Summary
Objectives
We investigated the agreement between different platelet tests to identify clopidogrel non response.
Background
Biological definition of clopidogrel non response remains controversial. Different platelet tests have been linked with recurrent ischemic events and proposed for daily practice.
Methods
We prospectively investigated the agreement of platelet tests to isolate clopidogrel non response in patients receiving high 150 mg clopidogrel maintenance dose after coronary stenting. Clopidogrel response was assessed with ADP-induced aggregation (ADP-Ag) (non response if > 70%), Platelet reactivity index VASP (PRI VASP) (non response if > 50%) and Verify Now Point-of-care assay (VN) (non response if PRU > 240 AU).
Results
Seventy consecutive patients were included. The rates of non-responders were respectively: 13% ( n = 9) with the ADP-Ag, 39% ( n = 27) with the PRI VASP and 33% ( n = 23) with the VN. We observed significant correlation between different platelet tests assessing clopidogrel response: r = 0.55 ( p < 0.0001) for ADP-Ag and PRI VASP, r = 0.64 ( p < 0.0001) for ADP-Ag and VN and r = 0.59 ( p < 0.0001) for PRI VASP and VN. However, using the most common thresholds, the agreement between the difference tests was poor: 0.35 for ADP-Ag and PRI VASP, 0.36 for ADP-Ag and VN and 0.46 for PRI VASP and VN.
Conclusion
This study showed that assessment of platelet function inhibition by clopidogrel is highly test-specific. Indeed, our results demonstrated a poor agreement between different platelet assays and suggested that identification of clopidogrel non responders is test-dependent.
Résumé
Background
La définition biologique de la non réponse au clopidogrel reste controversée. Différents tests plaquettaires ont été reliés au pronostic clinique et proposés pour la pratique quotidienne.
Objectif
Étudier la concordance entre différents tests plaquettaires pour l’identification de la non réponse au clopidogrel.
Méthodes
Nous avons de façon prospective évalué la concordance entre différents tests plaquettaires pour l’identification de la non réponse au clopidogrel chez des patients recevant une forte dose d’entretien de clopidogrel (150 mg) après stenting coronaire. La réponse au clopidogrel était évaluée par l’agrégation plaquettaire induite par l’ADP (ADP-Ag, non-réponse si supérieure à 70 %), l’indice de réactivité plaquettaire VASP (IRP VASP, non réponse si supérieure à 50 %) et le test minute VerifyNow P2Y12 (non réponse si P2Y12 reaction units > 240).
Résultats
Soixante-dix patients consécutifs ont été inclus. Le taux de non répondeurs était : 13 % ( n = 9) avec l’ADP-Ag ; 39 % ( n = 27) avec l’IRP VASP ; et 33 % ( n = 23) avec le test VerifyNow. Il existait une corrélation significative entre les différents tests : r = 0,55 ( p < 0,0001) pour ADP-Ag et IRP VASP ; r = 0,64 ( p < 0,0001) pour ADP-Ag et VerifyNow ; et r = 0,59 ( p < 0,0001) pour l’IRP VASP et le VerifyNow. En revanche, utilisant les seuils les plus communément admis, la concordance entre les différents tests était médiocre : kappa = 0,35 pour ADP-Ag et l’IRP VASP ; 0,36 pour l’ADP-Ag et le VerifyNow ; et 0,46 pour l’IRP VASP et le VerifyNow.
Conclusion
L’évaluation de la réponse au clopidogrel et l’identification des patients non répondeurs est très « test-dépendante ».
Introduction
Several studies have reported interindividual variability in platelet response to clopidogrel with clinical relevance . However, no method of quantification of platelet function inhibition by clopidogrel has consensually been recommended. There is a clinical need to have a reliable platelet assay for measuring platelet function after antiplatelet therapy for monitoring and potentially tailoring antiplatelet dosing regimens to individual patients. Light transmittance platelet aggregometry remains the gold standard for platelet function assessment, and elevated platelet activation in the context of impaired response to clopidogrel using this technique has been associated with recurrent ischemic events . For response to clopidogrel, classical aggregometry has been criticized for poor reproducibility and lack of specificity for the P2Y12 pathway. A new flow cytometric vasodilator-stimulated phosphoprotein (VASP) phosphorylation assay has been introduced to measure specific inhibition of clopidogrel’s biochemical target via the P2Y12 receptor. The platelet reactivity index VASP has been also associated with recurrent ischemic events after PCI . These laboratory tests demand specific knowledge and skills, requires specialized equipments, and is labour-intensive. Recently, point-of-care assay as Verify Now system was introduced to assess clopidogrel response with clinical relevance . However, there is not yet a consensus, neither about the “gold standard” test, nor about the definition of non-response. We therefore designed a prospective study to assess the agreement between the most common threshold of ADP-Ag, PRI VASP and Verify Now point-of-care test to detect clopidogrel non-responders among clopidogrel-treated patients undergoing coronary stenting for Non ST Elevation Acute coronary Syndrome (NSTE ACS).
