CLINICAL RESEARCH
European Heart Journal (2008) 29, 2877–2885
doi:10.1093/eurheartj/ehn419
Thrombosis and antithrombotic therapy
Comparison of four tests to assess inhibition
of platelet function by clopidogrel in stable
coronary artery disease patients
1
Faculty of Pharmacy, Université de Montréal, Montréal, Canada; 2Research Center, Hôpital du Sacré-Cœur de Montréal, 5400, boul. Gouin ouest, Montréal, Québec, Canada H4J
1C5; 3Department of Pharmacy, Hôpital du Sacré-Cœur de Montréal, 5400, boul. Gouin ouest, Montréal, Québec, Canada H4J 1C5; 4Faculty of Medicine, Université de Montréal,
Montréal, Canada; and 5Division of Cardiology, Hôpital du Sacré-Cœur de Montréal, 5400, boul. Gouin ouest, Montréal, Québec, Canada H4J 1C5
Received 14 March 2008; revised 19 August 2008; accepted 28 August 2008; online publish-ahead-of-print 30 September 2008
See page 2833 for the editorial comment on this article (doi:10.1093/eurheartj/ehn494)
Aims
We investigated the comparability of platelet function tests in quantifying platelet inhibition achieved by clopidogrel.
.....................................................................................................................................................................................
Methods
This pre-specified substudy of a randomized, double-blind trial included 116 patients with stable coronary artery
and results
disease requiring diagnostic angiography. Patients received clopidogrel for 1 (300 or 600 mg) or 7 days (300 þ 75
or 150 mg daily) before the procedure. Blood samples obtained before clopidogrel initiation and before diagnostic
coronary angiography were assayed using light transmission aggregometry [adenosine diphosphate (ADP) 5 and
20 mM as the agonist], whole-blood aggregometry (ADP 5 and 20 mM), PFA-100w (Collagen-ADP cartridge), and
VerifyNoww P2Y12. Although all assays studied were found sensitive to clopidogrel ingestion, none could distinguish
categorically between patients who had, or not, ingested clopidogrel. Agreement between assays to identify patients
with insufficient inhibition of platelet aggregation by clopidogrel was low.
.....................................................................................................................................................................................
Conclusion
The assessment of platelet function inhibition by clopidogrel is highly test-specific. Decision to increase clopidogrel
dosage may vary on the basis of the assay used, thus highlighting the need for unambiguous guidelines with respect to
assay selection, as platelet function assays are not interchangeable. At present, platelet function testing evaluating
clopidogrel efficacy cannot be recommended in routine clinical practice.
----------------------------------------------------------------------------------------------------------------------------------------------------------Keywords
Platelet function testing † Platelet aggregation † Platelet analyzers † Clopidogrel † Responsiveness
Introduction
Untoward platelet activation has been identified as an important
pathophysiological component of coronary artery disease (CAD)
and has led to the development of effective antiplatelet agents that
are now considered mainstay therapy for the prevention of acute
ischaemic events.1 Adding clopidogrel to daily aspirin treatment is
the basis of antiplatelet therapy in the context of percutaneous coronary interventions (PCIs), as it reduces the risk of stent thrombosis
to 1%.1 – 3 However, important interindividual variability in platelet
response to clopidogrel has been reported, resulting in a significant
proportion of patients displaying suboptimal inhibition of platelet
aggregation and an increased risk of thrombotic complications.4
There is disagreement in the recommendations issued by European and American experts on the conduct to adopt in patients
requiring antiplatelet therapy. While the European Society of
Cardiology states that routine assessment of platelet aggregation
inhibition in patients submitted to either aspirin or clopidogrel
therapy, or both, is not recommended, the American College of
Cardiology (ACC), the American Heart Association (AHA), and
the Society for Cardiovascular Angiography and Interventions
(SCAI) recommend that, in patients in whom stent thrombosis
may be catastrophic or lethal (unprotected left main, bifurcating
left main, or last patent coronary vessel), platelet aggregation
studies may be considered and the dose of clopidogrel increased
to 150 mg per day if ,50% inhibition of platelet aggregation is
*Corresponding author. Tel: þ1 514 338 2200, Fax: þ1 514 338 2694, Email: jean.gino.diodati@umontreal.ca
†The first two authors have contributed equally to this work.
Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org.
Downloaded from http://eurheartj.oxfordjournals.org/ at Pennsylvania State University on February 27, 2014
Marie Lordkipanidzé 1,2,3†, Chantal Pharand 1,2,3†, Thuy Anh Nguyen1,3,
Erick Schampaert 2,4,5, Donald A. Palisaitis 2,4,5, and Jean G. Diodati2,4,5*
2878
Methods
Patients
Patients were recruited from the pre-angiography outpatient clinic of
the Hôpital du Sacré-Cœur de Montréal in Canada and were included
if they presented suspected CAD requiring an elective diagnostic coronary angiography. Exclusion criteria were major bleeding disorders or
active bleeding; acute myocardial infarction, or unstable angina, with
ST-segment changes 1 mm in at least two contiguous electrocardiographic leads at rest or a troponin level .0.06 mg/L, within 14 days of
recruitment; stroke within the last 3 months; platelet count ,150 109/L, prothrombin time .1.5 times control, haematocrit ,35%, or
haemoglobin level ,100 g/L; alcohol or drug abuse; enrolment in
other investigational drug trials within the previous month; the use
of thienopyridines, glycoprotein IIb/IIIa inhibitors, warfarin, or acenocoumarol within the prior week; and allergic reaction or any contraindication to clopidogrel or aspirin. This study was approved by the
Institutional Scientific and Ethics Review Board, and patients gave
written informed consent for participation.
