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Journal of Thrombosis and Haemostasis, 4: 312–319REVIEW ARTICLEPlatelet function analyzer (PFA)-100 Ò closure time in theevaluation of platelet disorders and platelet functionC. P. M. HAYWARD,* P. HARRISON,à M.CATTANEO,§T.L.ORTEL– and A. K. RAO** ON BEHALFOF THE PLATELET PHYSIOLOGY SUBCOMMITTEE OF THE SCIENTIFIC AND STANDARDIZATIONCOMMITTEE OF THE INTERNATIONAL SOCIETY ON THROMBOSIS AND HAEMOSTASIS*Chair, Working Group on the PFA-100 Ò , ISTH-SSC Platelet Physiology Subcommittee; McMaster University and the Hamilton RegionalLaboratory Medicine Program, Hamilton, ON, Canada; àOxford Haemophilia Centre and Thrombosis Unit, Churchill Hospital, Oxford, UK;§Unità di Ematologia e Trombosi, Ospedale San Paolo, DMCO, Università di Milano, Milan, Italy; –Duke University Medical Center, Durham,NC, USA; **Chairperson, ISTH-SSC Platelet Physiology Subcommittee, 2001–2004; and Sol Sherry Thrombosis Research Center, TempleUniversity School of Medicine, Philadelphia, PA, USATo cite this article: Hayward CPM, Harrison P, Cattaneo M, Ortel TL, Rao AK on behalf of the Platelet Physiology Subcommittee of the Scientificand Standardization Committee of the International Society on Thrombosis and Haemostasis. Platelet function analyzer (PFA)-100 Ò closure time inthe evaluation of platelet disorders and platelet function. J Thromb Haemost 2006; 4: 312–9.Summary. Background: Closure time (CT), measured byplatelet function analyzer (PFA-100 Ò ) device, is now availableto the clinical laboratory as a possible alternative or supplementto the bleeding time test. Aim: On behalf of the PlateletPhysiology Subcommittee of the Scientific and StandardizationCommittee of the International Society on Thrombosis andHaemostasis (ISTH-SSC), a working Group was formed toreview and make recommendations on the use of the PFA-100CT in the evaluation of platelet function within the clinicallaboratory. Methods: The Medline database was searched toreview the published information on the PFA-100 CT in theevaluation of platelet disorders and platelet function. Thisinformation, and expert opinion, was used to prepare a reportand generate consensus recommendations. Results: Althoughthe PFA-100 CT is abnormal in some forms of plateletdisorders, the test does not have sufficient sensitivity orspecificity to be used as a screening tool for platelet disorders.A role of the PFA-100 CT in therapeutic monitoring of plateletfunction remains to be established. Conclusions: The PFA-100closure time should be considered optional in the evaluation ofplatelet disorders and function, and its use in therapeuticmonitoring of platelet function is currently best restricted toresearch studies and prospective clinical trials.IntroductionClosure time (CT), measured by platelet function analyzer(PFA-100 Ò ) device, is now available to the clinical laboratoryas a possible alternative or supplement to the bleeding timeCorrespondence: Catherine P. M. Hayward, Room 2N31, McMasterUniversity Medical Centre, Hamilton Health Sciences Corporation,1200 Main St. W, Hamilton, ON L8N 3Z5, Canada.Tel.: +1 905 521 2100 ext. 73373; fax: +1 905 521 2338; e-mail:haywrdc@mcmaster.ca[1–4]. On behalf of the Platelet Physiology Subcommittee of theScientific and Standardization Committee of the InternationalSociety on Thrombosis and Haemostasis (ISTH-SSC), thisdocument reviews recent literature and provides consensusrecommendations on using PFA-100 CT in the evaluation ofplatelet function within the clinical laboratory.MethodologyA working group of the Platelet Physiology Subcommittee ofthe ISTH-SSC on the PFA-100 CT was established to reviewinformation on the test and to make recommendations on theuse of the PFA-100 CT in the evaluation of platelet function byclinical laboratories. Relevant articles were identified bysearching the MEDLINE database for English papers on thePFA-100 CT, published before June 2005. Members of theworking group reviewed and summarized the publishedliterature, and provided expert opinions to establish concensusrecommendations.Results and discussionPrinciples of the CT measured by the PFA-100 deviceThe PFA-100 CT was introduced to provide a simple, rapidassessment of high shear-dependent platelet function by aprocedure that uses small amounts of citrated blood (0.8 mL/cartridge; maximal CT results: 300 s) [1,2,4,5]. Blood samplesare aspirated at high shear rates (5000–6000 s )1 ) through acapillary in the instrument cartridge and encounter a membranecoated with collagen and epinephrine (CEPI) or collagenand ADP (CADP) [1]. The membrane triggers plateletadhesion, activation and aggregate formation, leading toocclusion of the 150 lm central aperture and cessation ofblood flow [1]. Results are reported as the CT in seconds for theÓ 2006 International Society on Thrombosis and Haemostasis

