylomicraintheplasmaandinterferewithlight
transmissioninaggregationtesting.
Othersincludegarlic,turmericandcaffeine.
Plateletcount:Inindividualswithveryhighorlowplateletcounts,itmaybenecessarytoadjustthe
plateletcounttoachieveacountintheregionof200400x10
9
/L.Forveryhighcountsthecountcan
beadjustedwithPPP.Plateletcountsbelow200x10
9
/Lcangiverisetodiminishedaggregation
responses.Althoughitseemslogicaltoundertakeadditionalcentrifugationinsuchcasestoincrease
theplateletcount,inpracticethiscanleadtoactivationofplateletsandisnotrecommended.
Temperature:Bloodsamplesforplateletaggregationtestingshouldbestoredatroomtemperature.
pH:PlateletaggregationshouldbecarriedoutatphysiologicalpH.
FibrinogenConcentration:Plateletswillonlyaggregate(althoughtheymayagglutinate)iffibrinogenis
presentandsoitisimportanttocheckfibrinogenlevelsbeforeundertakingplateletaggregation
testing.
Anticoagulant:Currentguidelinessuggestthatsamplesforplateletaggregationtestingshouldbe
collectedintocitrate.However,morerecentdatasuggeststhatheparin,butnotcitrate,preserves
plateletresponsesforupto24hasdeterminedbyarangeoftechniques
Preparationof
PlateletRich
Plasma[PRP]
PlateletsareverysensitiveandcanbereadilyactivatedduringthepreparationofPRP.
Anticoagulant:Venousbloodwithminimalvenousocclusion,iscollectedinto3.2%/0.109Mcitrateina
ratioof1:9[1partanticoagulantto9partsblood.]
Wholebloodsamplesshouldbeprocessedwithin4hoursofcollection.
Bloodsamplesforplateletaggregationtestingshouldbestoredatroomtemperaturecooling
plateletscanleadtoactivation.
Transportsamplestothelaboratoryatroomtemperature.PRPispreparedbycentrifugationat20C
for1015minutesat150200g.ThePRPiscarefullyremovedandplacedintoastopperedplastictube.
PRPshouldbestoredatroomtemperature.PPPcanbepreparedbyfurthercentrifugationofthe
remainingplasmaat2700gfor15minutes.
Agonists
AdditionofaplateletagonisttothePRPleadstoplateletactivation,achangeintheirshapefrom
discoidtospinysphereswhichisassociatedwithatransientincreaseinopticaldensity.Theonly
exceptionstothisareepinephrineinwhichthereisnoshapechangeandristocetinwhichcauses
plateletagglutinationratherthanaggregationi.e.thereisnobindingoffibrinogen.
Therearetwotypesofagonists:
I)StrongAgonistse.g.Collagen,thrombin,TxA2:Thesedirectlyinduceplateletaggregation,TxA2
synthesisandplateletgranulesecretion.
II)WeakAgonistse.g.ADP&epinephrine:Theseinduceplateletaggregationwithoutinducing
secretion.
Plateletsecretioncansometimesfollowaggregationinducedbyaweakagonist,whenthesynthesisof
endogenousTxA2istriggeredbythecloseplatelettoplateletcontactthatoccursduringplatelet
aggregation.
Strongagonists,whenusedatlowconcentrations,mayactlikeweakagonists,butweakagonistseven
athighconcentrationswillnotactasstrongagonists.
Withsomeweakagonists[ADPandadrenaline]atcriticalconcentrations,theplateletaggregation
curvehasabiphasicappearance:aninitialwaveofaggregation(primarywave),followedbya
secondarywaveofaggregation,whichisusuallyirreversible[seeillustrationbelow.]Secondarywave
aggregationmaynotoccurandtheprimarywavemaydisaggregate.Athigheragonistconcentrations
(exceptwithepinephrine)thetwowavesofaggregationcombineandonlyasinglewaveisseenand
thebiphasicwaveformisabsent.
TheaggregationresponsetoanagonistisamplifiedbytheproductionofTxA2frommembrane
phospholipidsandbythesecretionofADPfromthedensegranules.ADPandTxA2areagonists,
which,byinteractingwiththeirspecificreceptors,amplifytheaggregationresponseoftheplatelet.
HUP3011HumanPathology1 PracticalWorkshopNotes
UnderstandingDiseaseProcesses
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III.CommonlyusedAgonistsinLightTransmissionAggregometry:
Commonly used agonists, their working concentration and mode of action are listed below. In
practice many laboratories use a number of agonists and various dilutions but vary the actual
agonists or agonist concentration depending upon the results of initial tests and the suspected
abnormality.Itisusefultoconsidertheroleofthesevariousagonistsbylookingatanimageofa
platelet and the various receptors that are activated by the agonists discussed below and how
theseinteractwiththeplatelet.

