Platelet function analyzer (PFA-100): A tool to quantify congenital or acquired platelet dysfunction

REVIEW ARTICLES Platelet function analyzer (PFA-100): A tool to quantify congenital or acquired platelet dysfunction BERND JILMA VIENNA, AUSTRIA

Abbreviations: α2β1-integrin = collagen receptor; ADP = adenosine diphosphate; BT = bleeding time; CADP = collagen plus adenosine-5´-diphosphate; CADP-CT = collagen adenosine closure time; CEPI = collagen plus epinephrine; CEPI-CT = collagen epinephrine closure time; COX = cyclo-oxygenase; CT = closure time; CV% = coefficient of variation (percent); DDAVP = 1-deamino-8-D-arginine vasopressin; GPIb = glycoprotein Ib (vWF-receptor); GPIIb/IIIa = glycoprotein IIb/IIIa (fibrinogen receptor); Hct = hematocrit; L-ASA = infusable aspirin; NC = nonclosure; PFA-100 = platelet function analyzer 100; RCo = ristocetin cofactor; RPFA = rapid platelet function assay; SPD = storage pool disease; vWD = von Willebrand disease; vWF = von Willebrand factor

I

n recent years considerable progress has been made in the determination of platelet function, and reviews have recently been published that provide an excellent overview of these novel platelet function tests.1,2 The purpose of the current review is to summarize the clinical studies testing the PFA-100 device and also in vitro experiments that help us understand what can be measured with the PFA-100. For this purpose, data were retrieved from publications cited in MEDLINE and other peer-reviewed papers referenced in these articles. The technical properties of this instrument have been described in detail previously,3,4 so only the basics of the mechanism of this device will be explained.

From the Department of Clinical Pharmacology-TARGET, University of Vienna. Supported in part by a grant from the Jubiläumsfonds der Österreichischen Nationalbank. Submitted for publication February 20, 2001; revision submitted May 8, 2001; accepted May 24, 2001. Reprint requests: Bernd Jilma, MD, Department of Clinical Pharmacology-TARGET, The Adhesion Research Group Elaborating Therapeutics, Vienna University, Währinger Gürtel 18-20, A-1090 Wien, Austria. Copyright © 2001 by Mosby, Inc. 0022-2143/2001 $35.00 + 0 5/1/117406 doi:10.1067/mlc.2001.117406 152

PRINCIPLE OF THE TEST SYSTEM

The instrument aspirates a blood sample under constant vacuum from the sample reservoir through a capillary and a microscopic aperture (147 µm) cut into the membrane. The membrane is coated with CEPI or CADP. The presence of these platelet activators and the high shear rates (5000 to 6000 s–1) under the standardized flow conditions result in platelet attachment, activation, and aggregation, building a stable platelet plug at the aperture.5 The time required to occlude the aperture is reported as CT, and measurements are finished after a maximum of 5 minutes. In vitro experiments have demonstrated that antibodies against GPIb, GPIIb/IIIa, vWF, and RGDS peptide—but not against fibrinogen—induce a dose-dependent prolongation of CTs.6 This study indicated that the primary adhesion process occurs through vWF-GPIb interaction and that platelet plug formation involves the interaction of vWF with GPIIb/IIIa. These findings were confirmed and extended by another study with various antibodies against vWF or GPIb.7 Two types of cartridges have been developed to differentiate between the aspirin-induced defect (which can be detected with the CEPI cartridge) and more severe platelet dysfunction (detected with the CADP cartridge). In clinical practice the CEPI cartridge is used first: when CEPI is abnormal, a second measurement is made with the CADP cartridge.4,8 Normal values of CT are provided in Table I. Most of the cited arti-

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cles included a control group; thus the percent differences mentioned in this review are in most cases defined as the difference versus controls in crosssectional trials or versus baseline in longitudinal studies. Influence of Hct and platelets. Samples with a 10% Hct or a platelet count of 10 × 109/L will result in nonclosure.9 Such samples may still be used for identification of patients in need of platelet transfusions. CEPICT increased more (~60%) than CADP-CT (~25%) when the Hct levels were lowered from 40% to 20% in vitro.9 Studies with a large sample size demonstrate inverse correlations of CT values with platelet counts and Hct (r2 = approximately 0.1 to 0.2). Further evidence for an inverse correlation between Hct and CT values has been observed in newborns,10 cirrhotic or uremic patients, and patients with sickle cell disease (see below).11 Blood sampling. Blood sampling through a 21-gauge versus a 23-gauge needle does not affect CT values.12 Samples drawn into vacuum blood-collection tubes were not different from those drawn with a syringe.7 Finally, PFA-100 results are comparable when blood is sampled by direct venipuncture or from an intravascular sheath.13 Anticoagulants. Whole blood anticoagulated with 129 mmol/L buffered citrate (3.8%) exhibited higher CT values than blood sampled into 100 to 106 mmol/L citrate14,15; this is likely related to its stronger calcium chelation properties. The PFA-100 system has built-in safeguards to detect an abrupt reduction in blood flow (“flow obstruction”) during a test. This feature warns the user of a potentially aberrant result caused by the obstruction of the blood path caused by microthrombi, clots, or particulates. Significantly more flow obstructions occurred in unbuffered 106 mmol/L citrated blood than in buffered blood (100 to 129 mmol/L citrate). This might be caused by platelet clumping around the aperture.14 Similarly, in one study, samples from patients taking 100 mg/d aspirin were unstable during the first 10 minutes after blood collection into 106 mmol/L citrate (substantial decrease in CT over time), whereas CT values did not decrease significantly over 1 hour in samples anticoagulated with 129 mmol/L buffered citrate.15 Hence, for aspirin testing there could be certain advantages to using 129 mmol/L rather than 106 mmol/L citrate as anticoagulant. Another anticoagulant that has successfully been tested recently is D-phenylalanyl-L-propyl-L-arginine chloromethyl ketone (5 µmol/L).13 Stability. Samples were shown to be stable for up to 4 to 6 hours at room temperature, and thereafter the CEPI-CT values preferentially increased.9,14 CV% values. Harrison et al9 measured CEPI-CT and