PFA-100 Ò in the evaluation of platelet disorders and platelet function 313CEPI and CADP cartridges, with values >300 s reported asnon-closure [1].Variables influencing PFA-100 CT resultsThe PFA-100 CT is highly dependent on von Willebrand factor(VWF) binding to the platelet membrane glycoprotein (GP)receptors Ib/IX/V and integrin a IIb b 3 (IIb/IIIa) under highshear [1,2,5–9]. CT has higher sensitivity for von Willebranddisease (VWD) compared with the bleeding time, and CT isabnormal in some congenital and acquired platelet functiondefects, but is not prolonged by coagulation factor deficiencies(reviewed in Harrison [5]) [4,7,10–19]. This review is primarilyfocused on the use of PFA-100 CT for evaluating plateletdisorders and platelet function.The manufacturer advises that each laboratory shouldestablish its own PFA-100 CT reference ranges, usingbuffered 0.109 M (3.2%) or 0.129 M (3.8%) citrate anticoagulatedblood [5,20,21]. Quality control procedures areimportant for test performance, but because the test requireswhole blood, there are no provided quality control materials[5]. Coefficients of variation range between 6% and 13% forCT with normal samples [8,22,23]. CT are significantly higherfor samples collected in 3.8% compared with 3.2% citrate,and 3.8% citrate increases the CEPI CT sensitivity to aspirin[23,24]. Samples anticoagulated with thrombin inhibitors (e.g.PPACK) have similar CT to 3.2% or 3.8% citrate anticoagulatedsamples [25]. CT should be determined within 4 hof sampling [8,23], and to avoid artefactual results, pneumatictubes should not be used for sample transport [26].Small diurnal variations in CT results have been noted, withmorning samples showing shorter CT, particularly with theCEPI cartridge [27]. CT reference ranges for males andfemales are similar although older males may have slightlyshorter CT [28,29]. Children and adults have similar CTvalues whereas neonates have shorter CT because of higherhematocrit and VWF levels [30–32].Like the bleeding time, the PFA-100 CT is prolonged bysignificant reductions in the platelet count or hematocrit[2,3,8,22,33]. For blood samples containing

314 C. P. M. Hayward et allowÕ, type1Ôplatelet discordantÕ, type 2A and type 3 VWD,and their failure to correct with VWF replacement[13,44,45]. In patients with severe aortic stenosis and anacquired VWD characterized by the loss of the largestmultimers, the PFA-100 CT is prolonged and shortensrapidly after surgery, along with the recovery of the largestmultimers [46]. There is also evidence that higher plateletcollagen receptor density (a 2 b 1 andtosomeextentGPVI)isassociated with shorter PFA-100 CT, especially in type 1VWD [9,36,37,47].Consumption of flavonoid rich foods (e.g. red wine, cocoaand chocolate) can prolong the CEPI CT [48]. Other dietaryeffects (e.g. fish oil consumption) on the PFA-100 CT have notbeen characterized.PFA-100 CT in congenital platelet disordersIn congenital platelet disorders, the PFA-100 CT varies withthe severity and nature of the platelet defect (Table 1). Therelatively severe function defects associated with deficiencies ordysfunction of the platelet membrane GP receptors a IIb b 3 (GPIIb/IIIa; Glanzmann thrombasthenia) or GP Ib/IX/V (Bernard–Souliersyndrome or platelet-type VWD) result inmarkedly prolonged PFA-100 CT and typically non-closurewith CEPI and CADP cartridges [7,8,16,34,49,50]. Plateletmembrane density of a 2 b 1 and GP VI influences PFA-100 CT[9,36,47], but there is currently no information on CT in plateleta 2 b 1 or GP VI deficiency or dysfunction, because of the paucityof well-characterized patients with defects in these proteins.Among the more common congenital platelet function disorders(including dense granule deficiency and conditions withdefective secretion), PFA-100 CT findings are variable and theyare more frequently detected with the CEPI cartridge than theCADP cartridge (which is often normal) (Table 1)[4,5,7,8,14,16,34,50–52].There is a need for more information on the PFA-100 CT incongenital platelet disorders as reported studies (summarized inTable 1) have evaluated relatively small numbers of individuals(