Agonist
Working
Concentration
Comment
ADP Lowdose:1,2.5,
5M

Highdose:10M
ADPbindstotheADPreceptoronthesurfaceofplatelets.Initialbindingresultsinthereleaseof
intracellularcalciumandachangeintheshapeoftheplateletleadingtotheprimarywaveof
aggregation.ThesecondarywavereflectsthereleaseofADPfromplateletstoragegranules.
LowdoseADPinducesonlyprimaryaggregationandtheeffectisreversible.ADPandarachadonic
acidareconsideredmildplateletagonists.
ADPbindstotwoGproteincoupledreceptors:P2Y1andP2Y12.BindingofADPtotheP2Y1receptor
inducesshapechangeandinitiatesprimarywaveplateletaggregationthroughcalciummobilisation.
TheP2Y12receptorisconsideredtobethemajorADPreceptorandresponsibleforfullplatelet
aggregationthoughttheinhibitionofadenylcyclase.
TheP2Y12receptorisalsothetargetforclopidogrel.WithbothADPandArachadonicacidthis
secondwaveofaggregationisinhibitedbyaspirinandNSAID's.
Collagen 1,4g/mL CollagenbindstotheGpVIandGpIa/IIareceptorsinducinggranulerelease,TBXA2generationand
thensustainedGPIIbIIIaactivation.
TheGpIa/IIareceptorisinvolvedinplateletadhesion.TheGpVIreceptorisinvolvedinplatelet
signalingandTBXA2generation.
AlagphaseisseenwithcollagenfollowingadditionoftheagonisttothePRPandusually<1minute.
Ristocetin Lowdose:.5mg/mL

Highdose:1.5,
5mg/mL
Ristocetin(butnotgenerallyinlowdosei.e.0.5mg/mL)causesplateletagglutination(andnot
aggregation)throughtheVWFandGPIbIXVcomplex.[Plateletaggregationrequiresthebindingof
fibrinogentotheplateletviatheGpIIbIIIacomplex.]
Adrenaline 5,10M Adrenalinebindstothe
2
adrenergicreceptoronthesurfaceofplateletsleadingtoinhibitionof
adenylcyclaseandthereleaseofcalciumions.AggregationofplateletswithAdrenalineissimilarto
thatofADPwithaninitialprimarywaveofaggregation,thereleaseofstoredADPfromtheplatelet
densebodiesandsecondwavesustainedaggregation.AswithADP,thissecondwaveofaggregation
isinhibitedbyaspirinandNSAIDs.Adrenalineisconsidered[asisADP]tobeaweakagonist.
However,defectsinsignalingthroughthe
2
adrenergichavebeenassociatedwithableeding
disorder.
Asmallproportionofthepopulationmaynotalwaysshowfullaggregationtoadrenalinedueto
naturalvariationsinadrenoreceptornumbers.Suchindividualshavenotrelatedplateletdefect.
Arachadonic
Acid
500g/mL ArachadonicAcidistheprecursorofthromboxaneA2[TXA2]withinplatelets.ArachadonicAcidis
convertedtoTXA2bycyclooxygenaseandthromboxanesynthase.TXA2isapotentinducerof
plateletaggregationcausinggranulerelease,moreTXA2generationandthensustainedGpIIbIIIa
activation
Thrombin Lowdose:50nmol/L

Highdose:
100nmol/L
Thrombinisthemostpotentphysiologicalactivatorofplateletsandproteaseactivatedreceptors1
(PAR1)and4(PAR4)areactivatedbythrombin.PAR1andPAR4aremembersofa7transmembrane
groupofGproteincoupledreceptorsthatareactivatedbyasinglecleavagewithintheNterminal
domaintogenerateanewNterminuswhichactivatestheGsubunitsandintracellularsignalling.
Thrombininducesplateletaggregationatlowconcentration[50nmol/L]butatthisdoseitis
insufficienttoinducefullaggregationbutbindstoplateletsandinducesashapechange.Atahigher
concentration(100nmol/L),thrombininducesfullaggregation.

HUP3011HumanPathology1 PracticalWorkshopNotes
UnderstandingDiseaseProcesses
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IV.Method
Plateletaggregometryisperformedasfollows:

Step
1 Plateletaggregometryisperformedat37C.
2 Theaggregometeriscalibratedby:
AcuvettecontainingPRPwhichequatesto0%lighttransmission
AsecondcuvettecontainingPPPwhichequatesto100%lighttransmission.
3 Plateletswillonlyaggregateiftheyareactivated(withanagonist)andincontactwitheachother
sotheymustbestirredwhilsttestingistakingplace.Absenceofstirringwillleadtoanabsenceof,
atleastasignificantreductionin,aggregation.
Acheckforspontaneousplateletaggregation[SPA]ismade.
SPAisrareinhealthyindividualsbutseeninsomecasesofVWD,insomepatientswithdiabetes,in
somelipiddisordersandinavarietyofotherdisorders]shouldbemadeinallpatientsbyplacing
undilutedPRPintheaggregometerandstirringfor15minutes.IncasesofSPA,dilutionofthePRP
mayabolishthisandiftheplateletcountremains>200x10
9
/Lthenaggregationtestingcan
proceed.
4 Ingeneral 270LofPRPisaddedtotheaggregometrycuvetteandwarmedat37Cuntilasteady
baselineisachieved.
30Loftheagonistisaddedtheresponserecorded.
Thetestsarerepeatedusingapanelofagonists.

