Abstract. 834 Am J Clin Pathol 2009;131: American Society for Clinical Pathology DOI: /AJCPTE3K1SGAPOIZ

Transcription

1 Coagulation and Transfusion Medicine / Platelet Aggregation in Whole Blood Assessment of Platelet Function on Whole Blood by Multiple Electrode Aggregometry in High-Risk Patients With Coronary Artery Disease Receiving Antiplatelet Therapy Rita Paniccia, MS, 1,2 Emilia Antonucci, MD, 1 Niccolò Maggini, BSc, 1 Eloisa Romano, MS, 1 Anna Maria Gori, MS, 1,2 Rossella Marcucci, MD, 1,2 Domenico Prisco, MD, 1,2 and Rosanna Abbate, MD 1,2 Key Words: Aspirin resistance; Clopidogrel resistance; Coronary syndrome; Light transmission platelet aggregation; Multiplate system; Point-of-care testing; Residual platelet reactivity; Whole blood platelet aggregation DOI: 1.139/AJCPTE3K1SGAPOIZ Abstract This study sought to compare Multiplate impedance platelet aggregometry (IPA) with light transmission aggregometry (LTA) and the PFA- for determining the prevalence of residual platelet reactivity (RPR) by the Multiplate IPA in 297 patients with acute coronary syndrome receiving dual antiplatelet therapy. Aggregations were induced by adenosine-5 diphosphate (ADP), arachidonic acid, and collagen. PFA- closure times were measured by collagen and ADP and epinephrine (CEPI) cartridges. Significant correlations were observed between Multiplate IPA and LTA after all stimulations (P <.1) and between Multiplate IPA (arachidonate and collagen) and PFA- CEPI closure time (P <.1 for both). Cutoff values of Multiplate IPA (for all stimulations) were calculated for the identification of RPR. Between the Multiplate IPA and LTA good agreement was found with all 3 agonists (P <.1 for all). Multiplate IPA might represent a reliable, handy, rapid tool to monitor antiplatelet therapy in clinical practice and for clinical investigations. Dual antiplatelet therapy with aspirin and clopidogrel is the treatment of choice to prevent thrombosis in patients undergoing percutaneous coronary intervention (PCI). 1 Different studies reported that the inhibitory effect of aspirin and clopidogrel on platelet function is not consistent, 2-5 and the presence of residual platelet reactivity (RPR) is associated with a higher prevalence of clinical adverse events in patients with acute coronary syndrome (ACS). 6-8 Light transmission aggregometry (LTA) in platelet-rich plasma (PRP) 9 is considered a standard method for assessing platelet reactivity to different agonists such as arachidonic acid (AA) and adenosine-5 diphosphate (ADP) after administration of aspirin and clopidogrel, respectively However, LTA is a manual and time-consuming method and requires dedicated laboratories. So, a simple and rapid method to evaluate platelet function in treated patients might be useful in clinical practice. Currently, different point-of-care (POC) methods are used to analyze platelet function in whole blood, such as the Platelet Function Analyzer (PFA-, Dade-Behring, Marburg, Germany), the VerifyNow system (Accumetrics, San Diego, CA), 1,12,14-18 and others Recently, a method that implements the principle of impedance platelet aggregometry (IPA) in whole blood by using a new device to measure platelet aggregation (multiple electrode platelet aggregometry; Multiplate analyzer, Dynabyte, Munich, Germany) has been introduced. 24 As a whole blood method, Multiplate IPA avoids the handling of blood samples, with the advantage that the cellular environment remains unchanged, and allows rapid evaluation of platelet aggregation by ready-to-use test cuvettes with 2 independent sensor units. In addition, the use of whole blood tests platelet function under more physiologic conditions. 834 Am J Clin Pathol 29;131: Downloaded 834 from DOI: 1.139/AJCPTE3K1SGAPOIZ

2 Coagulation and Transfusion Medicine / Original Article Comparisons of the Multiplate device with other POC devices have been conducted in a limited number of patients receiving dual antiplatelet therapy Thus, this study was planned to evaluate different pathways of platelet function in 297 patients with ACS undergoing dual antiplatelet therapy by the Multiplate IPA method and compare results with those for LTA on PRP and the PFA- system on whole blood. Materials and Methods Subjects The study population included 297 patients admitted to the Coronary Care Unit, Department of Heart and Vessels (Azienda Ospedaliero-Universitaria Careggi, Florence, Italy) for ACS. Of the 297 patients, 194 (65.3%) were men; the mean ± SD age was 69.3 ± 8.7 years (range, years). All patients underwent coronary angiography performed by the Judkins technique and PCI. Before PCI, all patients received a loading dose of 5 