PFA-100 Ò in the evaluation of platelet disorders and platelet function 315Aspirin and other non-steroidal anti-inflammatory drugs(NSAIDs) that inhibit platelet function by blocking cyclooxygenase1 (COX-1) and thereby thromboxane generationprolong the CEPI CT in about 95% of healthy individuals, buthave little to no effect on CADP CT [10,49,63] (reviewed in[5,64]). Studies of patients on aspirin therapy for coronary orperipheral vascular arterial disease indicate that only 20–50%have a prolonged CEPI CT [65–68]. Differences in reportedCEPI CT sensitivity for aspirin may reflect differences inpopulations studied (e.g. healthy controls comparedwith patients with higher VWF levels), aspirin dosage andformulation effects [67], and pretest variables, such as thecitrate concentration used for sample collection [24,69]. It isimportant to recognize that the aspirin/NSAID pattern ofPFA-100 CT abnormalities (CEPI prolonged, CADP normal)is not specific for drug-induced platelet dysfunction as similarabnormalities occur with congenital platelet disorders (Table 1)[4,8,14,34,50,51,53] and with consumption of flavonoid-richfoods [48]. When NSAIDs are discontinued, PFA-100 CTabnormalities revert by 6 days with aspirin [70] and by 24 hwith ibuprofen [71].The PFA-100 CT is relatively insensitive to therapy withticlopidine and clopidogrel, with detection of effects showingtime and dose dependence [20,25,57,72–76]. CADP and CEPICT detect synergism in the antiplatelet effects of clopidogreland aspirin [73,74].It has not yet been established if the PFA-100 CT is usefulfor monitoring therapy with antiplatelet agents in patientswith, or at risk for, coronary, cerebral, or peripheral vasculararterial disease [64]. Multiple studies have reported individualswho appear to be ÔresistantÕ to aspirin on the basis of normalCEPI CT on therapy [10,49,65–67,69,73]. These studiesindicate that CEPI CT can be normal even when COX-1 isadequately blocked. A significant inverse association existsbetween plasma VWF and CEPI CT during aspirin therapy[15], and as many patients with arterial disease have highplasma VWF levels, and short baseline CT, it is not surprisingthat a relatively high proportion have normal CEPI CT onaspirin [66,77–80]. Higher doses of aspirin have been reportedto increase the proportions of individuals with prolongedCEPI CT in some studies [67] but not in others [68,81] anduncoated aspirin prolongs the CT more than enteric-coatedaspirin [67]. Although some studies have suggested adverseevents are more prevalent among individuals with normalCEPI CT on aspirin therapy, the majority of studies are toosmall to draw firm conclusions and differences reported havenot reached statistical significance [64,65,78]. Large, prospective,randomized-clinical trials are needed to determine if thePFA-100 CT is useful to predict adverse events or maketherapeutic decisions on aspirin therapy. Evidence is requiredto support making clinical recommendations based on anormal CEPI CT in patients on aspirin therapy. It has beenrecommended that the term Ôaspirin resistanceÕ not be appliedto these patients and that PFA-100 CT not be measured onpatients on aspirin therapy outside of research studies(reviewed in [64,78]).There is limited information on the effects of many otherdrugs on the PFA-100 CT. The PFA-100 CT is prolongedapproximately 30% above baseline in subjects taking theserotonin-reuptake inhibitor paroxetine [82].PFA-100 CT and acquired platelet dysfunction duringcardiopulmonary bypassAcquired qualitative defects in platelet function can causebleeding after cardiac and cardiopulmonary bypass surgery.CADP CT is significantly prolonged after heparinization forbypass procedures, probably because of interference of highheparin concentrations on VWF binding to platelet GP Ib/IX/V[42]. CADP CT was reported to be further prolonged duringextracorporeal circulation, with rapid reversal of the abnormalityafter surgery [42]. In this study, preoperative CEPI CT wasabnormal because of aspirin therapy, and the magnitude of CTprolongation was not predictive of postoperative bleeding [42].PFA-100 CT and coronary syndromesOne study of patients, with acute chest pain and suspectedacute coronary syndromes, reported shorter CADP CT andincreased plasma VWF levels in the subset with myocardialinfarction [83]. Shorter CADP and CEPI CT, and higher VWFlevels, at presentation were also correlated with biochemicalevidence of greater myocardial necrosis [83]. One study ofPFA-100 CT, before and after exercise, in patients with stableangina who underwent coronary angiography reported thatreductions in CT (10 or more seconds from baseline) wereassociated with vessel stenoses whereas increases in CT (10 ormore second from baseline) were not [84]. Further prospectivestudies are needed to determine if the PFA-100 CT is useful formanagement of coronary syndromes.PFA-100 CT in uremia and liver diseaseCEPICTandCADPCTtendtobeprolongedinpatientswithuremia or liver