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Different methodologies for evaluating the effect of clopidogrel on platelet function in high‐risk coronary artery disease patients

2007; Elsevier BV; Volume: 5; Issue: 9 Linguagem: Inglês

10.1111/j.1538-7836.2007.02656.x

1538-7933

Rita Paniccia, Emilia Antonucci, Anna Maria Gori, Rossella Marcucci, Cristina Giglioli, David Antoniucci, Gian Franco Gensini, Rosanna Abbate, Domenico Prisco,

Acute Myocardial Infarction Research

Journal of Thrombosis and HaemostasisVolume 5, Issue 9 p. 1839-1847 Free Access Different methodologies for evaluating the effect of clopidogrel on platelet function in high-risk coronary artery disease patients R. PANICCIA, R. PANICCIA Department of Medical and Surgical Critical Care, Thrombosis Centre, Center for the Study at Molecular and Clinical Level of Chronic, Degenerative and Neoplastic Diseases to Develop Novel Therapies, University of Florence, FlorenceSearch for more papers by this authorE. ANTONUCCI, E. ANTONUCCI Department of Medical and Surgical Critical Care, Thrombosis Centre, Center for the Study at Molecular and Clinical Level of Chronic, Degenerative and Neoplastic Diseases to Develop Novel Therapies, University of Florence, FlorenceSearch for more papers by this authorA. M. GORI, A. M. GORI Department of Medical and Surgical Critical Care, Thrombosis Centre, Center for the Study at Molecular and Clinical Level of Chronic, Degenerative and Neoplastic Diseases to Develop Novel Therapies, University of Florence, FlorenceSearch for more papers by this authorR. MARCUCCI, R. MARCUCCI Department of Medical and Surgical Critical Care, Thrombosis Centre, Center for the Study at Molecular and Clinical Level of Chronic, Degenerative and Neoplastic Diseases to Develop Novel Therapies, University of Florence, FlorenceSearch for more papers by this authorC. GIGLIOLI, C. GIGLIOLI Department of Heart and Vessels, Azienda Ospedaliero-Universitaria Careggi, FlorenceSearch for more papers by this authorD. ANTONIUCCI, D. ANTONIUCCI Department of Heart and Vessels, Azienda Ospedaliero-Universitaria Careggi, FlorenceSearch for more papers by this authorG. F. GENSINI, G. F. GENSINI Department of Medical and Surgical Critical Care, Thrombosis Centre, Center for the Study at Molecular and Clinical Level of Chronic, Degenerative and Neoplastic Diseases to Develop Novel Therapies, University of Florence, Florence Department of Heart and Vessels, Azienda Ospedaliero-Universitaria Careggi, Florence Fondazione Don Carlo Gnocchi ONLUS, Centro S. Maria degli Ulivi-IRCCS, Florence, ItalySearch for more papers by this authorR. ABBATE, R. ABBATE Department of Medical and Surgical Critical Care, Thrombosis Centre, Center for the Study at Molecular and Clinical Level of Chronic, Degenerative and Neoplastic Diseases to Develop Novel Therapies, University of Florence, FlorenceSearch for more papers by this authorD. PRISCO, D. PRISCO Department of Medical and Surgical Critical Care, Thrombosis Centre, Center for the Study at Molecular and Clinical Level of Chronic, Degenerative and Neoplastic Diseases to Develop Novel Therapies, University of Florence, FlorenceSearch for more papers by this author R. PANICCIA, R. PANICCIA Department of Medical and Surgical Critical Care, Thrombosis Centre, Center for the Study at Molecular and Clinical Level of Chronic, Degenerative and Neoplastic Diseases to Develop Novel Therapies, University of Florence, FlorenceSearch for more papers by this authorE. ANTONUCCI, E. ANTONUCCI Department of Medical and Surgical Critical Care, Thrombosis Centre, Center for the Study at Molecular and Clinical Level of Chronic, Degenerative