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CADP-CT on three different machines and found a CV% of 6% to 8% for CEPI-CT and 9% to 10% for CADP-CT. Heilmann et al14 reported that duplicate measurement with CEPI and CADP cartridges exhibited a CV% of 12% to 13% and 10%, respectively. Marshall et al16 also reported a CV% of 12% for CEPI-CT as compared with 23% for the Simplate method. Day-to-day and diurnal variability. Individual day-today variability of CEPI-CT and CADP-CT averaged 9% and 12%, respectively.17 Dalby et al18 reported that measurements with the PFA-100 analyzer exhibited a diurnal variation, with about 30% longer CEPI-CT values at 5 PM than at 8:30 AM. As a consequence, it may be advisable to watch out for such a diurnal variation in other studies and to design studies accordingly. Additionally, the authors linked this observation to the wellknown morning peak of acute cardiovascular events. Necessity to perform duplicate testing. A low number of samples needed retesting (1.45%) because of an unacceptable variation of 20% between the duplicates (0.8%) or because of flow obstruction (0.65%). This suggests that in routine usage there is no need to perform duplicate testing.19 Dean et al20 recommended that if CT values are prolonged, this should be followed up with a full vWD workup in children, but if they fall in the normal range on two separate occasions, the work-up does not need to be done. PFA-100 versus aggregometry. The clinical sensitivity (94% to 95%) and specificity (88% to 89%) were virtually identical for PFA-100 and aggregometry in a study involving 176 patients and 206 control subjects.8 The sensitivities for PFA-100 and aggregometry to detect an aspirin-induced defect were 96% versus 100% and those to detect vWD and Glanzmann thrombasthenia were 96% versus 80%, respectively. In another trial, 100 patients undergoing cardiac artery bypass grafting were examined. All patients who had taken aspirin within the last 2 days had abnormal platelet aggregation, but only 33% had prolonged CEPI-CT values.21 However, it remains unclear whether results from PRPaggregation reflect the in vivo hemostatic capacity of platelets better than whole blood platelet function measurements with the PFA-100 device. An animal study has recently compared the effects of propofol infusions on platelet function measured with PFA-100 and aggregometry.22 Both methods turned out to be equally sensitive to the antiplatelet effects of this anesthetic drug. PFA-100 versus template BT. Francis et al22 screened 113 patients on whom a template BT was requested with the BT and PFA-100. In 74% of these patients, concordant results were obtained with both methods (either normal or abnormal values with both methods). The majority of patients (23/29) with discordant results had abnormal CEPI-CT but normal BT; 17 of

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Table I. Normal values of CTs induced by CEPI and CADP

Year

1995 1995 1997 1997 “ 1998 1998 “ 1998 1998 “ “ 1998 1999 1999 1999 1999 1999 1999 1999 1999 1999 1999 “ “ 1999 1999 2000 2000 “ 2000 2000 2000 “ “ “ 2000 2000 2000 2000 2000 2001 “ 2001 “ 2001 2001 “

Author

Mammen Kundu Marshall Heilmann “ Mammen Carcao & Rand “ Fressinaud Knöfler “ “ Harrison Boeck Cattaneo Cattaneo Escolar Meskal Feuring deMeijer Sestito Kereny Suzuki “ “ Favaloro Kottke-Marchant Homoncik von Pape “ Hezard Hergovich Dalby “ “ “ Ortel Schlammadinger Rein Homoncik Meyer Borzini “ Moeller “ Jilma-Stohlawetz Lippi “

Country

US US UK US “ US Can Can “ Fr Ger “ “ UK Ger Italy Italy Spain Bel Ger Neth Italy Hung Japan “ “ Austl US Aus Ger “ Fr Aus UK “ “ “ US Hung US Aus Fr Italy “ Ger “ Aus Italy “

Age (y)

— — 27-50 — — 18-67 0.3-17 25-54 Neonates 15-65 Neonates Infants Adults — Donors — — 45 ± 9 — 26-88 19-45 35-72 — — — — — — 23-32 — — — 19-35 — — — — — 32 ± 9 24-50 19-35 — Neonates Adults Donors — 19-49 1-15 —

n/sex

— 28/25 12m n = 36 — 206(62%f) n = 57 n = 31 n = 17 31m:65f n = 11 n = 17 n = 35 12m:8f 110m:199f n = 40 n = 40 11m:9f — 7m:3f 8m:7f 33m:29f n = 31 20f 36 pregnant 2nd trimester 18 — 4m:6f n = 20 n = 20 n = 29 15m 08:00h 08:30h 12:00h 17:00h — 4m:16f 12m:18f 30m n = 147 — — 162 Non-O blood group 30m:40f 34m:18f Non-O blood group

AC (mmol/L)

— — — 129 105 129 — — — 129 — — — 105 105 129 — 129 — 106 — — B105 — — — 105 B129 129 129 106 B129 129 — — — — 129 — — 129 — 126 125 — — 129 — —

Publications are ranked according to the publication year. All available data were used; missing information was not found in the publications. AC, Anticoagulant (citrate). Aus, Austria; Austl, Australia; B, buffered; Can, Canada; f, female; Fr, France; Ger, Germany; Hung, Hungary; m, male; min, mimimum; max, maximum; Neth, Netherlands; XO, crossover design; y, years.

