Comparison of VerifyNow-P2Y12 test and Flow Cytometry for monitoring individual platelet response to clopidogrel. What is the cut-off value for identifying patients who are low responders to clopidogrel therapy?

Abstract

Background: Dual anti-platelet therapy with aspirin and a thienopyridine (DAT) is used to prevent stent thrombosis after percutaneous coronary intervention (PCI). Low response to clopidogrel therapy (LR) occurs, but laboratory tests have a controversial role in the identification of this condition.

Methods: We studied LR in patients with stable angina undergoing elective PCI, all on DAT for at least 7 days, by comparing: 1) Flow cytometry (FC) to measure platelet membrane expression of P-selectin (CD62P) and PAC-1 binding following double stimulation with ADP and collagen type I either in the presence of prostaglandin (PG) E1; 2) VerifyNow-P2Y12 test, in which results are reported as absolute P2Y12-Reaction-Units (PRU) or % of inhibition (% inhibition).

Results: Thirty controls and 52 patients were analyzed. The median percentage of platelets exhibiting CD62P expression and PAC-1 binding by FC evaluation after stimulation in the presence of PG E1 was 25.4% (IQR: 21.4-33.1%) and 3.5% (1.7-9.4%), respectively. Only 6 patients receiving DAT (11.5%) had both values above the 1st quartile of controls, and were defined as LR. Evaluation of the same patients with the VerifyNow-P2Y12 test revealed that the area under the receiver-operating-characteristic (ROC) curve was 0.94 (95% CI: 0.84-0.98, p < 0.0001) for % inhibition and 0.85 (0.72-0.93, p < 0.005) for PRU. Cut-off values of ≤ 15% inhibition or > 213 PRU gave the maximum accuracy for the detection of patients defined as having LR by FC.

Conclusion: In conclusion our findings show that a cut-off value of ≤ 15% inhibition or > 213 PRU in the VerifyNow-P2Y12 test may provide the best accuracy for the identification of patients with LR.

Figures

Figure 1

Receiver Operator Characteristic (ROC) curves defining the sensitivity and specificity of the VerifyNow-P2Y12 test in patients receiving antiplatelet therapy. Sensitivity and specificity in discriminating responders from non-responders to treatment was based on the arbitrary definition of positive patients (Low responders to clopidogrel; n = 6) and negative patients (High plus Intermediate responders to clopidogrel; n = 46) based on the percentage of platelets exhibiting CD62P expression and PAC-1 binding after stimulation in the presence of PG E1. The two curves shown here, for results expressed as PRU (absolute P2Y12 Reaction Unit) and % of inhibition (% inhibition), respectively, define cut-off values of PRU > 213 and ≤ 15% inhibition, as those giving the highest accuracy (minimal false negative and positive results) in the VerifyNow-P2Y12 test.

Figure 2

Correlation between the results obtained by flow cytometric evaluation of platelet activation parameters and VerifyNow-P2Y12 test in patients receiving antiplatelet therapy. The percent of inhibition (% inhibition) was plotted against the percentage of platelets that expressed P-selectin (CD62P) after stimulation with 20 μM ADP and 5 μg/mL collagen type I for 110 min at room temperature in the presence of 1 μM PG E1. (§) First quartile of control values for CD62P expression. Patients receiving antiplatelet therapy with levels of CD62P expression and PAC-1 binding above this limit were defined as "Low-responder"; those with both values below this limit were defined as "High-Responder"; those with one value above and the other below the corresponding limit were defined as "Intermediate-responder". ($) Cut-off value for % of inhibition in the VerifyNow-P2Y12 test giving minimal false negative and positive results (see Figure 1).

Figure 3

Correlation between the results obtained by flow cytometric evaluation of platelet activation parameters and VerifyNow-P2Y12 test in patients receiving antiplatelet therapy. The percent of inhibition (% inhibition) was plotted against the percentage of platelets that expressed bound PAC-1 after stimulation with 20 μM ADP and 5 μg/mL collagen type I for 110 min at room temperature in the presence of 1 μM PG E1. (§) First quartile of control values for PAC-1 binding. The others abbreviations as reported in the Figure 2.