and Neoplastic Diseases to Develop Novel Therapies, University of Florence, FlorenceSearch for more papers by this authorA. M. GORI, A. M. GORI Department of Medical and Surgical Critical Care, Thrombosis Centre, Center for the Study at Molecular and Clinical Level of Chronic, Degenerative and Neoplastic Diseases to Develop Novel Therapies, University of Florence, FlorenceSearch for more papers by this authorR. MARCUCCI, R. MARCUCCI Department of Medical and Surgical Critical Care, Thrombosis Centre, Center for the Study at Molecular and Clinical Level of Chronic, Degenerative and Neoplastic Diseases to Develop Novel Therapies, University of Florence, FlorenceSearch for more papers by this authorC. GIGLIOLI, C. GIGLIOLI Department of Heart and Vessels, Azienda Ospedaliero-Universitaria Careggi, FlorenceSearch for more papers by this authorD. ANTONIUCCI, D. ANTONIUCCI Department of Heart and Vessels, Azienda Ospedaliero-Universitaria Careggi, FlorenceSearch for more papers by this authorG. F. GENSINI, G. F. GENSINI Department of Medical and Surgical Critical Care, Thrombosis Centre, Center for the Study at Molecular and Clinical Level of Chronic, Degenerative and Neoplastic Diseases to Develop Novel Therapies, University of Florence, Florence Department of Heart and Vessels, Azienda Ospedaliero-Universitaria Careggi, Florence Fondazione Don Carlo Gnocchi ONLUS, Centro S. Maria degli Ulivi-IRCCS, Florence, ItalySearch for more papers by this authorR. ABBATE, R. ABBATE Department of Medical and Surgical Critical Care, Thrombosis Centre, Center for the Study at Molecular and Clinical Level of Chronic, Degenerative and Neoplastic Diseases to Develop Novel Therapies, University of Florence, FlorenceSearch for more papers by this authorD. PRISCO, D. PRISCO Department of Medical and Surgical Critical Care, Thrombosis Centre, Center for the Study at Molecular and Clinical Level of Chronic, Degenerative and Neoplastic Diseases to Develop Novel Therapies, University of Florence, FlorenceSearch for more papers by this author First published: 09 June 2007 https://doi.org/10.1111/j.1538-7836.2007.02656.xCitations: 146 Rita Paniccia, Centro Trombosi-Malattie Aterotrombotiche, Piastra dei Servizi Azienda Ospedaliero-Universitaria Careggi, Viale Morgagni, 85, 50134 Florence, Italia.Tel.: +39 0557949421; fax +39 0557949418; e-mail: rita.paniccia@unifi.it AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Summary. Background: Two point-of-care (POC) systems have been recently proposed as rapid tools with which to evaluate residual platelet reactivity (RPR) in coronary artery disease (CAD) patients. Objectives and Methods: We compared Platelet Function Analyzer-100 (PFA-100) closure times (CTs) by collagen/adenosine 5´-diphosphate (ADP) (C/ADP CT) cartridge and the VerifyNow P2Y12 Assay (VerifyNow) with light transmission aggregation (LTA) induced by 2 and 10 μmol L–1 ADP in 1267 CAD patients on dual antiplatelet therapy who underwent percutaneous coronary intervention. We also performed the vasodilator-stimulated phosphoprotein (VASP) phosphorylation assay by cytofluorimetric analysis in a subgroup of 115 patients. Results: Cut-off values for identifying RPR were: ≥ 54% and ≥ 66% for LTA induced by 2 and 10 μmol L–1 ADP respectively, and ≥ 264 P2Y12 Reaction Units (PRU) for VerifyNow. The cut-off for PFA-100 C/ADP CT was ≥ 68 s. RPR was detected in 25.1% of patients by 2 μmol L–1 ADP-induced LTA (ADP-LTA), in 23.2% by 10 μmol L–1 ADP-LTA, in 24.4% by PFA-100, and in 24.7% by VerifyNow. PFA-100 results did not parallel those obtained with LTA. VerifyNow showed a significant correlation (ρ = 0.62, P < 0.001) and significant agreement (k = 0.34, P < 0.001) with LTA induced by 2 μmol L–1 ADP. The correlation was similar but the agreement was better between VerifyNow and 10 μmol L–1 ADP-LTA (ρ = 0.64, P < 0.0001; k = 0.43, P < 0.001). Significant relationships were found between VASP platelet reactivity index and both ADP-LTA and VerifyNow. PFA-100 C/ADP CT did not significantly correlate with any of the other assays. Conclusions: Our results show a significant correlation between LTA and VerifyNow but not the PFA-100 C/ADP assay. Clinical validation studies for POC systems are necessary. Introduction Platelet activation contributes to the development of coronary artery disease (CAD) [1], and dual antithrombotic treatment – aspirin plus clopidogrel – reduces the number of cardiovascular events in patients undergoing percutaneous coronary intervention (PCI) with coronary stenting [2]. Different studies have reported that the effect of aspirin and clopidogrel treatment is not consistent, and that the presence of residual platelet reactivity (RPR), measured by light transmission aggregation (LTA), on antiplatelet therapy is associated with a higher incidence of clinical adverse events [3-7]. This association is particularly relevant in high-risk patients such as those with acute coronary syndrome (ACS) undergoing PCI. Indeed, ACC/AHA/SCAI 2005 guidelines [8] suggest that in ACS patients at very high risk, in whom subacute thrombosis may be catastrophic or lethal, platelet aggregation studies may be considered and the dose of antiplatelet drug increased if less than 50% inhibition of platelet aggregation is demonstrated. In order to evaluate RPR, different methods have been employed. The established method to assess platelet reactivity in clopidogrel-treated patients – LTA – [4-11] requires a specialized laboratory, and is a manual and time-consuming test [10]. In recent years, 'point-of-care' (POC) devices evaluating platelet function have become available, such as the Platelet Function Analyzer-100 (PFA-100) and the VerifyNow systems [12, 13]. These POC instruments could allow rapid identification of RPR. However, few comparisons of these POC tests with LTA are available [14-19]. The aim of our study was to compare adenosine 5′-diphosphate (ADP)-induced LTA (ADP-LTA) with both the PFA-100 and the VerifyNow systems in a setting of very high-risk patients, i.e. patients with ACS undergoing PCI on dual antiplatelet therapy. Materials and methods Patients investigated The study population included 1267 consecutive adult patients admitted to the Coronary Care Units of the Department of Heart and Vessels (Azienda Ospedaliero-Universitaria Careggi, Florence, Italy) for ACS [849 patients (67.0%) with unstable angina and non-ST elevation myocardial infarction, and 418 with ST elevation myocardial infarction]. All patients underwent coronary angiography performed by the Judkins' technique and PCI. Before PCI, all patients received a loading dose of 500 mg of i.v. ASA and 300 mg of clopidogrel p.o., followed by 100–325 mg of ASA daily and 75 mg of clopidogrel daily. During the procedure, a bolus of 70 IU kg–1 of unfractionated heparin was used as anticoagulant. Patients receiving glycoprotein IIb/IIIa inhibitors or with a personal or family history of bleeding disorders, with a platelet count < 10 × 109 L–1 or >450 × 109 L–1, with hemoglobin levels < 10 g dL–1 or having had major surgery within 1 week of enrolment were excluded. The study was approved by the ethics review board, and written informed consent was obtained from all patients. Blood sampling Blood samples were obtained 24–48 h after PCI. Blood samples were anticoagulated with one-tenth volume 