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Table I. (continued) CEPI-CT

CADP-CT

Mean/median*

SD

SEM

Min

Max

— — 115-123 XO 124 113 132 117 106 81 120 81 108 107 — 110 121* 121* 113 — 92 128 — — 133 98 — 128 — 117 97 114 — 143 120* 114* 130* 141* — — — 137/123* 122 — — 112 104 140* 120 104

— — — — — — 23 21 17 20 — — — — 22 — — — — — 14 — — 18 21 — — — — — — — — — — — — — — — — 22 — — 15 17 46 25 21

— — — — — — — — — — — — — — — — — 5 — 7 — — — — — — — — — — — — 9 — — — — — — — 8 — — — — — 5 — —

— — 83 — — — — — — 77 — — — 74 — 61 69 — — — — — — — — — — — 69 84 76 — 96 87 91 99 97 — — 61 96 77 — — — — 79 — —

— — 161 — — — — — — 186 — — — 146 — 203 203 — — 47 — — — — — — — — 166 156 132 — 220 300 145 159 175 — — 180 276 191 — — — — 300 — —

Reference range

77-133 97-188 — 89-165 84-153 94-191 83-163 82-142 –108 80-160 — — — — –150 — –197 — 96-162 –137 — — 63-142 — — — 98-158 98-170 — –170 –150 94-191 — — — — — 94-193 65-142 — — — 90 75 — — — — —

Mean/median*

— — — 87 80 93 91 85 56 89 65 86 74 — 78 92 92* 87 — 97 — 97 — 93 77 — 95 — 92 89 89 — 106 93* 85* 95* 88* — — — 99/94* 90 — — 91 86 98* 88 87

SD

SEM

Min

Max

Reference range

— — — — — — 13 16 16 15 — — — — 12 — — — — — — 24 — 17 29 — — — — — — — — — — — — — — — — 15 — — 13 12 22 17 20

— — — — — — — — — — — — — — — — — 4 — 8 — — — — — — — — — — — — 6 — — — — — — — 4 — — — — — 2 — —

— — — — — — — — — 66 — — — 59 — 54 54 — — — — — — — — — — — 69 59 41 — 63 67 60 68 73 — — 52 68 65 — — — — 59 — —

— — — — — — — — — 126 — — — 112 — 187 187 — — 43 — — — — — — — — 121 119 107 — 145 175 189 154 135 — — 133 169 132 — — — — 160 — —

— 63-123 — 66-115 61-105 72-120 72-111 –111 –65 59-120 — — — — –100 — –140 — 71-99 –150 — — 55-118 — — — 66-124 77-133 — –120 –110 72-120 — — — — — 71-118 55-118 — — — — — — — — — —

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these 23 had platelet aggregation abnormalities compatible with recent aspirin intake. This finding supports the conclusion that the PFA-100 is more sensitive than the BT for such an “aspirin defect”.22 The sensitivities of both methods to detect vWD are discussed below. NORMAL RANGES

An overview of normal ranges is depicted in Table I. Once again it should be emphasized that the CT values are directly correlated with the citrate concentration. We and others23 have occasionally found CEPI-CT values of >300 seconds even in healthy volunteers (without bleeding history), possibly because of surreptitious intake of aspirin. Children and neonates. Whereas children have slightly (5% to 10%; P > .05) prolonged CT values as compared with healthy adults, neonates exhibit 25% to 35% shorter CT values than healthy volunteers, even after adjustment for Hct levels.12 At the same time, Knöfler et al10 found ~30% shorter CT values and blood flow velocities in newborns. In another trial, neonates had 10% shorter CADP-CT but similar CEPI-CT values when compared with an adult control group.24 It may be that the shorter CT values in neonates are caused by elevated vWF levels or large multimers of vWF in cord blood.25-27 In the Knöfler et al study, birth weight determined Hct (r2 = 0.67), and both of these factors correlated inversely with blood flow velocity in both cartridge types (r2 = 0.41 to 0.74). Similar results were obtained in another study.25 These observations further support the belief that higher Hct levels promote platelet plug formation. The umbilical cord arterial pH correlated with CT, potentially indicating that lower pH values are associated with more stressful labor and hence increased platelet activation. Pregnant women. The shortest CT values are observed during the second trimester (CEPI-CT, –26%; CADPCT, –17%; as compared with non-pregnant women).28 Sex and smoking. Smokers (n = 124) had ~3% (not significant) longer CEPI-CT than non-smokers (n = 121), despite 10% higher fibrinogen levels.19 CT values are independent of sex, and intake of oral contraceptives does not seem to affect CT values.19 This agrees with the lack of sex difference in vWF levels.29 Low platelet collagen receptor (α2β1-integrin) expression prolongs CT. Because both cartridges of the PFA-

100 device are coated with collagen, it seems logical that alterations in collagen receptor density might affect CT values. This notion is supported by two studies. First, the frequency of the 807C allele of the α2-integrin, which is associated with low collagen receptor levels (α2β1-integrin), was higher in patients with type 1 vWD than in normal subjects. At vWF:RCo levels <30 U/dL, the vWF deficiency overrides the allele

effect on CT. When vWF:RCo levels reach values between 40 and 80 U/dL, type 1 vWD patients homozygous for the 807C allele have significantly longer CT values. Differences in the degree of α2β1-integrin may thus contribute to the variable bleeding tendency observed among patients with type 1 vWD.30 Second, in our limited sample size, the only subject homozygous for the 807T allele (with high collagen receptor levels) had the shortest basal closure time and responded least to aspirin treatment.17 Finally, we observed that PFA-100 values correlated with α2β1-integrin density measured by flow cytometry.39 These data suggest that high collagen receptor expression is associated with short CT even in a normal population. PLATELET DISORDERS Glanzmann’s thrombasthenia and Bernard-Soulier syndrome. Three studies have reported that patients with

