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Cardiovascular Anesthesiology: Research Report

Point-of-Care Whole Blood Impedance Aggregometry Versus Classical Light Transmission Aggregometry for Detecting Aspirin and Clopidogrel: The Results of a Pilot Study

Velik-Salchner, Corinna MD*; Maier, Stephan MD*; Innerhofer, Petra MD*; Streif, Werner MD; Klingler, Anton PhD; Kolbitsch, Christian MD*; Fries, Dietmar MD§

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doi: 10.1213/ane.0b013e31818524c1
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Clinicians are increasingly confronted with patients admitted for elective or emergency surgery and taking antiplatelet drugs. Several platelet function tests have been used in clinical studies to quantify antiplatelet therapy and identify nonresponders.1–4 However, none of these tests, including the “gold standard” classical aggregometry, are suitable for clinical practice, where a simple instrument enabling quick measurement of platelet function is needed.5 The platelet function analyzer PFA-100 was introduced several years ago as a point-of-care instrument for platelet function and measures the time needed to form a platelet plug that occludes a given aperture during high shear stress. The PFA-100 is suitable for monitoring therapy with aspirin,6 desmopressin acetate therapy7 and as a screening test for detection of von Willebrand syndrome, but it failed to identify clopidogrel administration6,8 and might over-estimate the prevalence of aspirin resistance.9 Furthermore, the results depend on hematocrit and platelet count,10,11 thus limiting its intraoperative usefulness in patients exhibiting blood loss.11,12

The newly developed point-of-care instrument, the Multiplate analyzer (Multiplate, Dyna byte Medical, Munich, Germany), permits platelet aggregation to be measured after adding commonly used agonists by detecting changes in electrical resistance in whole blood. This eliminates the need for centrifugation of plasma or adjustment of platelet concentration as needed for classical aggregometry. Test cells are for single use, whereas the electrodes of forerunner models had to be manually cleaned after each use. Moreover, results are available within a few minutes. Instrument handling is easy, and platelet aggregation does not change between 30 and 240 min after vein puncture.13 The whole blood impedance technique has been compared to classical light transmission aggregometry (LTA)14 or single platelet counting13 using blood from healthy volunteers and blood donors, while data from surgical patients are lacking. Since discontinuation of antiplatelet therapy in cardiac patients puts at least selected patients at risk for thrombosis,15–17 patients often continue antiplatelet therapy as recommended by the consulting cardiologist. Therefore, we investigated consecutive cardiac patients admitted for aortocoronary bypass grafting and receiving no antiplatelet therapy, aspirin alone or in combination with clopidogrel. The aim of the study was to determine whether the Multiplate technique enables detection of these antiplatelet drugs and whether results are comparable with those of classical LTA or with results obtained with the PFA-100 device.

METHODS

Patient Selection and Study Design

After approval by the local Medical University Ethics Committee and written informed consent, 70 consecutive patients (male, n = 62; female, n = 8) scheduled for elective coronary artery bypass grafting were enrolled. Exclusion criteria were age <18 yr, emergency operation, lacking consent, severe liver or renal dysfunction, preoperative platelet count below 150 × 109/L and intake of antiplatelet drugs other than aspirin or clopidogrel. At the discretion of the consultant cardiologist, intake of aspirin and clopidogrel was continued intentionally until the day before surgery in cases of unstable angina pectoris or critical left main artery stenosis. In such cases, the benefit of continuing antiplatelet therapy to reduce myocardial ischemia was carefully weighed against the risk of bleeding.18–21 In addition to study-related blood sampling before induction of anesthesia, all patients were treated according to institutional routine.

For analysis of measurement results, patients were divided into three groups according to their intake of antiplatelet drugs: Patients in group A (n = 48) discontinued intake of antiplatelet drugs 7 days before surgery (control), while patients in group B (n = 11) were receiving aspirin (100 mg/d) and patients in group C (n = 11) receiving aspirin (100 mg/d) and clopidogrel (75 mg/d) until 1 day before surgery.