mg of aspirin intravenously and mg of clopidogrel by mouth, followed by 325 mg of aspirin daily and 75 mg of clopidogrel daily. During the procedure, a bolus of 7 IU/kg of unfractionated heparin was used as an anticoagulant. Patients receiving glycoprotein IIb/IIIa inhibitors or with a personal or family history of bleeding disorders, a platelet count of less than 1 3 /µl ( 1 9 /L) or more than /µl ( /L), a hemoglobin level of less than 1 g/dl ( g/l), a hematocrit values of less than 35% (.35), or having a major surgery within 1 week of enrollment were excluded. The control group included 23 healthy volunteers (1 men and 13 women) recruited from the hospital and university staff. None of the control subjects had a history of bleeding disorders, underwent major surgery within 1 month before sampling, or had taken aspirin, clopidogrel, or other drugs known to interfere with platelet function within 2 weeks previous to blood sampling. The clinical and laboratory characteristics of patients and control subjects are reported in Table 1. The study was approved by the ethics review board, and written informed consent was obtained from all subjects. Blood Sampling Blood samples were obtained 24 to 48 hours after PCI. Blood samples were anticoagulated with one tenth volume.19 mol/l buffered trisodium citrate in Vacutainer plastic tubes (Becton Dickinson, Plymouth, England). All tests were performed within 2 hours after blood sampling. Platelet Aggregation in Whole Blood by the Multiplate Analyzer Whole blood aggregation was performed by using the Multiplate analyzer, a new impedance aggregometer. The device consists of 5 channels for contemporary tests, an integrated computer, and guided automatic pipetting. Disposable ready-to-use test cells with 2 independent sensor units are used. One unit consists of 2 silver-coated highly conductive copper electrodes with a length of 3.2 mm. The method was described in detail elsewhere. 24 Briefly, the adhesion and aggregation of platelets on the sensor surface enhances the electrical resistance (impedance) between the 2 sensor electrodes. In the test cuvette, whole blood (3 µl) is diluted, 1:2 vol/vol, with.9% NaCl solution for AA tests and with.25 mmol/l CaCl 2 in saline solution for ADP and collagen tests and maintained at 37 C for 3 minutes. At the end of incubation of diluted whole blood, the agonist is added. The increase of impedance due to the attachment of platelets to the electrodes is detected for each sensor unit separately and transformed to arbitrary aggregation units (AU) Table 1 Clinical and Laboratory Characteristics of Patients With Acute Coronary Syndrome and Control Subjects * Patients (n = 297) Control Subjects (n = 23) M/F (No.) 194/13 1/13 Age (y) 69.3 ± ± 1.9 RBC count, 1 6 /µl ( 1 12 /L) 4.1 ±.8 (4.1 ±.8) 4.7 ±.5 (4.7 ±.5) WBC count, /µl ( 1 9 /L) 8,7 ± 2,7 (8.7 ± 2.7) 7,8 ± 1, (7.8 ± 1.) Hemoglobin, g/dl (g/l) 12.1 ± 1.2 (121 ± 12) 14.1 ± 1. (141 ± 1) Hematocrit, % 36.3 ± 7.2 (.36 ±.7) 39.3 ± 3.2 (.39 ±.3) Platelet count, 1 3 /µl ( 1 9 /L) 248 ± 75 (248 ± 75) 278 ± 35 (278 ± 35) Mean platelet volume, fl 8.7 ± ±.3 Smoking habit, % Hypertension, % 67.4 Diabetes, % 19.9 Dyslipidemia, % 26.2 Peripheral artery disease, % 18.8 * Data are given as mean ± SD unless otherwise indicated. Downloaded from Am J Clin Pathol 29;131: DOI: 1.139/AJCPTE3K1SGAPOIZ 835

3 Paniccia et al / Platelet Aggregation in Whole Blood that are plotted against time (AU*min). Approximately 8 AU correspond to 1 ohm. The aggregation measured with this device is quantified as area under the curve (AUC) of AU*min. Multiplate aggregation was performed in citrated anticoagulated whole blood by using the same agonists (2 µl) used for LTA: ADP, 1 µmol/l, final concentration; AA, 1mmol/L, final concentration; and collagen, 2 µg/ml, final concentration. Reference intervals (5th to 95th percentile of control sample distribution, n = 23) obtained in our laboratory were 32 to 14 AUC for 1 µmol/l ADP, 18 to 84 AUC for AA, and 42 to 83 AUC for 2 µg/ml collagen. Our laboratory imprecision measured as the coefficient of variation (CV) of Multiplate IPA was determined by assessing (5 times) samples from healthy control subjects and from patients with ACS receiving dual antiplatelet therapy. The mean CVs were as follows: 1 µmol/l ADP Multiplate IPA, 3.9% in control subjects and 6.2% in patients with ACS; 1 mmol/l AA Multiplate IPA, 5.1% in control subjects and 7.1% in patients with ACS; and 2 µg/ml collagen-ipa, 4.5% in control subjects and 6.6% in patients with ACS. Platelet Aggregation in PRP LTA was performed in all patients studied. PRP was prepared by centrifuging blood samples at 15g for 