Glanzmann’s thrombasthenia exhibit NC (n = 6, 3, and 1).9,31,32 In an additional study, Mammen et al8 described 5 patients with Glanzmann’s thrombasthenia, all of whom had markedly prolonged CT and BT. Together these data indicate 100% sensitivity to detect Glanzmann’s thrombasthenia. Of 2 patients described with Bernard-Soulier syndrome, 1 had NC and the other had prolonged CT values.9 Aspirin-like defect. In a field study, 86% of 68 patients with prolonged CEPI-CT (but normal CADP-CT) had an aspirin-like defect when examined with platelet aggregation.33 Storage pool disease. Cattaneo et al34 demonstrated 50% longer CEPI-CT but normal CADP-CT in patients with δ-storage pool deficiency or primary secretion defect (2/7 and 1/10 NC, respectively). The sensitivities of BT and CEPI-CT were 41% and 47%, respectively. In contrast, Fressinaud et al31 found very prolonged CT with both cartridges. In another study, Harrison et al demonstrated prolonged CEPI-CT values in all 6 patients with Hermansky-Pudlak syndrome and in all 6 patients with SPD, while CADP-CT identified only half of the patients with SPD.9 Kereny et al32 showed prolonged CEPI-CT (but normal CADPCT) in 4 of 5 albino children with Hermansky-Pudlak syndrome, all of whom had BT >15 minutes. In general, SPD is characterized by prolonged CEPICT but normal CADP-CT. vWF, ABO blood groups, vWD. In contrast to BT,35 several studies demonstrated a correlation of CT values and vWF levels even in a healthy population.17,23,34,36,37 Along similar lines, CT values are 10% to 20% longer in blood group O than in other blood groups,23,38-40 likely because vWF levels are lower in blood group O than in other blood groups.35 A detailed review of the performance of PFA-100 in

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vWD has been provided recently.41 The BT has long been recommended as a screening test for defects of primary hemostasis,42 although it is now considered useless in the perioperative setting.43 The sensitivity of the PFA-100 device to detect vWD is considered much better than the sensitivity of BT. The sensitivity of the PFA100 depends on the severity of vWD44 and reaches up to 100% for severe forms of vWD. The sensitivity of the PFA consistently proved to be higher (84% to 97%) than that of the BT (about 48% to 66% in larger studies).20,31,32,36 In one study, the CEPI cartridges were more sensitive for mild vWD.44 In contrast, the CADP cartridges were more sensitive in another study.31 The high sensitivity makes it a good screening test for vWD in adults and infants. Schlammadinger et al44 compared the performance of the PFA-100 to the O’Brien filter test and found both high-shear test systems superior to the BT, especially in mild vWD or type 2B vWD. Patients with acquired vWD (normal vWF levels in platelets) had shorter CT values than patients with type 3 vWD despite similar plasma vWF levels,36 indicating that both plasma and platelet vWF contribute to aperture closure in this system. In a field study, 83% of patients tested with prolonged CADP-CT had abnormalities in ristocetin-induced agglutination.33 In the same group of patients, 7 of 33 patients (or 2.3% of the total population tested) had decreased vWF levels. Only 3 patients (~1%) with prolonged CADP-CT had normal aggregation data and normal vWF levels. In conclusion, the PFA-100 system is sensitive to quantitative and qualitative abnormalities not only of plasma vWF but also of platelet vWF. APPLICATIONS FOR CLINICAL PHARMACOLOGY Desmopressin, vWF concentrates, and systemic inflammation. Desmopressin (0.3 µg/kg), which releases vWF

from the Weibel-Palade bodies of endothelial cells, shortened CEPI-CT by 40% to 50% and CADP-CT by 42% in patients with SPD. In contrast, aggregometry has been reported not to be sensitive to desmopressin.45 Desmopressin corrected CEPI-CT and CADP-CT in all 23 patients with type 1 vWD examined in the study by Fressinaud et al.46 For comparison, BT was corrected in all but one of these subjects. The estimated duration of effect (PFA and BT) was <6 hours based on 3 patients who were studied repeatedly. Desmopressin also normalized CEPI-CT and CADP-CT values in 9 of 11 and 7 of 11 patients with type 2 vWD, respectively. Similarly, DDAVP shortened CEPI-CT and CADP-CT in a 4-year-old child.47 Another mechanism that stimulates degranulation of the endothelial storage pool of vWF is systemic inflammation.48 Accordingly, we have recently shown that infusion of endotoxin into healthy humans shortened

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CT values of all subjects by 40% to 50%. In one of these subjects we observed that prolonged CT values and borderline vWF levels normalized during systemic inflammation. Hence CTs are influenced by fluctuating vWF levels in patients with systemic inflammation, which should be considered for correct interpretation of normal and abnormal CTs.37 In contrast to desmopressin, infusion of vWF concentrates to patients failed to shorten CT values in 8 of 9 patients who were unresponsive to DDAVP, despite normalization of vWF:RCo activity.46 This was also shown for 1 case of type III vWD.49 The authors provided two explanations for this observation. First, no concentrate exhibits an intact multimeric structure, and the large multimers are known to be most effective in primary hemostasis. Second, the concentrates do not correct the platelet vWF defect, which is essential for hemostasis.50,51 This notion is supported by the finding that type 1 patients with low platelet vWF did not respond well to desmopressin infusion. Together these data support the concept that PFA-100 is sensitive not only to plasma vWF but also to platelet vWF.36 In sum, the PFA-100 system can be used to discriminate between responders and non-responders to desmopressin but may not always correlate with vWF:RCo activity after infusion of vWF-concentrates. Finally, systemic inflammation has to be ruled out when accurate assessment of CT or vWF values is mandatory. Aspirin. In general, aspirin has no or little effect on CADP-CT.8,9,13,17,52 In vitro incubation of blood with 100 mg/L L-ASA for only 3 minutes is sufficient to achieve NC with CEPI cartridges.17 Interventional trials SINGLE DOSE, 1000 MG. Aspirin infusion dose-dependently prolongs CEPI-CT.17 Even after 1000 mg L-ASA intravenously, 2 of 10 subjects did not exhibit NC. Seventy-two hours after the end of infusion, CEPI-CT decreased from 283 to 167 seconds. A similar reduction was observed 3 to 4 days after discontinuation of aspirin therapy (before surgery).15 Yet in both studies, more than 4 days were necessary for complete recovery of normal platelet function. SINGLE DOSE, 325 MG. When 127 subjects were tested 2 to 30 hours after ingestion of 325 mg aspirin, 95% demonstrated prolonged CEPI-CT.8 SINGLE DOSE, 300 MG. Two hours after the intake of 300 mg aspirin, CEPI-CT was prolonged in 5 of 6 volunteers.9 MULTIPLE DOSE, 750 MG. Aspirin (750 mg, three times a day) for 5 days increased CEPI-CT from 123 to 217 seconds in Marshall’s trial.16 CEPI-CT and BT increased by 79% and 61% as compared with placebo. However, aspirin induced NC in 3 of 12 healthy subjects and failed to exhibit an effect in 1 of 12 subjects, despite complete inhibition of platelet cyclo-oxygenase.16