Laboratory Procedures

Blood Sampling

Blood samples were exclusively obtained with a 3 Fr radial catheter (BD, Swingdon, UK) immediately after inserting the arterial line and before the patients received any IV fluid therapy. All blood samples were analyzed after a resting phase of 30 min.

Hemoglobin levels, hematocrit values, platelet counts, and white blood cell counts were measured in 2.7-mL tubes containing 1.6 mg ethylene diamine tetra-acetic acid/mL blood (Sarstedt, Nuermbrecht, Germany) using the whole blood counter Sysmex XE 2100® (Sysmex, Kobe, Japan). For coagulation tests and LTA, blood samples were collected in 3.0-mL tubes containing 0.3 mL (0.106 mol/L) buffered (pH 5.5) sodium citrate (Sarstedt, Nuermbrecht, Germany). For whole blood impedance aggregometry and PFA-100, blood samples were collected in 3.8-mL tubes containing 0.38 mL (0.129 mol/L) buffered (pH 5.5) sodium citrate (Sarstedt, Nuermbrecht, Germany). Prothrombin time, activated partial thromboplastin time, fibrinogen concentration (Clauss method) and antithrombin were determined by standard laboratory methods using the appropriate tests from Dade Behring (Marburg, Germany). Standard human plasma and Control Plasma N® (Dade Behring, Marburg, Germany) were used to establish reference curves and quality control.

Assays for Platelet Function

Classical optical LTA.

Blood samples were centrifuged at 150 g for 15 min to obtain platelet-rich plasma (PRP, containing 250 × 109/L platelets), and platelet aggregation was performed with the PAP4ff® Thrombozytenaggregometer (Mölab, Hilden, Germany). Agonists were used according to laboratory routine for performing LTA assays at our institution and induced with collagen (LTA-COL) having a final concentration of 1.4 μg/mL PRP (Nycomed, Linz, Austria), arachidonic acid (LTA-AA) having a final concentration of 1.7 mM/mL PRP (Mölab, Hilden, Germany), adenosine diphosphate (LTA-ADP) at a final concentration of 2.7 μM/mL PRP (ADP, -Chrono-Par, Chrono Log, Haverton) and epinephrine (LTA-EPI) at a final concentration of 14 μM/mL PRP (Aventis, Vienna, Austria).

Point-of-care whole blood impedance aggregometry (M) (Multiplate, Dynabyte Medical, Munich, Germany).

Impedance aggregometry is based on the principle that blood platelets are nonthrombogenic in their resting state, but expose receptors on their surface after activation by agonists that allow them to attach to artificial surfaces.22 The Multiplate test cell provides two independent measuring units, each consisting of two silver-coated, conductive copper wires with a blood-contacting surface with a length of 3.2 mm and a diameter of 0.3 mm each. During analysis, the sample-reagent mixture is automatically stirred using a discardable PTFE (Poly-tetra-fluoro-ethylene)–coated magnetic stirrer (800 U/min). The instrument continuously measures the change in resistance, which is proportional to the amount of platelets adhering to the electrodes and transforms it to arbitrary “aggregation units” (AU); these are plotted against time (min) and give the area under the aggregation curve (AUC = AU*min) (Fig. 1), calculated from the mean values of the two curves. The analysis is accepted when the difference between the two curves is <20%. The device has five channels and thus enables parallel testing of five blood samples.13 Analysis uses 300 μL of whole blood at 37°C with the addition of 300 μL normal saline containing CaCl2 at a concentration of 3 mM. After an incubation time of 3 min, 20 μL of the selected agonist solution is added, giving final concentrations of collagen (M-COL) at 3.2 μg/mL (COL-Test, Dynabyte, Munich, Germany), arachidonic acid (M-AA) at 0.5 mM (ASPITEST, Dynabyte, Munich, Germany), adenosine diphosphate (M-ADP) at 6.4 μM (ADP-Test, Dynabyte, Munich, Germany), or thrombin receptor-activating peptide (M-TRAP) at 32 μM (TRAP-Test, Dynabyte, Munich, Germany).

