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Abstract

Platelet flow cytometry is an emerging tool in diagnostic and therapeutic hematology. It is eminently suited to study the expression of platelet surface receptors both qualitatively as well as quantitatively. It can serve as a useful marker for the documentation of in vivo platelet activation, and thus, fore-warn the risk of thromboembolism in patients with diabetes mellitus, coronary syndromes, peripheral vascular diseases, and pre-eclampsia. This technique can also be extended to study and compare the effect of various antiplatelet drugs on the level of activation of platelets and to establish any dose-effect relationship of these drugs. Topographical localization of platelet granules and study of platelet-platelet and platelet-leukocyte interaction is also possible by this procedure. All these parameters serve as pointers towards the presence of activated platelets in the circulation with its thromboembolic consequences. This is a simple reliable and cost effective technique which has a wide application in the diagnosis of various inherited and acquired platelet disorders. Study of platelet cluster of differentiation (CD) markers in various inherited disorders i.e. Bernard Soulier’s disease, von Willebrand disease, Glanzman’s disease, and Grey platelet syndrome may help categories the molecular lesions in these oft under-studied disorders.

Keywords: flow cytometry, platelets, CD63, CD62

Introduction

Platelets flow cytometry is an emergingtechnology in clinical and research hematology(1,2). It assesses the expression of surfacereceptors,componentsofgranules,boundligands,and interaction of plateletswith other platelets,neutrophilsandmonocytes(3–5).Withthehelpof monoclonal fluorescent antibodies, it hasbecome now possible to determine the absolutenumber of antibody labelled cells by flowcytometer(2).Thistechniqueismoreusefulthanother methods due to small amount of bloodsampleandminimummanipulationofthesampleduringprocessing(6).

Applications of flow cytometry Diagnosis of inherited thrombocytopathiesi.e. Bernard-Soulier’s syndrome, Glanzmann’sthrombasthenia, and storage pool diseasehas become easy through flow cytometry (7).Pathological and chronological evaluation ofacutecoronarysyndromes,acutecerebro-vascularischemia, peripheral vascular disease, diabetesmellitus, and pre-eclampsia can be seriallystudiedwithflowcytometrybydetectingactivatedplatelets, platelet derived microparticles, andplatelet-leukocyte aggregates in the circulating

Brief Communication

New Horizons in Platelets Flow Cytometry

Muhammad Saboor, Moinuddin Moinuddin, Samina ilyaS

Baqai Institute of Hematology, Baqai Medical University, 51 Deh Tor Gadap Road, Super Highway, Near Toll Plaza, P.O Box 2407, P. Code 74600, Karachi, Pakistan

Submitted: 21Nov2012Accepted:12Feb2013

blood.Itcanalsobeusedformonitoringplateletfunctions before and during angioplasty andcardiopulmonary bypass (2,7). Flow cytometricidentification of circulating platelet-monocyteaggregates can also serve as a sensitive markerfor in vivo platelet activation (8). In the fieldof therapeutics this technique can be usedto evaluate the efficacy of various antiplateletdrugs(9). Flow cytometry also enables to diagnoseheparin induced thrombocytopenia (4). This isthe method of choice for the detection ofaggregation response to agonists in patientstreated with antiplatelet drugs. This is moresensitive than platelet rich plasma aggregationquantifiedbyaggregometry(2,9). Platelets flow cytometry is an easy, reliableandsensitivetechniquefor:

● evaluation of platelet functionalstatus by studying the; expression ofneoepitopes e.g.CD63,CD62p,platelet–platelet aggregates, platelet-derivedmicroparticles, platelet–leukocyteaggregates. Conditions associated withincreased expression of these markersare acute coronary syndromes, acutecerebro-vascular ischemia, peripheralvascular disease, diabetes mellitus, andpre-eclampsia.

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● diagnosis of inherited plateletdisorders; Bernard–Soulier’s syndrome,Glanzmann’s thrombasthenia, Storagepooldiseases.

● d i a gno s i s o f h epa r i n - i nduc edthrombocytopenia.

● monitoring of antiplatelet therapy; ADPreceptorblockers,GPIIb-IIIaantagonists,cyclooxygenaseinhibitors.

● determinationofrateofplateletturnover;Reticulatedplateletcount.

● immuno -p l a t e l e t c oun t i n g b yimmunologicaltechnique.

● applications in blood banking andtransfusion medicine; quality controlof platelet concentrates, identificationof leuko−contamination in plateletconcentrates, immunophenotyping ofhuman platelet antigen-1a (HPA-1a),detectionofmaternal,andfetalanti-HPA-1aantibodies,plateletcrossmatch.