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MULTIPLE DOSE, 100 MG. Constant intake of 100 mg aspirin per day for 11 days increased CEPI-CT by a maximum of 110% but induced NC only in 50% of all volunteers.17 Subjects with NC and those with CEPI-CT <300 seconds were not different with regard to inhibition of thromboxane production (mean –90%). Cross-sectional trials. In a field study, 68% of 88 patients who reported taking aspirin had prolonged CEPI-CT values.33 Another study showed that 93% of patients undergoing long-term aspirin therapy (100 mg/d) had CEPI-CT >170 seconds and 48% >300 seconds before discontinuation of aspirin for surgery. Unfortunately, no information on other relevant co-medication such as non-steroidal antiphlogistic drugs was given.15 Only 31% of 48 patients with coronary artery disease had prolonged CEPI-CT values, and 10% had CEPI-CT of >300 seconds on constant treatment with 100 mg/d aspirin.53 Only 10/14 patients with antiphospholipid syndrome who were taking aspirin had prolonged CEPI-CT (NC only in 3/14).9 Hence the PFA-100 provides a tool to identify patients who take aspirin and those who are insensitive to aspirin. The reason for non-responsiveness may include noncompliance or pharmacodynamic non-responsiveness, which could be caused by overcompensation via other hemostatic mechanisms that do not depend on intact thromboxane metabolism. We were able to show that basal CEPI-CT predicts maximal response (CEPI-CT) to aspirin after intake of either 50 or 100 mg aspirin (r2 = 0.72; P < .005).17 Along similar lines, vWF-Ag levels determine basal CEPI-CT (r2 = 0.56) and aspirin-induced CEPI-CT (r2 = 0.81). Finally, we identified in our small sample that 1 subject, who had the highest density of collagen receptor (807T/T polymorphism of the α2-integrin), had the shortest basal CEPI-CT and responded most poorly to aspirin intake. Meloxicam and indomethacin. The PFA-100 has been used to discriminate between nonsteroidal antiphlogistic drugs with mixed or preferential inhibition of COX1 and COX2, because platelet production of thromboxane is exclusively mediated by COX-1. The preferential COX-1 inhibitor indomethacin inhibited thromboxane B2 production by 95% and prolonged CEPI-CT to >300 seconds in 13 of 15 subjects. In contrast, the more COX-2–selective inhibitor meloxicam reduced thromboxane B2 levels by 66% but increased CEPI-CT by only 10%.54 This suggests that only marked inhibition of thromboxane B2 production affects CEPI-CT. Glycoprotein IIb/IIIa inhibitors. In vitro incubation with abciximab showed that NC was reached at a concentration of 2.25 mg/mL (with a steep increase between 1.25 and 1.75 mg/mL). An abciximab infusion (c7E3 Fab 0.25 mg/kg bolus, followed by 10 µg/h for 12 hours) induced NC in all 10 patients until 4 hours after the end of infusion.13

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Similarly, infusion of the same abciximab dose regimen uniformly induced abnormal CT values in 15 patients undergoing percutaneous transluminal coronary angioplasty (93% of GPIIb/IIIa receptors were blocked at 10 minutes).52 The effect began to fade at 24 hours (receptor blockade 71%, 10/15 patients had abnormal CADP-CT values), and after 48 hours, only 1 of 11 patients still had abnormal CADP-CT values. Further, the switch from a negative to a positive PFA-100 system result occurred in a narrow range of blocked receptors (60% to 75%, measured by flow cytometry). Similar results were obtained in a different trial.55 In this series of patients undergoing angioplasty, the only postprocedural myocardial infarction was observed in 1 patient who did not achieve adequate platelet inhibition (CT 137 seconds, 10 minutes after the abciximab bolus). This raises the question of whether the level of platelet inhibition is in fact predictive of future clinical events, which should be addressed in a sufficiently powered clinical trial. Gurbel et al56 measured fibrinogen binding (RPFA), CT, and platelet aggregation (ADP) before and after 1 month of oral GPIIb/IIIa administration (roxifiban). In patients with high baseline platelet activity, as determined by aggregometry in response to ADP, the respective indexes changed by –89% (RPFA), +156% (CT), and –62% (aggregation with 5 µmol/L ADP). In patients with average baseline platelet activity, the following changes occurred: –64% (RPFA), +109% (CT), and –65% (aggregation with 5 µmol/L ADP). The authors concluded that their findings challenge the “one-sizefits-all” approach for chronic antiplatelet regimens and suggested individual dosing of these drugs in the future. Above all, this study compared the sensitivity of three different assays in response to oral GPIIb/IIIa inhibitors. Remarkably, not even a 90% inhibition of fibrinogen binding was associated with maximal CT prolongation (228 ± 32 seconds rather than >300 seconds). This could indicate that platelet plug formation may not simply be predicted from studies of fibrinogen receptor occupancy. Together with the case report of rare afibrinogenemia,9 this indicates that in high-shear conditions, PFA100 is sensitive to the inhibition of GPIIb/IIIa ligands other than fibrinogen. One case report indicates that inhibition of platelet function by an orally active GPIIb/IIIa inhibitor (roxifiban) shows marked day-to-day variability during a 4week observation period.57 Hence the PFA-100 analyzer can be used to monitor therapy with GPIIb/IIIa inhibitors. Are CT values predictive? A relevant question is, how can arithmetic percent deviations of CT values be transferred in the clinical context? For example, which percent deviation of CT values predisposes to