1 minutes. PRP was removed, and then platelet-poor plasma was prepared by further centrifugation at 3,g for 3 minutes. Aggregation studies were performed by using an APACT-4 aggregometer (LABiTec, Ahrensburg, Germany) in 25-µL minicuvettes stirred at 1, rpm at 37 C. The % line was set using platelet-poor plasma and the % baseline established with PRP (adjusted from /µl up to /µl). ADP (1 µmol/l, final concentration), AA (1 mmol/l, final concentration), and collagen (2 µg/ml, final concentration) were used as agonists. After 1 minutes, the maximal percentage of aggregation was recorded. Our laboratory imprecision measured as the CV of LTA was determined assessing (5 times) samples from 1 healthy control subjects and from 1 patients with ACS receiving dual antiplatelet therapy. The mean CVs were as follows: ADP LTA, 4.7% in control subjects and 6.8% in patients with ACS; AA LTA, 4.4% in control subjects and 6.3% in patients with ACS; and collagen LTA, 2.3% in control subjects and 6.7% in patients with ACS. Reference intervals (5th to 95th percentile of control sample distribution, n = 98) obtained in our laboratory were 64% to % for 1 µmol/l ADP LTA, 7% to % for AA LTA, and 56% to % for 2 µg/ml collagen LTA. Treated patients were considered to have RPR when their platelets showed aggregation of 7% or more induced by 1 µmol/l ADP, 7,16 aggregation of 2% or more induced by 1 mmol/l AA, 15,28 and aggregation of 56% or more induced by 2 µg/ml collagen in PRP. 29 Platelet Function Measurement by the PFA- System The PFA- system simulates high shear platelet function within test cartridges. 14 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 (CEPI) or collagen/adp (CADP). Blood samples can be kept for at least 3 hours after collection at room temperature without significant changes in CT. A citrated whole blood sample,.8 ml, was pipetted into the sample reservoirs of a CEPI cartridge and then loaded into the PFA- device. The PFA- analysis was performed for 111 patients. Testing of the blood samples from 1 control subjects and 1 patients with ACS receiving dual antiplatelet therapy was repeated 4 times to determine our laboratory CV for the CEPI CT and CADP CT tests. For CEPI CT, the mean CV was 5.4% in control subjects and 9.9% in patients with ACS. For CADP CT, the mean CV was 4.3% in control subjects and 9.3% in patients with ACS. Reference intervals (5th to 95th percentile of control sample distribution, n = 98) obtained in our laboratory were 65 to 23 seconds for CT with the CEPI cartridge and 62 to 139 seconds for CT with the CADP cartridge. RPR was defined as a CEPI CT of 23 seconds or less and CADP CT as 68 seconds or less. 15 Statistical Analysis Statistical analysis was performed by using SPSS (version 11.; SPSS, Chicago, IL). A value of 3 seconds was attributed to all samples with a PFA- CT of more than the maximum predefined value of 3 seconds. The relationship between different methods was evaluated by using the Spearman correlation test. Receiver operator characteristic (ROC) curves were determined to assess the optimal cut-off point of the Multiplate IPA for the prediction of RPR by using the LTA as the gold standard method. The 2 2 agreement tables between LTA, the Multiplate analyzer, and the PFA- were used for qualitative analysis. Agreement between the different tests was determined by κ statistics, and 95% confidence intervals (CI) were calculated. Results A total of 32 simultaneous measurements of platelet aggregation induced by ADP, AA, and collagen with the Multiplate IPA and LTA were obtained. For PFA-, a total of 134 measurements of CT by the CADP and CEPI cartridges were obtained. Table 2 gives the Multiplate IPA, LTA, and PFA- values for the 2 study groups. Correlations of the Multiplate IPA With LTA and PFA- Significant correlations between the Multiplate IPA and LTA were observed after stimulation with ADP, AA, and collagen Figure 1A. In addition, a significant correlation was observed 836 Am J Clin Pathol 29;131: Downloaded 836 from DOI: 1.139/AJCPTE3K1SGAPOIZ