Methods
Study protocol
Consecutive patients admitted for NSTE ACS in our institution were eligible for this prospective study after successful coronary stenting. NSTE ACS was defined as clinical symptoms compatible with acute myocardial ischemia within 12 h before admission and at least one of the following: a new finding of ST changes at least two leads, elevated levels of cardiac markers or coronary artery disease as documented by a history of revascularization or myocardial infarction. The exclusion criteria were a history of bleeding diathesis, persistent ST elevation ACS, NYHA class IV, PCI or coronary bypass grafting (CABG) < 3 months, contraindications to antiplatelet therapy, platelet count < 100 G/L, and creatinin clearance < 25 mL/min. Patients received oral loading doses of 250 mg aspirin and 600 mg clopidogrel at least 12 hours before stenting. At discharge, all patients received aspirin 75 mg and clopidogrel 150 mg and assessment of clopidogrel response was performed at 1 month. The study protocol was approved by the ethics committee of our institution, and patients gave written informed consent for participation.
Blood samples and platelet parameters
Blood samples for testing platelet reactivity were drawn one month after discharge from a peripheral blood sample.
ADP-Induced platelet aggregation (ADP-Ag)
The blood-citrate mixture was centrifuged at 120 g for 5 min. The resulting platelet rich plasma (PRP) was kept at room temperature for use within 1 h. The platelet count was determined in the PRP sample and adjusted to 2.5 × 10 8 mL -1 with homologous platelet-poor plasma (PPP). Platelets were stimulated with ADP (10 μmol/L) and aggregation was assessed with a PAP4 Aggregometer (Biodata Corporation, Wellcome, Paris, France). Aggregation was expressed as the percentage change in light transmittance from baseline with PPP as reference. Here, we report data on maximal intensity of platelet aggregation. We performed one measurement for each sample. The coefficient of variation of maximal intensity of platelet aggregation with ADP was measured at 6.5%. Non-response to clopidogrel was defined as ADP-Ag > 70%, as previously proposed .
Platelet Reactivity Index VASP (PRI VASP)
To determine the VASP phosphorylation state of whole blood, we used a standardized flow cytometric assay (Platelet VASP ® ; Diagnostica Stago [Biocytex], Asnières, France), which is an adaptation of the method of Schwarz et al. . A platelet reactivity index (PRI VASP) was calculated from the median fluorescence intensity (MFI) of samples incubated with PGE1 or PGE1 and ADP according to the formula: PRI VASP = [MFI (PGE1) − MFI (PGE1+ADP)/MFIPGE1] × 100. Non-response to clopidogrel was defined as PRI VASP > 50%, as previously proposed .
Point of care Verify Now Assay
We used the VerifyNow P2Y12 (Accumetrics, San Diego, CA, USA) point-of-care system, a rapid platelet-function cartridge-based assay with specific cartridges for the P2Y12 pathway. The Verify Now P2Y12 is designed to directly measure the effects of drugs on the P2Y12 receptor. VerifyNow-P2Y12 assay uses prostaglandin E1 in addition to ADP to increase intraplatelet cAMP. VerifyNow-P2Y12 assay results are expressed in P2Y12 Reaction Units (PRU) and % inhibition P2Y12 from baseline activation via TRAP (% inhibition P2Y12). The Verify Now analyser is designed to measure this agglutination as an increase in light transmittance. We used the PRU to define non-response to clopidogrel with a PRU > 240 AU .
Statistical analysis
Statistical analysis was performed with the SAS Software (v 8.01; SPSS Inc., Chicago, IL, USA). Continuous variables are expressed as mean ± S.D. or median and interquartile range. Categorical variables are expressed as frequencies and percentages. The Wilcoxon rank-sum test was used to compare continuous variables. Comparison between categorical variables were performed using the v2-test or the Fischer’s exact test when frequencies were below five. The agreements between ADP-Ag, PRI VASP and PRU were determined by linear regression (Pearson’s correlation coefficient) and Bland-Altman analysis. For each comparison, agreement between the two tests was calculated using the kappa statistic. A kappa statistic value of < 0.40 represents poor-to-fair agreement, a value of 0.41–0.60 reflects moderate agreement, a value of 0.61–0.80 is considered substantial agreement, and a kappa value of 0.81–1.00 is considered excellent agreement. P < 0.05 was considered significant.
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