F1-7
Figure 1.:
Typical Multiplate® tracing: during a measurement period of 6 min the change in electrical impedance (AU*min) is calculated from the mean values of the two curves.

The platelet function analyzer (PFA-100, Dade Behring, Marburg, Germany) is designed to determine platelet function in whole blood (800 μL) in vitro during high shear stress by measuring the time needed to close a defined 150-μm aperture in a membrane coated with COL and EPI (PFA100-EPI) or ADP (PFA100-ADP) as activating agents.7

Statistical Analysis

Data are given as medians (1st, 3rd quartiles) and were analyzed with nonparametric tests because of a lack of normal distribution. Differences in baseline values were analyzed with the Kruskal-Wallis Test and post hoc Wilcoxon’s ranked sum test for unpaired observations for comparison of intergroup differences. Correlations between classical aggregometry, the Multiplate and the PFA-100 assays were analyzed using Spearman’s correlation coefficients. However, correlation analysis shows only that results are related to each other, and does not allow the accuracy of various test methods to be assessed. To assess the accuracy of the three different platelet function test methods in discriminating patients not taking from those taking antiplatelet drugs, we calculated receiver operating characteristic (ROC) curves.23 A ROC curve is the plot of a test’s true-positive rate (sensitivity) versus its false-positive rate (1-specificity) and is determined by calculating the sensitivity and specificity of a test for each possible result.24 The area under the ROC curve (AUC) is a quantitative measure of selectivity (1.0 = best selectivity; 0.5 = worst selectivity) and is a function of the sensitivity and specificity of a test.24 Areas under the ROC curves were compared using an algorithm suggested by DeLong et al.25 A P value <0.05 was considered statistically significant.

RESULTS

Patients were comparable with regard to demographic and baseline coagulation variables (Table 1).

T1-7
Table 1:
Results of Laboratory Analysis and Demographics (No Significant Differences Among Groups Were Detected)

Multiplate Point-of-Care Whole Blood Impedance Aggregometry (M)

(Fig. 2A) Patients not taking antiplatelet drugs showed significantly larger M-COL (median, [1st, 3rd quartiles]) values (374 AU*min, 231,469) than did patients receiving aspirin alone (164 AU*min, [86–211] P = 0.0009) or those receiving aspirin and clopidogrel (118 AU*min, [101–244] P = 0.004). As expected, M-AA aggregation was significantly greater in patients not taking antiplatelet drugs (200 AU*min, [86–345]) than in those taking aspirin alone (45 AU*min, [28–60] P = 0.0004) or on aspirin and clopidogrel (44 AU*min, [26–221] P = 0.008). M-ADP showed a strong trend to distinguish between patients taking no antiplatelet drugs (258 AU*min, [158–389] or taking aspirin 261 AU*min, [159–393]) from those taking aspirin and clopidogrel together (88 AU*min, [48–231] P = 0.054). M-TRAP values showed no intergroup differences.

F2-7
Figure 2.:
(A) Results of platelet function tests performed with the Multiplate® whole blood impedance aggregometry in patients not taking antiplatelet drugs, patients taking aspirin 100 mg/d and those taking 100 mg aspirin/d and 75 mg clopidogrel/d until the day before surgery. *P < 0.05 no antiplatelet drugs vs aspirin, #P < 0.05 no antiplatelet drugs vs aspirin and clopidogrel, for comparison of calculated differences. (B) Results of platelet function tests performed with classical light transmission aggregometry in patients not taking antiplatelet drugs, patients taking aspirin 100 mg/d and those taking 100 mg aspirin/d and 75 mg clopidogrel/d until the day before surgery. *P < 0.05 no antiplatelet drugs vs aspirin, #P < 0.05 no antiplatelet drugs vs aspirin and clopidogrel, ΔP < 0.05 aspirin vs aspirin and clopidogrel for comparison of calculated differences. (C) Results of platelet function tests performed with the PFA-100® in patients not taking antiplatelet drugs, patients taking aspirin 100 mg/d and those taking 100 mg aspirin/d and 75 mg clopidogrel/d until the day before surgery. *P < 0.05 no antiplatelet drugs vs aspirin, #P < 0.05 no antiplatelet drugs vs aspirin and clopidogrel, for comparison of calculated differences.