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bleeding? In terms of inherited platelet defects or vWD, it seems quite evident that a simultaneous increase in CEPI-CT and CADP-CT carries a risk for bleeding. Similarly, bleeding is a relevant adverse effect of GPIIb/IIIa inhibitors, which prolong both CEPI-CT and CADP-CT mostly >300 seconds. Hence, NC, particularly when it occurs with both cartridges, can be considered a relevant risk factor for bleeding. How about aspirin? In terms of bleeding it has been shown that cyclooxygenase inhibitors enhance fecal blood loss even in healthy volunteers.58 Thus, even isolated prolongation of CEPI-CT may be associated with bleeding risk. In contrast, it remains to be defined whether a certain percent prolongation in CT values is associated with a certain percent increase in the bleeding risk. In general, severe bleeding even caused by thrombocytopenia is rare and occurs only in patients with a concomitant coagulopathy or anatomic defects in the vascular system.59 Thus it is probably necessary to define cut-off CT values for bleeding risk for different patient groups individually. Conversely it may be asked what CT values are needed to effectively prevent the occurrence of ischemic events in cardiovascular therapy? Although it appears plausible that patients with cardiovascular disease could profit from monitoring of platelet inhibition, hard evidence for this concept is still lacking. Smaller studies suggest that in angioplasty procedures it is necessary to obtain >80% inhibition of platelet aggregation 60 or NC with the PFA-100 system 55 to prevent postprocedural cardiovascular complications. Yet welldesigned large55 prospective trials are still lacking to support this concept. Thienopyridines (ticlopidine and clopidogrel). Ticlopidine (in combination with aspirin) did not affect CADPCT values in a small cross-sectional study13 that was confirmed by another trial.52 This seems to be in contrast to effects of thienopyridines on BT61 or aggregometry.13 Two cases of prolonged CEPI-CT and CADP-CT were reported in patients receiving ticlopidine or clopidogrel therapy. Thus, available data suggest that the current setup of the PFA-100 device is not suitable for monitoring therapy with thienopyridines, although the combination of aspirin and thienopyridines may slightly prolong CT values.13,62 Yet data from a longitudinal interventional trial evaluating the long-term effects of thienopyridines are still lacking. Contrast media. When contrast media were added 10% by volume to (105 mmol/L buffered) citrated blood they prolonged CADP-CT. This effect was more marked with ionic media than with non-ionic media: tiatrizoate prolonged CADP-CT to >300 seconds, whereas iohexol-treated samples exhibited median CADP-CT values of 170 seconds.63 Heparin. Patients with pulmonary embolism who were

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Fig 1. Simulation of prolonged CT and two different kinetics.

treated with heparin (0.66 IU/mL antifactor Xa activity) had normal CT values.52 Similarly, heparin did not prolong CT in 10 patients with coronary artery disease.13 Hydroxyethylstarch and gelatin. Hydroxyethylstarch (200 kd) was found to prolong CADP-CT in otherwise healthy subjects with minor surgery.64 Another study showed a 20% to 30% prolongation in CADP-CT 2 days after major abdominal surgery in three groups of patients treated with 70 kd or 200 kd hydroxyethylstarch or gelatin.65 Anesthetic drugs. Propofol prolongs CT values in animals,22 and high concentrations of ropivacaine have similar effects in vitro.67 The concentrations of ropivacaine studied correspond to those that could occur in the epidural space. Thus the authors concluded that potential antiplatelet effects of ropivacaine in the epidural space may decrease the efficacy of an early or prophylactic epidural blood patch. Yet data in human subjects are not available. Nutrition. Intake of 19 g cocoa powder but not a comparable dose of caffeine prolonged CEPI-CT by 44% and CADP-CT by only 13%. Thus cocoa had an aspirin-like effect at 6 hours.67 Because it is conceivable that other dietary products may also affect CT values, it seems advisable to perform elective PFA-100 measurements after an overnight fast. APPLICATIONS FOR PATIENT MONITORING AND DIAGNOSTICS

The following paragraphs summarize applications in clinical settings according to the frequencies in which they may occur. Menorrhagia. Menorrhagia, excessive menstrual bleeding, affects many women of child-bearing age and is a frequent cause of hysterectomy. Recently a preliminary study in 43 women with menorrhagia showed that 25% of these women had an aspirin-like defect, 2 suffered from vWD, and 3 had a platelet defect.68 Currently a larger

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multicenter trial is being conducted, because identification of underlying hemorrhagic diathesis in menorrhagia may potentially prevent hysterectomy in a considerable number of premenopausal women. Surgical patients and surgery. In the absence of a history of excessive bleeding, the BT fails as a screening test and is, therefore, not indicated as a routine preoperative test.43 Limited data are available regarding the usefulness of the PFA-100 device in the perioperative setting. The majority (82%) of patients undergoing aortic or mitral valve replacement (n = 90) had abnormal platelet function and 11% had an “aspirin effect.” In contrast, abnormal platelet function and an aspirin effect were seen in 16% and 43% of patients undergoing coronary artery bypass grafting (n = 306), respectively.69 Interestingly, chart reviews of patients with CADP-CT >300 seconds revealed that 3 of such 6 patients having valve replacement bled excessively. Long preoperative CT correlated with increased blood loss in patients undergoing open heart surgery (r = 0.41).70 No correlation was found between preoperative or postoperative CT values and blood loss in a very small study with 12 patients undergoing cardiopulmonary bypass,71 although the power of that trial is limited. Another study with 146 patients undergoing coronary artery bypass grafting found no correlation between PFA values and blood loss.38 However, this study enrolled just a small number of patients with only moderately prolonged CEPI-CT (n = 6) or CADP-CT (n = 4) values. Hence platelet dysfunction seems to be quite prevalent in patients with valve disease but less so in patients undergoing coronary artery bypass grafting. The former group of patients could be prone to excessive bleeding, which may be foreseen by PFA-100 analysis. Uremia and liver cirrhosis. One report showed ≥60% and ≥34% prolonged CT in uremic and cirrhotic patients, respectively. In vitro adjustment of the Hct levels only partially corrected this defect.11 Hematologic abnormalities Patients with hemophilia. Adults