● identification of platelet-associatedantibodies; neonatal alloimmunethrombocytopenia, dengue hemorrhagicfever(10,11).

AdaptedfromMichelsonetal.(12) Basis of laboratory diagnosis of many ofthese disorders is the characteristics alterationsin the CD markers. Some of these changes aredescribedbelow; αIIbβ3 is normally present on the surfaceof the resting platelets, after platelet activationconformational changes occur in the αIIbβ3.PAC-1 is a specific monoclonal antibody thatbinds to confirmationally changed αIIbβ3 afteractivation (13). PAC-1-negative platelets areconsideredasrestingplateletswhileitspositivityisanindicationofplateletactivation. CD62pisfoundintheα-granulesofplatelets.PresenceofCD62ponthesurfaceoftheplateletsindicates platelets in activated state while theirabsenceimpliesrestingstateoftheplatelets(14).CD63 is a 53 kDa lysosomalmembrane proteindetectedonthesurfaceofactivatedplateletsafterreleasereaction(15,16). Number of young or reticulated plateletsdepends upon the rate of thrombopoiesis aswell as their removal from the circulation.Thiazole orange (TO) is a fluorescent dye thatbindsintracellularribonucleicacid(RNA)oftheplatelets. Flow cytometric analysis of thiazoleorangepositiveplateletsisameasureoftherate

ofthrombopoiesis(17). Indo-1 and Fluo-3 are fluorescent probesusedforthemeasurementofintracellularcalciumafter platelet activation (6). Changes in plateletmembraneactinandmyosincanalsobemeasuredbyflowcytometry(18).

Technical considerations

Principle of flow cytometry Platelets are labeled with fluorescentmonoclonalantibodies.Cellsuspensionispassedthrough the flow chamber equipped with afocusedlaserbeamthatactivatesthefluorophore.Fluorescence is measured for the identificationof platelets. Intensity of the emitted light isdirectlyproportionaltothenumberofantibodiesattachedtotheplateletreceptors/antigens(12).

Selection of anticoagulant for platelets flow cytometry Suitableandappropriateuseofanticoagulantismandatoryfortheprocessingofsamplesforflowcytometry. Sodium citrate is the anticoagulantof choice for platelet flow cytometric analysis(19,20).Ethylenediaminetetraaceticacid(EDTA)and heparin affect the glycoprotein structureand cause artifactual platelets activation.EDTA also causes swelling of the platelets andproduceschangesinαIIbβ3complex(21).EDTAanticoagulated samples also show significantlylower number of antiCD41a (clone 96.2C1)positiveplateletsthansamplescollectedincitratetubes(20).

Fixation of platelets The use, type and concentration of thefixative for platelet fixation are controversial.Processing of the unfixed sample immediatelyafter collection, results in the lowest in vitroactivation (22). However if it is not possibleto process the sample immediately plateletsare fixed to prevent in vitro platelet activation.Immediate fixation is essential for the study oftimedependentchangesinplateletactivation. Expressionofglycoprotein lb-IX-Vcomplexonthesurfaceofplateletstartstodeclinewithin30secondsafteractivation,andreachesthenadirin5minutes.Itreturnstoitspre-activationlevelafter 45 minutes of activation (23). Cleavageand internalizationofGPIb-IX-Vcomplex is thebasis of this appearing-disappearing cycle afteractivation (24). Expression of αIIbβ3 complexonplateletssurfacemembranealsodeclineswiththepassageoftime(23). Formaldehyde and paraformaldehyde in

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different concentrations have been used asplatelet fixative by several researchers.Platelet studies must be done within 2 hourswithout fixation. After fixation with 0.5−1.0%formaldehyde or paraformaldehyde plateletsamplescanbeanalyzedafter2hours(25). Cahill et al. (26) reported that fixation ofplateletswithformaldehydebeforelabellingwithmonoclonal antibodies increases the expressionof CD62p andCD63 on the surface of platelets.Formaldehyde should therefore not be used asafixativeforplateletactivationstudies(26). Hu et al. (25) on the other hand observedno change in the expression of CD62p duringresting state of platelets when fixed with 0.2%or 0.5% formaldehyde. Contrary to the findingsof Cahill et al. (26) Hu et al. (25) remarkedthat 1.0% formaldehyde or paraformaldehydeslightlydecreasedCD62ppositivityoftherestingplatelets. Stimulation with ADP after fixationwith formaldehyde causes slight increase inthe positivity of CD62p markers (26). Thisdiscrepancycanberesolvedbyfixingtheplateletsafterlabelling.Itwasobservedthatwhenfixationwas done after labelling the platelets with theantibody, fixative did not change the positivityofCD41,CD62p,andPAC-1(22). As with formaldehyde use ofparaformaldehyde as a platelet fixative alsogives conflicting results. Atar et al. (27),stated that platelet flow cytometric analysis ofparaformaldehyde fixed inactivated plateletscan be done within five days. Platelets fixationwith 0.5% paraformaldehyde before antibodylabelling shows marked increase in CD62Ppositivity. CD41 and PAC-1 activity is alsoincreasedwhencomparedwithunfixedplatelets,while CD42b activity is reduced after fixation(22).Fixationofplateletswithparaformaldehydeafter labelling results in less than0.2% increasein activation (28). Since red cell lysis releasesadenosinediphosphate(ADP)whichisaplateletagonist, it isrecommendedthatlysisofredcellsand excessive centrifugation should be avoidedduringplateletstudies(20).