4 Coagulation and Transfusion Medicine / Original Article Table 2 Results Obtained by LTA, PFA-, and Multiplate IPA in Patients With Acute Coronary Syndrome and Control Subjects ADP, 1 µmol/l Arachidonic Acid, 1 mmol/l Collagen, 1 µg/ml Patients (n = 297) Multiplate IPA (AUC) Mean ± SD 26.8 ± ± ± 18.3 Median (range) 22. (-91) 7 (-76) 26. (1-111) LTA (%) Mean ± SD 49. ± ± ± 22.6 Median (range) 5. (2-94) 14. (3-91) 26. (5-96) CADP Closure Time CEPI Closure Time PFA- (s) (n = 111) Mean ± SD 16.3 ± ± 65.5 Median (range) 9 (5-3) 3 (8-3) ADP, 1 µmol/l Arachidonic Acid, 1 mmol/l Collagen, 1 µg/ml Control subjects (n = 23) Multiplate IPA (AUC) Mean ± SD 67.7 ± ± ± 13.6 Median (range) 7 (32-15) 57. (18-85) 75. (42-84) LTA (%) Mean ± SD 87.7 ± ± ± 5.3 Median (range) 9. (72-) 9. (64-97) 9 (79-) CADP Closure Time CEPI Closure Time PFA- (s) Mean ± SD 83.8 ± ± 26.6 Median (range) 81. (59-125) 125. (89-22) ADP, adenosine-5 diphosphate; AUC, area under the curve; CADP, collagen/adp; CEPI, collagen/epinephrine; IPA, impedance platelet aggregation; LTA, light transmission aggregation. A ADP Multiplate IPA (AUC) ADP LTA (%) AA Multiplate IPA (AUC) AA LTA (%) Collagen Multiplate IPA (AUC) Collagen LTA (%) B ADP Multiplate IPA (AUC) PFA- CADP (CT) AA Multiplate IPA (AUC) PFA- CEPI (CT) Collagen Multiplate IPA (AUC) PFA- CEPI (CT) Figure 1 Spearman rank correlations between Multiplate impedance platelet aggregation (IPA) and light transmission aggregation (LTA; A) and PFA- (B). A, Whole blood aggregation of Multiplate quantified as area under the curve (AUC) of arbitrary units (AU*min) and platelet-rich plasma LTA induced by 1 µmol/l adenosine-5 diphosphate (ADP), 1 mmol/l arachidonic acid (AA), and 2 µg/l collagen in all subjects examined. Left, ρ =.73; P <.1; center, ρ =.5; P <.1; right, ρ =.66; P <.1. B, Whole blood aggregation of Multiplate induced by 1 mmol/l AA and 2 µg/l collagen and PFA- CT by collagen/epinephrine (CEPI) cartridge (center and right). Left, ρ =.; P <.1; center, ρ =.58; P <.1; right, ρ =.68; P <.1. Downloaded from Am J Clin Pathol 29;131: DOI: 1.139/AJCPTE3K1SGAPOIZ 837

5 Paniccia et al / Platelet Aggregation in Whole Blood between the ADP Multiplate IPA and PFA- CADP CTs; significant correlations between the AA Multiplate IPA and PFA- CEPI CTs and between the collagen Multiplate IPA and PFA- CEPI CTs were also observed Figure 1B. Significant correlations were also found between the ADP LTA and PFA- CADP CTs (ρ =.51; P <.1), the AA LTA and PFA- CEPI CTs (ρ =.74; P <.1), and the collagen LTA and PFA- CEPI CTs (ρ =.77; P <.1). Classification of Patients and Agreement of the Multiplate IPA With LTA and PFA- In terms of RPR, the percentage of patients with RPR determined by LTA was 2.2% for ADP LTA, 2.9% for AA LTA, and 23.9% for collagen LTA. To define the best cutoff for the Multiplate IPA that allows identification of patients with RPR, ROC analysis was performed by using LTA as the reference method. The cutoffs found were as follows: 37 AUC for the ADP Multiplate IPA (AUC ROC =.93; 95% CI, ; P <.1), 18 AUC for the AA Multiplate IPA (AUC ROC =.85; 95% CI,.79-.9; P <.1), and 42 AUC for the collagen Multiplate IPA (AUC ROC =.9; 95% CI, ; P <.1). Consequently, the percentage of patients with RPR determined by the Multiplate IPA was 19.9% for the ADP Multiplate IPA, 13.1% for the AA Multiplate IPA, and 2.9% for the collagen Multiplate IPA. The classification of patients with RPR based on the preceding cutoff values is shown in Table 3. Concerning the classification of the values for Multiplate IPA with LTA by using ADP, in relation to the presence or absence of RPR, 272 (91.6%) of the 297 patient samples tested were concordant. Good significant agreement was observed between the results obtained by the 2 tests (κ =.74; 95% CI, ; P <.1). By using AA, in relation to the presence or absence of RPR, 264 (88.9%) of the 297 patient samples tested were concordant. Good significant agreement was observed between the results obtained by the 2 tests (κ =.61; 95% CI, ; P <.1). By using collagen as the agonist, in relation to the presence or Table 3 Classification of Results (n = 297) by Multiplate IPA and LTA Multiplate IPA LTA ADP, 1 µmol/l RPR 7% RPR <7% Total RPR 37% RPR <37% Total AA, 1 mmol/l RPR 2% RPR <2% Total RPR 18% RPR <18% Total Collagen, 2 µg/ml RPR 56% RPR <56% Total RPR 42% RPR <42% Total AA, arachidonic acid; ADP, adenosine-5 diphosphate; IPA, impedance platelet aggregation; LTA, light transmission aggregation; RPR, residual platelet reactivity. absence of RPR, 264 (88.9%) of the 297 patient samples tested were concordant; the significant agreement between the results obtained by the 2 tests was good (κ =.68; 95% CI, ; P <.1). The sensitivity and specificity values of the Multiplate system by assuming LTA as the reference method and by using cutoff values obtained from ROC curves are reported in Table 4. Considering the Multiplate system and PFA- results, the classification in relation to the presence or absence of RPR is reported in Table 5. In terms of RPR, the percentages of patients with RPR were 12.5% for the PFA- CADP CT and 22.9% for the PFA- CEPI CT. For the ADP Multiplate IPA compared with the PFA- CADP CT, in relation to the presence or absence of RPR, 85 (76.6%) of the 111 patient samples tested were concordant, but no agreement was observed (κ =.19; not significant). Between the AA Multiplate IPA and the PFA- CEPI CT, a concordance of 81.1% (9/111 concordant samples) Table 4 Multiplate Method Results in Relation to Light Transmission Aggregation Results * Test ADP, 1 µmol/l AA, 1 mmol/l Collagen, 2 µg/ml Sensitivity (%) 78 (68-89) 55 (42-67) 7 (59-81) Specificity (%) 95 (92-98) 98 (96-99) 95 (92-98) Accuracy (%) Positive predictive value (%) 8 (69-9) 87 (67-92) 81 (71-9) Negative predictive value (%) 95 (92-97) 89 (85-93) 91 (87-94) P <.1 <.1 <.1 AA, arachidonic acid; ADP, adenosine-5 diphosphate. * Values in parentheses are the 95% confidence intervals. 