Classical Optical LTA

(Fig. 2B) Patients taking no antiplatelet drugs showed significantly greater LTA-COL (median, [1st, 3rd quartiles]) values (58%, [50–72]) than did patients taking aspirin alone (41%, [23–48] P = 0.0004) or taking aspirin and clopidogrel (28%, [20–53; P = 0.004]). As expected, LTA-AA aggregation was significantly greater in patients taking no medication (66%, [38–74]) than in those on aspirin alone (15%, [13–18] P = 0.0002) or aspirin and clopidogrel (19%, [13–62] P = 0.046). LTA-ADP values did not differ between patients taking no antiplatelet drugs (42%, [31–54]) and patients taking aspirin alone (48%, [34–60]), but were significantly lower in patients taking aspirin and clopidogrel (29%, [16–41]) than in those not taking antiplatelet medication (P = 0.023) or those taking aspirin alone (P = 0.017).

LTA-EPI was not able to distinguish among the three groups.

PFA 100

(Fig. 2C) PFA100-EPI closure times (median, [1st, 3rd quartiles]) were significantly shorter in patients not taking antiplatelet drugs (132 s, [111–174]) than in patients taking aspirin alone (251 s, [143–300] P = 0.0292) or aspirin and clopidogrel (274 s, [164–300] P = 0.008). PFA100-ADP closure times were similar in all groups.

Comparison of the Methods for Platelet Function Testing (PFT)

Pairwise Spearman correlation showed a significant correlation between M-COL and LTA-COL values (r2 = 0.27, P = < 0.0001), between M-AA and LTA-AA (r2 = 0.2, P = 0.0005) and between M-ADP and LTA-ADP (r2 = 0.08, P = 0.025). PFA-EPI showed a significant correlation only to LTA-AA (r2 = 0.13, P = < 0.001) (data not shown).

ROC Curves

The area under the ROC curves used to discriminate patients taking aspirin alone until the day before surgery and patients not taking antiplatelet drugs varied from 0.84 to 0.87 for M-COL, M-AA, LTA-COL, and LTA-AA (Table 2, Fig. 3A). At 0.74 the AUC for the PFA-100-EPI was lower than for the other four assays (Fig. 3C). Pairwise comparison of the AUC values showed no statistically significant differences among M-COL, M-AA, LTA-COL, LTA-AA, and PFA-100-EPI, and these results were significantly better than were the AUC values for M-ADP, M-TRAP, LTA-ADP, LTA-EPI, and PFA-100-ADP (P = <. 0.01), which were not able to distinguish between patients taking no antiplatelet drugs and patients taking aspirin. For discrimination of patients taking no antiplatelet drugs from patients taking aspirin and clopidogrel until the day before surgery, AUC values varied from 0.73 to 0.78 for LTA-ADP, M-ADP, M-COL, LTA-COL, M-AA, LTA-AA, and PFA-100-EPI (Table 3, Fig. 3B). A pairwise comparison between the AUC values for M-COL, M-ADP, LTA-COL, LTA-ADP for discrimination of patients taking aspirin and clopidogrel from patients not taking antiplatelet drugs showed no statistically significant differences. M-TRAP, LTA-EPI and PFA-100-ADP were not able to distinguish between patients not taking antiplatelet drugs and patients taking aspirin and clopidogrel. AUC values for M-AA, LTA-AA, and PFA-100-EPI were also significant (Table 3), but these tests are likely to be influenced by aspirin alone since the area under the ROC curves for discrimination of patients taking aspirin from patients taking aspirin and clopidogrel were significant only for LTA-ADP (AUC 0.80, P = 0.017, sensitivity 82%, specificity 73%) and M-ADP (AUC 0.75, P = 0.045, sensitivity 64%, specificity 91%) (data not shown).