Thefollowingaggregationtraceshowstheeventsinclassicbiphasicaggregation:

1.Baseline
2.Additionofagonistthisresultsinachangeinplateletchangeandhenceadropinthe
baselineabsorbance
3.Primarywaveaggregation
4.Releaseofnucleotides
5.Secondarywaveaggregation
AdrenalineandlowdoseADPclassicallygiveabiphasicaggregationcurvewhereaswithanumber
ofotheragonistsonlyasinglewaveisseenanditisnotpossibletodistinguishtheprimarywave
fromthesecondarywave.
HUP3011HumanPathology1 PracticalWorkshopNotes
UnderstandingDiseaseProcesses
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V.InterpretationofPlateletAggregationTraces
Calculatingtheslopeortherateofaggregation:

Historically,percentage[%]maximalaggregationhasbeenreportedwhenanalysingaggregation
curves.
Tocalculatethe%maximalaggregation,thedistancebetweenthebaseline[0%aggregation
plateletrichplasma]andplateletpoorplasma[100%aggregation][Y]isdividedbythemaximal
aggregation[X].
SointheexampleaboveiftheY=100mmandX=87mmthenpercentagemaximalaggregation=
X/Y=87%.

Theinterpretationofplateletaggregationtracescanbedifficult.

Commonaggregationtracesthatarelikelytobeencounteredinaclinicalsettingare:
Glanzmann'sThrombasthenia[orafibrinogenaemia]
BernardSoulierSyndrome[orVonWillebrandDisease]
StoragePoolDisorder[orreleasedefect]
Aspirin[oraspirinlikedefect]
Clopidogreltreatment

Representativetracesforsomedisordersandshownbelow.Ineachcasethecontrolisshown
inblueandthepatientinred.

HUP3011HumanPathology1 PracticalWorkshopNotes
UnderstandingDiseaseProcesses
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Example1:
Inthepatientshownbelow,theonlyabnormalityisalackofagglutinationwithristocetin.Possible
diagnosesaretherefore,VonWillebrandDiseaseorBernardSoulierSyndrome.PlatelettypeVWD
duetoamutationinthehingeregionoftheplateletGpIbreceptorwouldgivesimilarfindings.

Example2:
This is the converse of the patient shown above and the only agglutination [and this is not
complete] is seen with the ristocetin. There is no aggregation with ADP, adrenaline or collagen.
PossiblediagnosesincludeGlanzmann'sthrombastheniaorafibrinogenaemia.Remember,platelet
agglutination with ristocetin occurs independently of fibrinogen.
In the traces shown below it is clear that only partial agglutination is seen with ristocetin
emphasising that for aggregation to occur, binding of fibrinogen to the GpIIb/IIIa receptor is
necessary.

HUP3011HumanPathology1 PracticalWorkshopNotes
UnderstandingDiseaseProcesses
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Example3:
Inthispatientreversible,firstwaveaggregationisseenwithADP,adrenalineandcollagenand
onlypartialagglutinationwithristocetin.Thepictureisclearlydifferentfromthetwotracesabove
1)or2):theresultssuggestafailureofgranulereleaseandandisconsistentwitheitherplatelet
storagepooldisorderoradefectinnucleotiderelease.


Itsusefultosummarisethe'commonly'describedabnormalitiesseenwithlighttransmission
aggregometryalthoughinpracticemanyoftheseareextremelyrare.Thetablebelowsummarises
these:
Disorder CharacteristicFindingsonLTA
Glanzmann'sThrombasthenia
ORafibrinogenaemia
Absentormarkedlyimpairedaggregationtoallagonistsexceptristocetin.
Ristocetininducedagglutinationshowsonlyprimarywaveaggregationcannot
occurbecausefibrinogencannotbind.
Afibrinogenaemiagivessimilarresults.
BernardSoulierSyndromeORVon
WillebrandDisease
Absentormarkedlyreducedplateletagglutinationwithristocetin.
StoragePoolDisorderORPlatelet
ReleaseDefect
PrimaryaggregationonlywithADP,adrenalineandcollagenandonlypartial
agglutinationwithristocetinsuggestingafailureofgranulereleaseoradeficiency
ofplateletgranules.
Aspirin[ordefectsintheCOXpathway] Absentaggregationtoarachadonicacid.
PrimarywaveaggregationonlywithADP.
Decreasedorabsentaggregationwithcollagen.
Clopidogrel AbsentaggregationwithADP
2BVWD/Platelettype[pseudo]VWD Aggregationwithlowdoseristocetine.g.0.5mg/mL.