838 Am J Clin Pathol 29;131: Downloaded 838 from DOI: 1.139/AJCPTE3K1SGAPOIZ

6 Coagulation and Transfusion Medicine / Original Article and significant agreement were found (κ =.42; 95% CI, ; P <.1). Between the collagen Multiplate IPA and the PFA- CEPI CT, a concordance of 85.6% (95/111 concordant samples) and significant agreement were found (κ =.; 95% CI, ; P <.1). About the classification of LTA with PFA-, in relation to the presence or absence of RPR, for the ADP LTA compared with the CADP CT and for the AA LTA with the CEPI CT, results similar to those described in our previous reports 15,16 were obtained (data not shown). In addition, by using collagen as the inducer of LTA and PFA- CEPI CT, in relation to the presence or absence of RPR, 13 (92.8%) of the 111 samples tested were concordant. Significant, good agreement was observed between the results obtained by the 2 tests (κ =.82; 95% CI, ; P <.1). Discussion To our knowledge, this is the first study in which platelet function in response to ADP, AA, and collagen was evaluated by the Multiplate device and compared with LTA in highrisk coronary patients undergoing PCI and receiving dual antiplatelet therapy. Previously, Tóth et al 24 validated this renewed technique by studying the pattern of platelet function in healthy subjects in comparison with single platelet counting and evaluating the in vitro inhibitory effect of aspirin on ADP- and collagen-induced aggregation. The present findings show that this new POC whole blood impedance aggregometer has the capability to detect residual platelet reactivity with good agreement with LTA, which has been found in clinical studies to be associated with increased occurrence of ischemic events. 2-4,7,3-33 LTA by ADP, collagen, and AA targets the platelet activation pathways inhibited by clopidogrel and aspirin. However, the use of LTA is restricted to dedicated laboratories. Most important, its major drawback is the difficulty of standardization, which does not allow the comparison of assay results among research teams. In particular, the preparation of PRP and the adjustment of the platelet count in PRP may determine disparities in platelet function results among laboratories. 34 The Multiplate system implements the principle of impedance aggregometry to measure platelet function in a small quantity of whole blood by using disposable, readyto-use test cells with a prearranged dual-sensor unit. The IPA measured in whole blood requires no preparation of the blood sample, thus offering the advantage of a more physiologic milieu for platelets and reducing preanalytic error. By this method, the detection of platelet (dys)function has become more rapid than with conventional aggregometry, maintaining good precision of the method. Actually, Table 5 Classification of Results (n = 297) by Multiplate IPA and PFA- Multiplate IPA PFA- CADP ADP, 1 µmol/l RPR <68 s RPR 68 s Total RPR 37% RPR <37% Total PFA- CEPI AA, 1 mmol/l RPR 23 s RPR <23 s Total RPR 18% RPR <18% Total Collagen, 2 µg/ml RPR 42% RPR <42% Total AA, arachidonic acid; ADP, adenosine-5 diphosphate; CADP, collagen and ADP; CEPI, collagen/epinephrine; IPA, impedance platelet aggregation; LTA, light transmission aggregation; RPR, residual platelet reactivity. the imprecision of the method for the Multiplate was less than 5% in control subjects and 7% in patients undergoing antiplatelet treatment, and these values were similar to those obtained in house for LTA. Moreover, but not less important, this method does not use expensive reagents and instruments. For clinical use in acquired hemorrhagic conditions, the Multiplate IPA has been found to be able to detect impaired hemostasis after cardiopulmonary bypass surgery and to identify patients before and after cardiac surgery with enhanced risk of bleeding and of blood product transfusion. 