T2-7
Table 2:
Area Under the ROC Curves, 95% CI Values, Cutoff Values and Corresponding Sensitivity and Specificity As Measured by Multiplate® Whole Blood Impedance Aggregometry, by Classical Light Transmission Aggregometry and by the PFA-100® in Patients Taking Aspirin 100 mg/d Until the Day Before Surgery in Comparison to Patients Taking No Antiplatelet Drugs
F3-7
Figure 3.:
Receiver operating characteristic (ROC) curves for classical light transmission aggregometry, Multiplate® whole blood aggregometry and the PFA-100®; (A) using collagen and arachidonic acid in patients taking aspirin 100 mg/d until the day before surgery (B) using adenosine diphosphate and collagen in those taking aspirin 100 mg/d and clopidogrel 75 mg/d until the day before surgery and (C) using the PFA-100®-epinephrine cartridge in patients taking aspirin 100 mg/d until the day before surgery.
T3-7
Table 3:
Area Under ROC Curves, 95% CI Values, Cutoff Values and Corresponding Sensitivity and Specificity According to the AUC Values Measured by Multiplate Whole Blood Impedance Aggregometry, by Classical Light Transmission Aggregometry and by the PFA-100 in Patients Taking Aspirin 100 mg/d and Clopidogrel 75 mg/d Until the day Before Surgery in Comparison to Patients Taking No Antiplatelet Drugs

DISCUSSION

This is the first study evaluating the point-of-care Multiplate whole blood aggregometer to detect antiplatelet drugs as compared to classical LTA in cardiac patients preoperatively. Results show that both aggregation methods exhibit similar accuracy in detecting antiplatelet drugs (Fig. 3), which, however, was lower for clopidogrel than for aspirin. Because of its frequent use we also performed PFA-100 measurements, which showed accuracy in detecting aspirin’s effects to be inferior to those of both aggregation methods, and confirmed the known fact that the PFA-100 fails to identify clopidogrel.8 Interestingly, the PFA-100 EPI results correlated only with classical aggregometry induced by AA.

Generally, when testing platelet function the agonists ADP, COL, EPI, AA, and TRAP are used to activate platelets (Table 4). Aspirin irreversibly acetylates platelet cyclooxygenase-1, thereby decreasing synthesis of thromboxane A2, a potent platelet aggregator. Accordingly, when using classical aggregometry or the Multiplate, the agonists COL26,27 and AA, which activate platelets primarily via thromboxane formation, showed significantly weaker platelet aggregation in patients taking aspirin alone or combined with clopidogrel. As expected in the presence of aspirin alone, aggregation was maintained after stimulation with TRAP and ADP. EPI is one of the weakest agonists, shows an enhancing effect for other activators,28 and the presence of aspirin blocks aggregation completely,29 even at the strongest EPI concentrations. However, this was not observed in the present study when using LTA and EPI, while the PFA-100-EPI measurements showed significantly prolonged closure times in patients taking aspirin as compared with those not taking antiplatelet drugs. ADP plays a major role in thrombosis and hemostasis. It interacts with the P2Y1 and P2Y12 receptors on the platelet surface causing shape change and, at higher concentrations, aggregation, and degranulation of platelets. Clopidogrel irreversibly inhibits the platelet P2Y12 receptor for ADP by forming a disulfide bridge between the thiol group of the active metabolite and a cysteine residue of the platelet ADP receptor. Additionally, clopidogrel inhibits platelet aggregation after stimulation with thromboxane, COL and low concentrations of thrombin by inhibiting ADP’s effects and reducing platelet granule secretion.30–32 In our present study, only classical aggregometry-ADP and Multiplate-ADP were able to discriminate between patients taking aspirin from those taking aspirin and clopidogrel. TRAP mimics the strong aggregation effect of thrombin on platelets; the response is thus only slightly reduced in the presence of antiplatelet drugs,29 as observed in the present study (Table 5).