and children with hemophilia do not have prolonged CT,7,12,31,47 indicating that deficiency in factor VIII and IX does not affect CT values. Afibrinogenemia. Patients with afribinogenemia were found to have normal or only slightly prolonged CT values,9 consistent with the lack of effect of antibodies against fibrinogen on CT.6 Sickle cell anemia and thalassemia. Both patient groups have slightly prolonged rather than shortened CT values.9 Ortel et al33 also reported prolonged CADP-CT values in 29% of 24 patients with sickle cell anemia. These patients had lower Hct levels (22% Hct) than sickle cell patients with normal CT values (29% Hct).

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Antiphospholipid syndrome. One of 6 patients with antiphospholipid syndrome had shortened CADP-CT.9 Transfusion medicine Screening of apheresis platelet donors.

Donors who have taken aspirin or other medications that alter platelet function within the previous 3 days are usually deferred, because the platelets obtained by apheresis are often the single source of platelets given to a patient (AABB Technical Manual, 12th ed; 1996; p. 117; annotation: number of days may differ between countries). Currently most centers rely on the donors’ history and on a questionnaire. Yet we have previously explored the suitability of the PFA-100 device to screen for an aspirin-like defect in 206 apheresis donors.39 Our results showed that one fifth of apheresis donors had abnormal CEPI-CT values and 11% exceeded >300 seconds (NC). Decreased serum thromboxane B2 levels were found in 9% of all donors, which is compatible with surreptitious intake of cyclooxygenase inhibitors or with an aspirin-like defect. The high prevalence of an aspirin-like defect may justify screening of platelet donors with the PFA-100 immediately before apheresis and not relying on questionnaires alone. Platelet concentrates. Borzini et al72 have provided pioneer data for the quality control of platelet concentrates during storage. They have resuspended stored platelet concentrates (final concentration 230 × 109/L) with washed O group packed red cells (final Hct 58%) and thawed AB group fresh frozen plasma. Both cartridges demonstrated a significant deterioration in hemostatic function during storage of platelets—that is, prolongation of CT. Furthermore, Borzini et al calculated a predictive index to overcome the problem of infinite CTs (using two experimental parameters measured by the PFA-100, total flow volume and initial flow rate), as follows: PFA-PI = (total flow volume [in µL]:flow rate [in µL/min] × closure time [in seconds]/100, where PI stands for predictive index. Although infinite CT values were reached after 4 days of platelet storage, there was a continual increase in the predictive index beyond this period. In another interesting report, the hemostatic function of cryopreserved platelets was measured in vitro.73 After cryopreservation, platelet function was greatly diminished for both ThromboSol- and dimethyl sulfoxide–treated platelets and was partly restored after washing of ThromboSol-treated platelets. Although both studies used the predictive index, it should be mentioned that total flow volume and flow rate are parameters that currently cannot be considered standard procedure in all countries. It may be worthwhile to follow up the validity of this interesting index in other settings, such as the effects of aspirin on platelet function.

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It could also be of possible interest to use an algorithm to evaluate the kinetics of the CT besides the CT value itself. For example, the same CT may derive by different closure kinetics (see Fig 1). Thus it could be possible to calculate the area under the curves for flow rates over time. However, it remains to be demonstrated whether this or another procedure of evaluating the kinetics of the CT curves can enhance sensitivity of the PFA-100 for platelet dysfunction.

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CONCLUSIONS

The PFA-100 device provides an attractive test not only for the specialized hemostasis laboratory but also for small, non-specialized hematology laboratories, pharmaceutical research and development groups, and hospital departments interested in platelet function. The beauty of the system is its inherent simplicity and ease of use without the need for operators to have specialized knowledge or training and its remarkably good precision and reliability. Hence the PFA-100 facilitates rapid diagnosis in a cost-effective way and could help to monitor antiplatelet therapy in the future.