0.109 mol L–1 buffered trisodium citrate within Vacutainer plastic tubes (Becton Dickinson, Plymouth, UK). An additional 1.8-mL aliquot of blood was taken into the special citrated Vacutainer tube (Vacuette, Grainer Bio-One, Monroe, USA) for VerifyNow analysis. An adjunctive citrated blood sample was taken in a subgroup of 115 patients to perform the vasodilator-stimulated phosphoprotein (VASP) phosphorylation assay by cytofluorimetric analysis [20]. All assays were performed within 2 h after blood sampling. Platelet aggregation in platelet-rich plasma Platelet-rich plasma (PRP) was prepared by centrifuging blood samples at 250 × g for 10 min. PRP was removed, and then platelet-poor plasma was prepared by further centrifugation at 3000 × g for 3 min. Aggregation studies were performed with an APACT-4 aggregometer (LABiTec, Ahrensburg, Germany) in 250-μL minicuvettes stirred at 1000 r.p.m. at 37 °C. The 100% line was set using platelet-poor plasma, and the 0% baseline was established with PRP (adjusted from 180 × 103 μL–1 up to 300 × 103 μL–1). ADP (2 and 10 μmol L–1 final concentration) and arachidonic acid (AA) (1 mmol L–1 final concentration) were used as agonists. After 10 min, the maximal percentage of aggregation was recorded. Our laboratory imprecision measured as coefficient of variation (CV) of LTA was determined by assessing (five times) samples from 10 control healthy subjects and from 10 CAD patients on dual antiplatelet therapy. The mean CVs were: ADP-LTA, 4.7% in controls and 6.8% in CAD patients; and AA-LTA, 4.4% in controls and 6.3% in CAD patients. The reference intervals (5–95th percentile of control distribution, n = 98) obtained in our laboratory were 52–100% for 2 μmol L–1 ADP-LTA, 64–100% for 10 μmol L–1 ADP-LTA, and 70–100% for AA-LTA. PFA-100 system The PFA-100 system (Dade-Behring, Marburg, Germany) simulates high shear platelet function within test cartridges [12]. Platelet function is measured as a function of the time [closure time (CT)] that platelets take to occlude an aperture in a membrane coated with collagen/epinephrine (C/EPI) or collagen/ADP (C/ADP). Because clopidogrel does not affect CT with the C/EPI cartridge [15], only the C/ADP test was considered in this study. Blood samples from 10 control subjects and 10 CAD patients on dual antiplatelet therapy were measured four times to determine our laboratory CV of the C/ADP CT test. The mean CVs for C/ADP CT were 4.3% in controls and 9.3% in CAD patients. A citrated whole blood sample (0.8 mL) was pipetted into the sample reservoirs of the C/ADP cartridge and then loaded into the PFA-100 device. The reference interval (5–95th percentile of control distribution, n = 98) obtained in our laboratory was 62–139 s for CT with the C/ADP cartridge. VerifyNow system The VerifyNow system (Accumetrics, San Diego, CA, USA) is a turbidimetry-based optical detection device that measures platelet-induced aggregation [13] in a system containing fibrinogen-coated beads. The instrument measures changes in light transmission and thus the rate of aggregation in whole blood. In the cartridge of the VerifyNow P2Y12 Assay, there is a channel in which the inhibition of the ADP P2Y12 receptor is measured [18]. This channel contains ADP as platelet agonist and prostaglandin E1 (PGE1) as a suppressor of intracellular free calcium levels to reduce the non-specific contribution of the ADP binding to P2Y1 receptors. In a separate chamber, a modified thrombin receptor-activating peptide (iso-TRAP) is used as agonist, and a baseline value (BASE) of platelet function is obtained. Platelet aggregation by iso-TRAP occurs independently of P2Y12 receptors. Results are