T4-7
Table 4:
Summary of Agonists Used for Light Transmission and Impedance Aggregometry
T5-7
Table 5:
List of Abbreviations

Because classical LTA is not routinely available, a bedside PFT that facilitates parallel testing of samples within a few minutes may be useful for the clinician. Confirming our findings, a recently published study by Sibbing et al. showed a significant correlation (r2 = 0.5, P = < .0001) for ADP-induced platelet aggregation when using classical LTA or the Multiplate whole blood aggregometer in 149 patients scheduled for coronary angiography and receiving a single dose of 600 mg clopidogrel.33 Seyfert et al. investigated healthy volunteers and blood donors not taking antiplatelet drugs14 and found no correlation between classical aggregometry and the Multiplate technique, while a good correlation (r2 = 0.85) was found by Tantry et al. when comparing classical aggregometry and modified thrombelastography for detection of aspirin resistance.9 The classical aggregometry method, originally described in 1963,34 used in PRP was developed by Born and Cross. When comparing the classical aggregometry and the Multiplate technique it should be remembered that the classical aggregometry and whole blood impedance aggregometry use completely different physical methods to detect platelet aggregation. Classical aggregometry measurements are derived from a change in light transmittance detected by a photometer and result from platelet shape change and platelet aggregation, whereas the Multiplate detects a change in resistance, which is proportional to the quantity of platelets adhering to the electrodes. Classical aggregometry is performed in PRP prepared by centrifugation of anticoagulated blood. Unfortunately, classical aggregometry is poorly standardized across laboratories because of the huge variation in centrifugation and concentrations of agonists used.35 For this reason, the method is time-consuming and laboratory-dependent, and larger platelets can be lost by centrifugation.5 Although classical aggregometry is considered the gold standard for PFT,36 this method is not in widespread clinical use. Nevertheless, classical aggregometry is a reliable method for detecting antiplatelet drugs such as aspirin, clopidogrel or both.37 Using this method, correlations with blood loss were reported in cardiac surgery38 and an increased risk for death, myocardial infarction and cerebrovascular accidents in patients identified as aspirin-insensitive.1 Such data are not currently available for whole blood aggregometry.

Since the first electronic aggregometry22 suitable for whole blood and for PRP was developed, several studies have shown its usefulness.17,39–42 Unfortunately, all PFT are associated with nonphysiological conditions and the influence of the endothelial cells, exposure of collagen and von Willebrand factor are lacking in available assays. However, the use of whole blood for PFT might be preferable because it prevents centrifugation injury to platelets and enables platelet function to be determined in the presence of red and white blood cells, as occurs in vivo. Platelets physiologically interfere with other blood cells, such as e.g., erythrocytes, leukocytes as well as microparticles that reveal procoagulant activity.43 It has been demonstrated that the presence of leukocytes and erythrocytes plays an important role because of their ability to produce ADP and convert ADP to adenosine monophosphate.31,44 To clarify whether these blood cells artificially hinder platelet aggregation when tests are performed in whole blood or whether results reflect the in vivo situation, further studies, especially using flow cytometry, are needed.

Some limitations of our study need to be mentioned. First, our results were obtained in a small patient population. Thus, further data are needed to confirm our preliminary results. However, it seems difficult to collect such data within an acceptable study period because only few patients meet the criteria for intended continuous intake of antiplatelet drugs. Second, our data refer to a comparison made in the preoperative period only, and in this pilot study we collected no data on the association between assay results and blood loss or transfusion requirements. Finally, the Multiplate analyzer has so far not been validated under conditions of low platelet count and/or low hematocrit, as occurring during and after surgery, and reference levels are available only for normal platelet numbers. Thus, additional studies are needed to clarify these issues.

In conclusion, our preliminary data show the Multiplate device is able to monitor the effects of aspirin and clopidogrel on platelet function in a manner comparable to that of classical LTA. For the clinician, a clear advantage of the Multiplate device is its ability to be used as a point-of-care instrument. Thus, this method might be useful for estimating platelet function in patients presenting for immediate surgery and taking antiplatelet drugs. Further studies are needed to determine the degree of platelet inhibition as measured by classical aggregometry and the Multiplate device, at which it is safe to perform invasive procedures, including surgery.

ACKNOWLEDGMENTS

The authors are indebted to Christian Reif for excellent technical assistance.

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