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44. Schlammadinger A, Kerenyi A, Muszbek L, Boda Z. Comparison of the O’Brien filter test and the PFA-100 platelet analyzer in the laboratory diagnosis of von Willebrand’s disease. Thromb Haemost 2000;84:88-92. 45. Cattaneo M, Lombardi R, Bettega D, Lecchi A, Mannucci PM. Shear-induced platelet aggregation is potentiated by desmopressin and inhibited by ticlopidine. Arterioscler Thromb 1993;13:393-7. 46. Fressinaud E, Veyradier A, Sigaud M, Boyer-Neumann C, Le BC, Meyer D. Therapeutic monitoring of von Willebrand disease: interest and limits of a platelet function analyser at high shear rates. Br J Haematol 1999;106:777-83. 47. Rand ML, Carcao MD, Blanchette VS. Use of the PFA-100 in the assessment of primary, platelet-related hemostasis in a pediatric setting. Semin Thromb Hemost 1998;24:523-9. 48. Gralnick HR, McKeown LP, Wilson OM, Williams SB, Elin RJ. von Willebrand factor release induced by endotoxin. J Lab Clin Med 1989;113:118-22. 49. Meskal A, Vertessen F, van der Plaken, Berneman ZN. The platelet function analyzer (PFA-100) may not be suitable for monitoring the therapeutic efficiency of von willebrand concentrate in type III von willebrand disease. Ann Hematol 1999;78:426-30. 50. Castillo R, Escolar G, Monteagudo J, Aznar-Salatti J, Reverter JC, Ordinas A. Hemostasis in patients with severe von Willebrand disease improves after normal platelet transfusion and normalizes with further correction of the plasma defect. Transfusion 1997;37:785-90. 51. Fressinaud E, Federici AB, Castaman G, Rothschild C, Rodeghiero F, Baumgartner HR, et al. The role of platelet von Willebrand factor in platelet adhesion and thrombus formation: a study of 34 patients with various subtypes of type I von Willebrand disease. Br J Haematol 1994;86: 327-32. 52. Hezard N, Metz D, Nazeyrollas P, Droulle C, Elaerts J, Potron G, et al. Use of the PFA-100 apparatus to assess platelet function in patients undergoing PTCA during and after infusion of c7E3 Fab in the presence of other antiplatelet agents. Thromb Haemost 2000;83:540-4. 53. Feuring M, Haseroth K, Janson CP, Falkenstein E, Schmidt BM, Wehling M. Inhibition of platelet aggregation after intake of acetylsalicylic acid detected by a platelet function analyzer (PFA-100). Int J Clin Pharmacol Ther 1999;37:584-8. 54. de Meijer A, Vollaard H, de Metz M, Verbruggen B, Thomas C, Novakova I. Meloxicam, 15 mg/day, spares platelet function in healthy volunteers. Clin Pharmacol Ther 1999; 66:425-30. 55. Madan M, Berkowitz SD, Christie DJ, Jennings LK, Smit AC, Sigmon KN, et al. Rapid assessment of glycoprotein IIb/IIIa blockade with the platelet function analyzer (PFA100) during percutaneous coronary intervention. Am Heart J 2001;141:226-33. 56. Gurbel PA, McKenzie ME, Serebruany VL. Initial platelet activity may predict efficacy after chronic oral glycoprotein IIb/IIIa blockade: should we still consider uniform treatment regimens [letter]? Thromb Res 2000;99:105-7. 57. Serebruany VL, McKenzie ME, Levin DJ, Gurbel PA. Monitoring platelet inhibition during chronic oral platelet glycoprotein IIb/IIIa blockade: are we missing something? Thromb Haemost 1999;83:356-7. 58. Green D, Davies RO, Holmes GI, Kohl H, Lee RB, Reynolds N, et al. Effects of diflunisal on platelet function and fecal blood loss. Clin Pharmacol Ther 1981;30:378-84. 59. Vostal JG, Reid TJ, Mondoro TH. Summary of a workshop on in vivo efficacy of transfused platelet components and platelet substitutes. Transfusion 2000;40:742-50.

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60. Steinhubl SR, Kottke-Marchant K, Moliterno DJ, Rosenthal ML, Godfrey NK, Coller BS, et al. Attainment and maintenance of platelet inhibition through standard dosing of abciximab in diabetic and nondiabetic patients undergoing percutaneous coronary intervention. Circulation 1999; 100:1977-82. 61. Klinkhardt U, Kirchmaier CM, Westrup D, Graff J, Mahnel R, Breddin HK, et al. Ex vivo-in vitro interaction between aspirin, clopidogrel, and the glycoprotein IIb/IIIa inhibitors abciximab and SR121566A. Clin Pharmacol Ther 2000;67:305-13. 62. Fischetti D, Sciahbasi A, Leone AM, Niccoli G, Schiavoni G, Trani C, et al. Ticlopidine and aspirin fail to suppress the increased platelet aggregability that follows percutaneous coronary interventions. J Thromb Thrombolysis 2000;10: 265-9. 63. Dalby MCD, Dewar A, Davidson SJ, Burman JF, Davies SW. Evaluation of angiographic contrast media and platelet function with impedance aggregometry and the PFA-100 ‘platelet function analyser’. Platelets 1999;10:293-7. 64. Stogermuller B, Stark J, Willschke H, Felfernig M, Hoerauf K, Kozek-Langenecker SA. The effect of hydroxyethyl starch 200 kD on platelet function. Anesth Analg 2000;91:823-7. 65. Huttner I, Boldt J, Haisch G, Suttner S, Kumle B, Schulz H. Influence of different colloids on molecular markers of haemostasis and platelet function in patients undergoing major abdominal surgery. Br J Anaesth 2000;85: 417-23.

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66. Porter J, Crowe B, Cahill M, Shorten G. The effects of ropivacaine hydrochloride on platelet function: an assessment using the platelet function analyser (PFA-100). Anaesthesia 2001;56:15-8. 67. Rein D, Paglieroni TG, Wun T, Pearson DA, Schmitz HH, Gosselin R, et al. Cocoa inhibits platelet activation and function. Am J Clin Nutr 2000;72:30-5. 68. Lukes AS, Ortel T, Thames B, Walker C, Laskowitz J, James AH. Screening for underlying bleeding disorders in women with menorrhagia. Obstet Gynecol 2001;97:S22. 69. Francis JL. Platelet dysfunction detected at high shear in patients with heart valve disease. Platelets 2000;11:133-6. 70. Wahba A, Sander S, Birnbaum DE. Are in vitro platelet function tests useful in predicting blood loss following open heart surgery? Thorac Cardiovasc Surg 1998;46:228-31. 71. Dietrich GV, Schueck R, Menges T, Kiesenbauer NP, Fruehauf AC, Marquardt I. Comparison of four methods for the determination of platelet function in whole blood in cardiac surgery. Thromb Res 1998;89:295-301. 72. Borzini P, Lazzaro A, Mazzucco L. Evaluation of the hemostatic function of stored platelet concentrates using the platelet function analyzer (PFA-100). Haematologica 1999; 84:1104-109. 73. Borzini P, Lazzaro A, Mazzucco L, Schiavo R, Connor J, Siena S. Platelet cryopreservation using second-messenger effector and low-dose (2%) dimethyl sulfoxide. In vitro evaluation of post-thawing platelet activity with the platelet function analyzer. Haematologica 2000;85:885-7.