38,39 Few data concerning the sensitivity of the Multiplate device for von Willebrand syndrome are available.,41 Feasibility of the Multiplate IPA for Monitoring Antiplatelet Therapy The most important results of this study are that by using ADP as the inducer, the Multiplate IPA is able to detect the inhibitory effect of clopidogrel treatment on platelet function and that the Multiplate IPA values in whole blood correlate well with results of ADP LTA assessed in PRP. A strong, significant correlation coefficient of.73 has been found, similar to that observed by Sibbing et al 27 in 149 patients affected by coronary artery disease. In addition, this value was similar to that observed in other studies in which LTA was compared with different clopidogrelsensitive tests such as vasodilator-stimulated phosphoprotein phosphorylation 42 and the POC system, VerifyNow. 16 Downloaded from Am J Clin Pathol 29;131: DOI: 1.139/AJCPTE3K1SGAPOIZ 839

7 Paniccia et al / Platelet Aggregation in Whole Blood In the present study, the prevalence of 1 µmol/l ADP RPR in patients with ACS was 2.2% with LTA and 19.9% with the Multiplate IPA, and good, significant agreement (κ =.74; P <.1) between the 2 tests was found by using the cutoff value of 37 AUC calculated by us with ROC analysis. Assuming LTA as reference method, the ADP Multiplate IPA showed high specificity (95%) for identifying patients with ADP RPR, with a negative predictive value of 95%. This is the first study that compares the AA Multiplate IPA in whole blood with conventional LTA in PRP in a clinical setting showing, in terms of presence or absence of RPR, a concordance of 88.8% with significant, good agreement between the 2 tests (κ =.61; P <.1). Lordkipanidzé et al, 21 comparing impedance aggregation by a 4-channel Chronolog aggregometer, found a weaker correlation (ρ =.243; P <.5); likely the different correlation value has to be ascribed to the different characteristics of the device. The 2 impedance sensors of disposable test cells of the Multiplate aggregometer serve as an internal control to reduce the occurrence of systematic errors; during testing, the device correlates point-to-point measurements of the curves assessed by the 2 electrode pairs and the difference of the 2 AUCs is calculated 27 ; the result is flagged if the values are outside the acceptance range (correlation coefficient, <.98, difference to the mean curve, >2%). Notably, the Multiplate device expresses platelet aggregation as the AUC, which depends not only on the total height of the aggregation curve, but also on its time course. In addition, the Multiplate device, which uses disposable cuvettes and electrodes, guarantees the consistency of results not influenced by incompletely removed platelets attached on electrodes. Up to now, no investigation searched for a correlation between LTA and IPA by using collagen. In the present study, a strict relationship was found (ρ =.66; P <.1), with a large concordance of results (88.8%), similar to that obtained with AA but with higher agreement; this finding appears relevant for application in clinical studies, because in a large clinical study, we demonstrated that RPR to collagen in high-risk patients is an independent predictor of adverse cardiovascular events, with an accuracy of about 9%. 32 It is important to note that in this study, good agreement was also found between collagen LTA and PFA- CEPI CT, to indicate the role of collagen for identifying patients with ACS with RPR. In this study, a good relationship was observed by comparing Multiplate IPA data with those for the PFA- system (with AA and collagen for the first time), and the prevalence of RPR in patients with ACS receiving dual antiplatelet therapy (about 2%) by using the Multiplate device was comparable to that obtained by us 15,16 and by others 42,43 by using LTA. Study Limitations A limitation of this study is that we could not compare the Multiplate with other POC assays such as the VerifyNow system, Platelet Mapping assay (Hemoscope, Niles, IL), or Impact Cone and Plate(let) analyzer (Diamed, Cressier, Switzerland) (these methods reviewed by Harrison et al 12 and Gurbel et al 44 ). It is possible, but unlikely because this is a comparison study, that the presence of other drugs could interfere and somehow bias the results. There is also some concern about the variability in LTA measurements, but this potential error has been reduced by using a limited, well-trained staff with set procedural protocols. Conclusions Further studies of the assessment of platelet function by these different POC systems are necessary to evaluate their usefulness and comparability in clinical practice. The introduction of POC systems might give the chance to perform rapid and easy screening of platelet function in patients with ACS