expressed as P2Y12 Reaction Units (PRU), BASE and percentage of inhibition (% INHIB), which is calculated as 1 – PRU/BASE × 100. The reference interval (5–95th percentile of control distribution, n = 98) obtained in our laboratory was 244–382 PRU. Samples from five control subjects and five CAD patients on dual antiplatelet therapy and VerifyNow Assay Wet Quality Control (level 1, normal, and level 2, abnormal) were assessed four times to determine our laboratory CV of VerifyNow. The mean CVs were 3.5% in controls, 3.2% in CAD patients, and 2.5% and 3.4% for level 1 and 2 quality controls. VASP phosphorylation flow cytometric assay Platelet responsiveness to clopidogrel was monitored by an ex vivo analysis of the platelet reactivity index (PRI) according to a standardized flow cytometric assay (Platelet VASP, Diagnostica Stago, Biocytex, Marseille, France) [20]. Samples were incubated with PGE1, with or without ADP (10 μmol L–1 final concentration), for 10 min and fixed with paraformaldehyde. After 10 min, platelets were permeabilized, and labeled with a primary monoclonal antibody against serine 239-phosphorylated VASP (clone 16C2) (VASP-P) or its isotype, followed by a secondary fluorescein isothiocyanate-conjugated polyclonal goat anti-mouse antibody. All procedures were performed at room temperature. Samples were analyzed within 4 h on a Becton Dickinson FACScanto at a medium rate. The platelet population was identified using both its forward and side scatter distribution and from the specific surface marker CD61-PE; 15 000 platelets were gated. The extent of VASP phosphorylation was measured by mean fluorescence intensity (MFI) values. For each sample, PRI (%) values were calculated, using the VASP-P measure, according to the following formula: where T1 and T2 are either MFI or percentage of fluorescence-positive platelets in the presence of PGE1, without or with ADP. Negative isotypic control values were subtracted from the corresponding fluorescence values before calculation of PRI. Statistical analysis Statistical analysis was performed using spss statistical software package (version 11.0; SPSS Inc., Chicago, IL, USA). The relationship between different methods was evaluated with the Spearman correlation test. The 2 × 2 agreement tables between the LTA and POC systems were used for the qualitative analysis. Agreement between the different tests was determined by kappa statistics, and 95% CIs were calculated. Statistical significance was considered as a two-tailed probability < 0.05. Results In Table 1, clinical and laboratory characteristics of the patients investigated are reported. LTA, PFA-100 and VerifyNow data are given in Table 2. Table 1. Clinical and laboratory characteristics of patients studied n 1267 Male/female, n 924/343 Age (years) 68.2 ± 11.0 Smoking habit (%) 37.5 Hypertension (%) 64.0 Diabetes (%) 25.0 Dyslipidemia (%) 23.2 Peripheral artery disease (%) 13.0 Erythrocytes (× 1012 L–1) 4.0 ± 1.2 Leukocytes (× 109 L–1) 8.9 ± 2.8 Hemoglobin (mg dL–1) 12.6 ± 2.0 Platelets (× 109 L–1) 215 ± 85 Table 2. Results obtained by platelet aggregation, Platelet Function Analyzer-100 (PFA-100) and VerifyNow P2Y12 Assay in patients (n = 1267) 2 μmol L–1 ADP-LTA (%) n = 1267 10 μmol L–1 ADP-LTA (%) n = 1267 1 mmol L–1 AA-LTA (%) n = 1267 PFA-100 C/ADP CT (s) n = 626 VerifyNow P2Y12 Assay (PRU) n = 1267 Mean ± SD 34.4 ± 19.3 51.8 ± 19.3 18.4 ± 18.7 102.2 ± 59.6 198.8 ± 89.1 Median (range) 31.0 (1–100) 54.0 (1–100) 11.0 (1–100) 83 (45–300) 195.0 (2–422) AA-LTA, arachidonic acid-induced light transmission aggregation; ADP-LTA, ADP-induced light transmission aggregation; PRU, P2Y12 Reaction Unit; C/ADP