Effect of incubation Platelet markers CD41 and CD61 are notaffectedby incubationor stimulationwithADP.When incubated for 20 minutes, 2 hours, and3 hours, no statistical difference in CD41 andCD61wasfound(29). Activation of platelets with ADP results inan increase in the number of CD63 andCD62ppositive platelets. Persistent increase in thenumber of CD63 positive platelets reflects the

stability of this molecule after activation withADP(29,30). In vivo increase in CD63 is also reportedby several authors in various clinical conditionscharacterized by persistent platelet activation.Peripheral artery disease,myocardial infarction,congestiveheartfailure,acutecerebralinfarction,atheroscleroticischemicstroke,andnon-embolicischemicstrokearesomeofthecommonclinicalconditions that are frequently associated withincreasedCD63activityinvivo(31–36). Flow cytometry can also provide anavenue to assess the effect of varying dosesof antiplatelet drugs on suppressing plateletactivation.Inpatientswithpersistentthromboticcomplications, despite continued administrationof antiplatelet drugs may be benefitted if aneffective dose is administered which may bedetermined by titrating the dose and offering acustomized dose as prophylactic antithrombotictherapy. InastudybySabooretal.(29)itwasfoundthat incubation of platelet rich plasma withADP resulted in an initial increase in CD62ppositivity;thisrisewashowevertransient.Whenactivated platelets were incubated for 2 and3 hours, the mean fluorescence percentpositivity of CD62p progressively andsignificantly decreased. Reduction in CD62ppositivity was greater after 3 hours than after2hoursofincubation.AlthoughCD62ppositivityalso decreased during incubation, its valuesremained higher after 3 hours of incubationthan the base line values. Buggle et al. (30)also found reduction in the CD62p positiveplateletsatdifferent intervals instrokepatients.Matzdorff et al. (37) also reported decreasednumber of CD62p positive platelets in their invitroexperiments.

Advantages of flow cytometry

Some of the advantages of flow cytometryover other laboratory procedures that makeflow cytometry an ideal method of plateletanalysis are:

● Platelets are analyzed in theirphysiological state.

● Insignificant in vitro platelet activationdue to minimum manipulation of thesample.

● Verysmallamountofbloodrequired.● Simultaneous determination of resting

aswellasactivatedstateoftheplatelets.

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● Detection of neoepitopes expression onthesurfaceofplatelets.

● Highly sensitive and specific procedurefor the detection of resting andactivated platelets and platelet derivedmicroparticles.

● Accurate analysis of platelets of patientswithseverethrombocytopenia.

● Fluorescenttechniqueeliminatestheriskofexposuretoradioactivematerial.

Conclusion

It is concluded that platelet flow cytometrycanbeutilizedforthediagnosisofvariousplateletsdisorders, evaluation of platelets functionalstatus, monitoring of antiplatelet therapy andresearchpurposes.Immediatesampleprocessingwithout fixation, appropriate anticoagulantare the key players of achieving good results ofplateletsflowcytometry.

Acknowledgement

WearethankfultoProf.SyedAzharAhmedforhisguidance.

Conflict of interest

Nil.

Funds

Baqai Institute of Hematology, Baqai MedicalUniversity provided financial and technicalsupport.

Authors’ Contributions

Conceptionanddesign:MSCritical revision of the article for the importantintellectual content and final approval of thearticle:MMDraftingofthearticle:SI

Correspondence

MuhammadSaboorMPhil(Hematology)AssistantProfessorBaqaiInstituteofHematologyBaqaiMedicalUniversityKarachiTel:+009-23332279927Fax:+009-22134410293Email:msaboor81@gmail.com

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