at high risk. In the scenario of the assessment of platelet function in whole blood, the Multiplate IPA represents a reliable, handy, rapid, and open system and allows the choice of the appropriate agonist for clinical investigations. From the Departments of 1 Medical and Surgical Critical Care, Thrombosis Center, University of Florence, Florence, Italy; and 2 Heart and Vessels, Azienda Ospedaliero-Universitaria Careggi, Florence. Multiplate aggregometer and cuvettes were kindly provided by Dynabyte, Munich, Germany. This organization had no role in study design, data collection, analysis and interpretation, and manuscript preparation or approval. Address reprint requests to Dr Paniccia: Dip Area Critica Medico-Chirurgica, Centro Trombosi, Malattie Aterotrombotiche, Piastra dei Servizi Azienda Ospedaliero-Universitaria Careggi, Viale Morgagni, 85, 5134 Firenze, Italia. References 1. Mehta SR, Yusuf S. Short- and long-term oral antiplatelet therapy in acute coronary syndromes and percutaneous coronary intervention. J Am Coll Cardiol. 23;41(4 suppl S):79S-88S. 2. Campbell CL, Steinhubl SR. Variability in response to aspirin: do we understand the clinical relevance? J Thromb Haemost. 25;3: Michelson AD, Linden MD, Furman MI, et al. Evidence that pre-existent variability in platelet response to ADP accounts for clopidogrel resistance. J Thromb Haemost. 27;5: De Miguel A, Ibanez B, Badimón JJ. Clinical implications of clopidogrel resistance. Thromb Haemost. 28;: Gasparyan AY, Watson T, Lip GY. The role of aspirin in cardiovascular prevention: implications of aspirin resistance. J Am Coll Cardiol. 28;51: Am J Clin Pathol 29;131: Downloaded 8 from DOI: 1.139/AJCPTE3K1SGAPOIZ

8 Coagulation and Transfusion Medicine / Original Article 6. Geisler T, Langer H, Wydymus M, et al. Low response to clopidogrel is associated with cardiovascular outcome after coronary stent implantation. Eur Heart J. 26;27: Buonamici P, Marcucci R, Migliorini A, et al. Impact of platelet reactivity after clopidogrel administration on drug-eluting stent thrombosis. J Am Coll Cardiol. 27;49: Crescente M, Di Castelnuovo A, Iacoviello L, et al. PFA- closure time to predict cardiovascular events in aspirin-treated cardiovascular patients: a meta-analysis of 19 studies comprising 3,3 patients. Thromb Haemost. 28;99: Born GV. Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature. 1962;194: Michelson AD. Platelet function testing in cardiovascular disease. Circulation. 24;11:e489-e493. doi:1.1161/1. CIR F Michelson AD, Frelinger AL III, Furman MI. Current options in platelet function testing. Am J Cardiol. 26;98:4N-1N. 12. Harrison P, Frelinger AL III, Furman MI, et al. Measuring antiplatelet drug effects in the laboratory. Thromb Res. 27;12: Shantsila E, Watson T, Lip GYH. Laboratory investigation of platelets. In: Gresele P, Fuster V, Lopez JA, et al, eds. Platelets in Hematologic and Cardiovascular Disorders: A Clinical Handbook. New York, NY: Cambridge University Press; 28: Harrison P, Segal H, Blasbery K, et al. Screening for aspirin responsiveness after transient ischemic attack and stroke: comparison of 2 point-of-care platelet function tests with optical aggregometry. Stroke. 25;36: Paniccia R, Antonucci E, Gori AM, et al. Comparison of different methods to evaluate the effect of aspirin on platelet function in high-risk patients with ischemic heart disease receiving dual antiplatelet treatment. Am J Clin Pathol. 27;128: Paniccia R, Antonucci E, Gori AM, et al. Different methodologies for evaluating the effect of clopidogrel on platelet function in high-risk coronary artery disease patients. J Thromb Haemost. 27;5: Dichiara J, Bliden KP, Tantry US, et al. Platelet function measured by VerifyNow identifies generalized high platelet reactivity in aspirin treated patients. Platelets. 27;18: Harrison P, Segal H, Silver L, et al. Lack of reproducibility of assessment of aspirin responsiveness by optical aggregometry and two platelet function tests. Platelets. 28;19: Agarwal S, Coakley M, Reddy K, et al. Quantifying the effect of antiplatelet therapy: a comparison of the platelet function analyzer (PFA-) and modified thromboelastography (mteg) with light transmission platelet aggregometry. Anesthesiology. 26;15: Ivandic BT, Schlick P, Staritz P, et al. Determination of clopidogrel resistance by whole blood platelet aggregometry and inhibitors of the P2Y12 receptor. Clin Chem. 26;52: Lordkipanidzé M, Pharand C, Schampaert E, et al. A comparison of six major platelet function tests to determine the prevalence of aspirin resistance in patients with stable coronary artery disease. Eur Heart J. 27;28: Hochholzer W, Trenk D, Frundi D, et al. Whole blood aggregometry for evaluation of the antiplatelet effects of clopidogrel. Thromb Res. 27;119: Ivandic BT, Giannitsis E, Schlick P, et al. Determination of aspirin responsiveness by use of whole blood platelet aggregometry. Clin Chem. 27;53: Tóth O, Calatzis A, Penz S, et al. Multiple electrode aggregometry: a new device to measure platelet aggregation in whole blood. Thromb Haemost. 