CT, closure time by Collagen/ADP cartridge. The stratification of patient platelet reactivity assessed by LTA, PFA-100 and VerifyNow into quartiles is shown in Table 3. Figure 1 shows a stratification of patient results obtained with different assays in comparison with the control range. We considered as patients with residual platelet reactivity (RPR) those with results in the fourth quartile for ADP-LTA [6] and VerifyNow, and in the first quartile for PFA-100 C/ADP CT [21, 22]. The ranges of values in the fourth quartile were 54–100% for 2 μmol L–1 ADP-LTA, 66–100% for 10 μmol L–1 ADP-LTA, and 264–422 PRU for VerifyNow. For PFA-100 C/ADP CT, the range of values in the first quartile was 45–68 s. Table 3. Stratification of platelet reactivity into quartiles Quartiles I II III IV 2 μmol L–1 ADP-LTA (%) (n = 1267) n = 327 n = 314 n = 309 n = 317 Mean ± SD 12.3 ± 4.8 25.1 ± 3.4 40.2 ± 6.1 61.6 ± 8.4 Median (range) 13 (1–19) 25 (20–31) 39 (32–53) 58 (54–100) 10 μmol L–1 ADP-LTA (%) (n = 1267) n = 327 n = 340 n = 286 n = 314 Mean ± SD 26.4 ± 9.4 47.4 ± 4.4 60.1 ± 4.5 75.3 ± 8.0 Median (range) 28 (1–39) 47 (40–54) 60 (55–65) 73 (66–100) PFA-100 C/ADP CT (s) (n = 626) n = 153 n = 137 n = 150 n = 147 Mean ± SD 60.3 ± 5.1 75.7 ± 3.9 95.0 ± 7.7 138.2 ± 25.3 Median (range) 61 (45–68) 76 (69–82) 94 (84–108) 130 (110–300) VerifyNow P2Y12 Assy (PRU) (n = 1267) n = 318 n = 319 n = 317 n = 313 Mean ± SD 85 ± 36.6 168 ± 17.9 231 ± 19.7 315 ± 35.2 Median (range) 90 (2–135) 168 (136–196) 230 (197–263) 312 (264–422) ADP-LTA, ADP-induced light transmission aggregation; PFA-100, Platelet Function Analyzer-100; PRU, P2Y12 Reaction Unit. Figure 1Open in figure viewerPowerPoint Stratification of patient results in quartiles alongside control data for different assays. The gray area corresponds to the range of normal distribution for each test obtained from a normal population of 98 healthy subjects. (A) 2 μmol L–1 (ADP) adenosine 5′-diphosphate-induced light transmission aggregation (ADP-LTA) data. (B) 10 μmol L–1 ADP-LTA data. (C) Platelet Function Analyzer-100 collagen/ADP closure time results. (D) VerifyNow P2Y12 Assay data. With use of the above-mentioned cut-off values, the percentages of patients with RPR were: 25.1% and 23.2% for 2 and 10 μmol L–1 ADP-LTA; 24.4% for PFA-100; and 24.7% for VerifyNow. Figure 2A shows the correlation between 2 μmol L–1 ADP-LTA and PFA-100 C/ADP CT (ρ = −0.07 ns). In relation to the presence or absence of RPR (as defined above), 376 (60.1%) of the 626 samples tested were concordant, with 335 patients without RPR and 41 with RPR, whereas among the 250 discordant results, 138 patients had RPR by 2 μmol L–1 ADP-LTA alone and 112 by PFA-100 alone (Table 4A). No agreement between the two tests was observed. Figure 2B shows the correlation between 10 μmol L–1 ADP-LTA and PFA-100 C/ADP CT (ρ = −0.11, P < 0.01). In relation to the presence or absence of RPR (as defined above), 380 (60.7%) of the 626 samples tested were concordant, with 345 patients without RPR and 35 with RPR, whereas among the 246 discordant results, 118 patients had RPR by 10 μmol L–1 ADP-LTA alone and 128 by PFA-100 alone (Table 4B). No agreement between the two tests was observed. Figure 2Open in figure viewerPowerPoint (A) Comparison and agreement of results obtained by 2 μmol L–1 adenosine 5′-diphosphate (ADP)-induced light transmission aggregation (ADP-LTA) and Platelet Function Analyzer-100 collagen/ADP closure time (PFA-100 C/ADP CT). The horizontal arrowed line represents the cut-off value for residual platelet reactivity (RPR) by LTA (≥ 54%). The vertical arrowed line represents the cut-off value for RPR by PFA-100 C/ADP CT (≤ 67 