cirrhosis [85,86], possibly from associatedanemia, as the abnormalities correct with in vitro elevation ofthe hematocrit [85].PFA-100 CT in monitoring therapies for bleedingWhile PFA-100 CT can detect hemostatic effects of sometherapies given to treat or prevent bleeding, there have been nostudies designed to determine if improved CT predictsimproved clinical outcomes with regard to hemorrhage andfew studies of its utility in monitoring therapy for qualitativeplatelet defects. Correction of a prolonged CEPI CT withdesmopressin (DDAVP) therapy has been reported in smallstudies of patients with storage pool disease and primarysecretion defects associated with increased plasma VWF [14].DDAVP also significantly shortens the CADP and CEPI CTof healthy individuals [58]. In type 1 VWD, prolonged CEPIand/or CADP CT show correction after DDAVP-inducedÓ 2006 International Society on Thrombosis and Haemostasis

316 C. P. M. Hayward et alincreases in plasma VWF but not in patients with discordant orlow levels of platelet VWF [11,13,44,87]. In severe VWD, PFA-100 CT often does not correct with VWF replacement, possiblybecause of abnormalities in concentrate multimer profile and/or lack of intraplatelet VWF [11,13,44,45].No studies have investigated PFA-100 CT in patients givenDDAVP for bleeding secondary to antiplatelet therapy,although CT shortening occurs in healthy individuals givenDDAVP after antiplatelet agents [17,58]. One study of 30healthy volunteers, given 500 mg of aspirin for 3 days (whichprolonged CEPI CT in 90% of volunteers), reportednormalization of PFA-100 CT by 30 min in all subjects givenintravenous DDAVP, and in 93% given nasal DDAVP [17].Another study of 10 healthy volunteers, treated with DDAVPafter aspirin and eptifibatide, reported accelerated normalizationof CEPI and CADP CT that was most evident severalhours after DDAVP was given [58].It must be emphasized that none of the studies reportingnormalized PFA-100 CT during DDAVP therapy for certaincongenital or acquired qualitative platelet defects (in lieu or inaddition to the bleeding time) or type 1 VWD were designed toevaluate if normalized CT correlated with improved clinicaloutcomes.Few studies have evaluated the utility of the PFA-100 CT inmonitoring other therapies. With platelet transfusion therapy,shortening of the PFA-100 CT by more than 40 s, or itsnormalization, has been reported to correlate better withcessation of bleeding than corrected platelet count increments[88]. Data are lacking on the utility of the PFA-100 CT tomonitor hemostasis in other clinical situations (e.g. uremia).Changes in PFA-100 CT with other therapiesAlthough the clinical significance is unknown, PFA-100 CT canbe prolonged by perioperative colloid or crystalloid administration[89] and by plasmapheresis [90]. In vitro,thePFA-100CTis prolonged by low-molecular weight dextran sulfate [91].RecommendationsThe PFA-100 CT is now an optional test for clinical laboratoriesto consider as part of their diagnostic evaluation of plateletdisorders and platelet function. To date, the evidence on thePFA-100 CT in different congenital platelet disorders indicatesthat the test does not have sufficient sensitivity or specificity tobe used as a screening tool in determining which individualsneed further testing for platelet disorders. However, data areavailable only on a limited number of patients with defineddisorders. Prolonged CT can reflect other abnormalities (e.g.VWD), and as such, abnormal results require further diagnosticevaluations. Normal CT can help exclude some severe plateletdefects (e.g. Glanzmann thrombasthenia and Bernard–Souliersyndrome) and moderate-to-severe VWD, but if clinicalsuspicion is strong, further testing should be performed. A rolefor the PFA-100 CT in therapeutic monitoring remains to beestablished, and therefore its use in such monitoring is currentlybest restricted to research studies and prospective clinical trials.Based on the current knowledge, adequately powered clinicalstudies that compare results to meaningful clinical outcomes arerequired to establish a role for the PFA-100 CT in predictingclinical outcomes and/or monitoring therapy.AcknowledgementsWe acknowledge Dr William L. Nichols (Mayo Clinic,Rochester, MN, USA) for providing information on CTresults from reference [16] and Dr Paquita Nurden (HopitalCardiologique, UMR 5533 CNRS, Pessac, France) forproviding unpublished data for Table 1. P. Harrison is aconsultant for Sysmex UK and was a consultant for Dade-Behring until 30/11/2004.References1 Kundu SK, Heilmann EJ, Sio R, Garcia C, Davidson RM, OstgaardRA. 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