26;96: Mueller T, Dieplinger B, Poelz W, et al. Utility of whole blood impedance aggregometry for the assessment of clopidogrel action using the novel Multiplate analyzer: comparison with two flow cytometric methods. Thromb Res. 27;121: Von Pape KW, Dzijan-Horn M, Bohner J, et al. Control of aspirin effect in chronic cardiovascular patients using two whole blood platelet function assays: PFA- and Multiplate [in German]. Hamostaseologie. 27;27: Sibbing D, Braun S, Jawansky S, et al. Assessment of ADP-induced platelet aggregation with light transmission aggregometry and multiple electrode platelet aggregometry before and after clopidogrel treatment. Thromb Haemost. 28;99: Gum PA, Kottke-Marchant K, Poggio ED, et al. Profile and prevalence of aspirin resistance in patients with cardiovascular disease. Am J Cardiol. 21;88: Giusti B, Gori AM, Marcucci R, et al. Role of glycoprotein Ia gene polymorphisms in determining platelet function in myocardial infarction patients undergoing percutaneous coronary intervention on dual antiplatelet treatment. Atherosclerosis. 28;196: Cuisset T, Frere C, Quilici J, et al. High post-treatment platelet reactivity is associated with a high incidence of myonecrosis after stenting for non-st elevation acute coronary syndromes. Thromb Haemost. 27;97: Marcucci R, Paniccia R, Antonucci E, et al. Residual platelet reactivity is an independent predictor of myocardial injury in acute myocardial infarction patients on antiaggregant therapy. Thromb Haemost. 27;98: Gori AM, Marcucci R, Migliorini A, et al. Incidence and clinical impact of dual nonresponsiveness to aspirin and clopidogrel in patients with drug-eluting stents. J Am Coll Cardiol. 28;52: Gori AM, Marcucci R, Paniccia R, et al. Thrombotic events in high risk patients are predicted by evaluating different pathways of platelet function. Thromb Haemost. 28;: Linnemann B, Schwonberg J, Mani H, et al. Standardization of light transmittance aggregometry for monitoring antiplatelet therapy: an adjustment for platelet count is not necessary. J Thromb Haemost. 28;6: Mengistu AM, Wolf MW, Boldt J, et al. Evaluation of a new platelet function analyzer in cardiac surgery: a comparison of modified thromboelastography and whole-blood aggregometry. J Cardiothorac Vasc Anesth. 28;22: Görlinger K, Jambor C, Hanke AA, et al. Perioperative coagulation management and control of platelet transfusion by point-of-care platelet function analysis. Transfus Med Hemother. 27;34: Steinlechner B, Dworschak M, Birkenberg B, et al. Platelet dysfunction in outpatients with left ventricular assist devices. Ann Thorac Surg. 29;87: Rahe-Meyer N, Winterhalter M, Hartmann J, et al. An evaluation of cyclooxygenase-1 inhibition before coronary artery surgery: aggregometry versus patient self-reporting. Anesth Analg. 28;17: Rahe-Meyer N, Winterhalter M, Boden A, et al. Platelet concentrates transfusion in cardiac surgery and platelet function assessment by multiple electrode aggregometry. Acta Anaesthesiol Scand. 29;53: Downloaded from Am J Clin Pathol 29;131: DOI: 1.139/AJCPTE3K1SGAPOIZ 841

9 Paniccia et al / Platelet Aggregation in Whole Blood. Inglott S, Burgess C, Liesner R, et al. The evaluation of impedance aggregometry to measure response to ristocetin in children in type I von Willebrand disease pre and post intranasal DDAVP [abstract]. Haemophilia. 26;12(suppl 2):3. Abstract PO Lison S, Dick A, Giebl A, et al. The evaluation of whole blood platelet aggregation in von Willebrand disease [abstract]. Haemostasiology. 29;1:PP Gurbel PA, Bliden KP, Etherington A, et al. Assessment of clopidogrel responsiveness: measurements of maximum platelet aggregation, final platelet aggregation and their correlation with vasodilator-stimulated phosphoprotein in resistant patients. Thromb Res. 27;121: Angiolillo DJ, Fernández-Ortiz A, Bernardo E, et al. High clopidogrel loading dose during coronary stenting: effects on drug response and interindividual variability. Eur Heart J. 24;25: Gurbel PA, Bliden KP, Hayes KM, et al. The relation of dosing to clopidogrel responsiveness and the incidence of high posttreatment platelet aggregation in patients undergoing coronary stenting. J Am Coll Cardiol. 25;45: Am J Clin Pathol 29;131: Downloaded 842 from DOI: 1.139/AJCPTE3K1SGAPOIZ