s). (B) Comparison and agreement of results obtained by 10 μmol L–1 ADP-LTA and PFA-100 C/ADP CT. The horizontal arrowed line represents the cut-off value for RPR by LTA (≥ 66%). The vertical arrowed line represents the cut-off value for RPR by PFA-100 C/ADP CT (≤ 67 s). Table 4. Classification of results (n = 1267) by platelet aggregation, Platelet Function Analyzer-100 (PFA-100) (A, B), VerifyNow P2Y12 Aassy (VerifyNow) (C, D) and by VerifyNow and PFA-100 (E) (A) 2 μmol L–1 ADP-LTA Classification n No RPR RPR (>54%) 524 102 PFA-100 No RPR 473 393 80 RPR (< 68 s) 153 131 22 (B) 10 μmol L–1 ADP-LTA Classification n No RPR RPR (≥ 66%) 463 163 PFA-100 No RPR 473 345 128 RPR (< 68 s) 153 118 35 (C) 2 μmol L–1 ADP-LTA Classification n No RPR RPR (54%) 949 318 VerifyNow No RPR 954 795 159 RPR (≥ 264 PRU) 313 154 159 (D) 10 μmol L–1 ADP-LTA Classification n No RPR RPR (≥ 66%) 973 294 VerifyNow No RPR 954 831 123 RPR (≥ 264 PRU) 313 142 171 (E) VerifyNow Classification n No RPR RPR (>264 PRU) 415 211 PFA-100 No RPR 473 315 158 RPR (< 68 s) 153 100 53 ADP-LTA, ADP-induced light transmission aggregation; RPR, residual platelet reactivity; PRU, P2Y12 Reaction Unit. Figure 3A shows the correlation between 2 μmol L–1 ADP-LTA and VerifyNow data (ρ = 0.62, P < 0.001). In relation to the presence or absence of RPR (as defined above), 954 (75.3%) of the 1267 samples tested were concordant, with 795 patients without RPR and 159 with RPR, whereas among the 313 discordant results, 159 patients had RPR by 2 μmol L–1 ADP-LTA alone and 154 by VerifyNow alone (Table 4C). A significant, although poor, agreement between the two tests was observed (κ = 0.34, 95% CI 0.29–0.35, P < 0.0001). Figure 3B shows the correlation between 10 μmol L–1 ADP and VerifyNow data in the 1267 patients (ρ = 0.64, P < 0.0001). These two methods gave concordant results in 1002 samples (79.1%), with 831 patients without RPR and 171 patients with RPR, whereas among the 265 discordant results, 123 patients had RPR by 10 μmol L–1 ADP-LTA alone and 142 by VerifyNow alone (Table 4D). A significant, moderate, agreement was observed between the results obtained by the two tests (k = 0.43, 95% CI 0.36–0.42, P < 0.0001). Figure 3Open in figure viewerPowerPoint (A) Comparison of and agreement between results obtained by 2 μmol L–1 adenosine 5′-diphosphate (ADP)-induced light transmission aggregation (ADP-LTA) and the VerifyNow P2Y12 Assay. The horizontal arrowed line represents the cut-off value for residual platelet reactivity (RPR) by ADP-LTA (≥ 54%). The vertical arrowed line represents the cut-off value for RPR by the VerifyNow P2Y12 Assay (≥ 264 PRU). (B) Comparison of and agreement between results obtained by 10 μmol L–1 ADP-LTA and the VerifyNow P2Y12 Assay. The horizontal arrowed line represents the cut-off value for RPR by ADP-LTA (≥ 66%). The vertical arrowed line represents the cut-off value for RPR by the VerifyNow P2Y12 Assay (≥ 264 PRU). Taking as reference 2 μmol L–1 ADP-LTA, VerifyNow showed a sensitivity of 50.0%, a specificity of 83.8% (159 false-negative and 154 false-positive results), a positive predictive value of 50.8%, and a negative predictive value of 83.3%. Taking as reference 10 μmol L–1 ADP-LTA, VerifyNow (cut-off value = 264 PRU) showed a sensitivity of 58.2%, a specificity of 85.4% (123 false-negative and 142 false-positive results), a positive predictive value of 54.6%, and a negative predictive value of 87.1%. Figure 4 shows the relationship between VerifyNow values and PFA-100 C/ADP CT (ρ = −0.09, P < 0.05). In relation to the presence or absence of RPR (as defined above), 368 (58.8%) of the 626 samples tested were concordant, with 315 patients without RPR and 53 with RPR, whereas among the 258 (41.2%) discordant results, 158 patients had RPR by VerifyNow alone and 100 by PFA-100 alone (Table 4E). No agreement between the two tests was observed. Figure 4Open in figure viewerPowerPoint Comparison of and agreement between results obtained by Platelet Function Analyzer-100 (PFA-100) collagen/adenosine 5′-diphosphate (ADP) closure time (C/ADP CT) and the VerifyNow P2Y12 Assay. The horizontal arrowed line represents the cut-off value for residual platelet reactivity (RPR) by the VerifyNow P2Y12 Assay (≥ 264 PRU). The vertical arrowed line represents the cut-off value for RPR by PFA-100 C/ADP CT (≤ 67s). We also evaluated the performance of VerifyNow taking as reference the cut-off value of 10 μmol L–1 ADP-LTA of 70%, reported by different authors [3, 6, 7, 9]. With use of this cut-off value, the percentage of patients with RPR was 18.8% (238/1267), and the results of the two tests were concordant in 1000 samples (78.9%), with 858 patients without RPR and 142 patients with RPR, whereas among the 267 discordant results, 96 patients had RPR by 10 μmol L–1 ADP-LTA alone and 171 by VerifyNow alone. A significant, although poor, agreement was observed between the results obtained by the two tests (k = 0.38, 95% CI 0.33–0.39, P < 0.0001). In this evaluation, VerifyNow showed a sensitivity of 59.7%, a specificity of 83.4% (96 false-negative and 171 false-positive results), a positive predictive value of 45.5%, and a negative predictive value of 89.9%. Finally, considering only the results obtained by VerifyNow in those patients with both AA-LTA > 20% [3] of maximal aggregation and 10 μmol L–1 ADP-LTA > 66% of maximal aggregation (n = 186; 14.7% of total patients), the PRU mean value was 284 ± 78.3 (median = 299; range: 2–422 PRU). In this group, 150/193 (68.3%) patients had a PRU value above the cut-off of 264 PRU. In 115 patients, we performed the VASP phosphorylation assay by cytofluorimetric analysis. Median PRI (%) values in patient samples were 66.0 (range 9–91.7%). Patients with RPR by ADP 10 μmol L–1-LTA had significantly higher levels of PRI [73.0% (54.5–91.7%) vs. 65.4% (9.0–88.1%)] (P < 0.01), whereas only a trend towards a significant difference in PRI levels between patients with or without RPR by 2 μmol L–1 ADP-LTA was observed (data not shown). Significantly higher PRI levels were found in patients with RPR by PRU than in the other patients [73.0% (59.6–91.7%) vs. 65.4 (9.0–88.1%)] (P < 0.001). In 115 patients, PRI showed a significant correlation with 2 μmol L–1 ADP-LTA (ρ = 0.47, P < 0.001), 10 μmol L–1 ADP-LTA (ρ = 0.50, P < 0.0001) and PRU values (r = 0.52, P < 0.001). Discussion The results of this study have demonstrated significant correlations between ADP-LTA and VerifyNow in patients on dual antiplatelet therapy. This is the largest available study of high-risk vascular patients comparing the performance of LTA and POC systems in evaluating platelet function. It is important to note that the patients investigated were from a single institution and that their platelet function was assessed in a single laboratory, so maintaining uniformity of handling and procedures for platelet function assays. Two POC systems were compared with LTA, the classic method for investigating platelet function, which has been found to provide an index function associated with increased risk of clinical events in high-risk patients [3, 5-7, 23-25]. Several methods have been used to assess clopidogrel-induced antiplatelet effects [11]. However, none of these tests has been fully standardized and fully agreed upon for the measurement of clopidogrel responsiveness [26]. A