Study protocol and blood sampling
The present study was a planned and pre-specified substudy of a randomized, prospective, double-blind, and placebo-controlled trial evaluating the effect on platelet aggregation of four different dosing
regimens of clopidogrel given before elective diagnostic coronary
angiography with or without PCI.7 From 20 September 2004 to 18
April 2006, 116 subjects were randomly assigned in a double-blind
fashion to one of four groups of clopidogrel dosing regimens 1 week
prior to PCI: 300 mg (n ¼ 29) or 600 mg (n ¼ 28) on the day prior
to PCI; 300 mg followed by 75 mg daily (n ¼ 31); or 150 mg daily
(n ¼ 28) started 1 week before PCI. In addition to the randomized
study medication, all patients received 80 mg of enteric-coated
aspirin daily for at least 7 days before the procedure and throughout
the study period.
Four platelet function assays were assessed simultaneously in all
patients: LTA and electrical impedance whole-blood aggregometry
(WBA) after stimulation with ADP, VerifyNoww P2Y12, and Platelet
Function Analyzer (PFA-100w). Blood was drawn twice, before
clopidogrel initiation and just before elective coronary angiography.
The first 2 mL of blood, drawn by venipuncture through a 21 gauge
needle, was discarded. Then, blood was drawn into evacuated tubes
containing 3.2% sodium citrate. All blood samples were processed
within 2 h of collection.
Platelet aggregation assessment
Light transmission aggregometry
The assessment of platelet function by LTA was considered the gold
standard in this study.6 Platelet aggregation was assessed in PRP at
378C by LTA. PRP was obtained by the centrifugation of citrated
whole blood for 10 min at 1000 r.p.m. (89 g). Platelet-poor plasma
was obtained by the centrifugation of the remaining blood for
10 min at room temperature at 3000 r.p.m. (805 g). Platelet count in
the PRP varied from 250 to 450 109/L. No adjustment of platelet
count with PPP was performed, as it has been demonstrated that
the adjustment of PRP with PPP could have deleterious effects on
platelet function assessment.8,9 Aggregation was measured with a
ChronoLog Aggregometer (ChronoLog 540 model, Havertown, PA,
USA) after stimulation with 5 and 20 mM of ADP (Sigma Aldrich, Oakville, Ontario, Canada), using platelet-poor plasma as reference. All
analyses were performed by a single highly trained and experienced
technician. Aggregation curves were recorded for 5 min and analysed
according to international standards. The results are reported as
either maximal platelet aggregation or platelet inhibition, defined as
the relative change in platelet aggregation from baseline to the time
of angiography. Platelet inhibition is calculated as (12residual aggregation/baseline aggregation) 100.
Whole-blood aggregometry
WBA measures electrical impedance (maximal amplitude) between
two electrodes immersed in whole blood 5 min after the addition
of a platelet agonist (ADP 5 and 20 mM), using a ChronoLog
Aggregometer (ChronoLog 560 model, Havertown, PA, USA). The
results are reported as either residual platelet aggregation, measured
as maximal amplitude of impedance (V), or platelet inhibition,
defined as the relative change in impedance from baseline to the
time of angiography. Platelet inhibition is calculated as (12residual
impedance/baseline impedance) 100.
VerifyNoww P2Y12
The VerifyNoww P2Y12 (Accumetrics, San Diego, CA, USA)
point-of-care system is based on turbidimetric optical detection of
platelet aggregation in whole blood. Blood was drawn into evacuated
tubes containing 3.2% sodium citrate provided by the manufacturer.
After withdrawal, whole blood was transferred into cartridges containing a combination of 20 mM of ADP and 22 nM of PGE1, the latter
being added to specifically measure the effect of clopidogrel following
P2Y12, but not P2Y1, ADP receptor activation. As aggregation occurs,
the system converts luminosity transmittance results into P2Y12
reaction units (PRU). Results are reported as either residual platelet
aggregation, measured in PRU, or platelet inhibition, defined as the
relative change in platelet aggregation from baseline to the time of
angiography. Platelet inhibition is calculated as (12residual aggregation/
baseline aggregation) 100.
Platelet function analyser (PFA-100)w
PFA-100w (Dade Behring, Deerfield, IL, USA) is a point-of-care assay
that assesses platelet aggregation under high shear, mimicking
platelet-rich thrombus formation after injury to a small vessel wall
under flow conditions. Whole blood was transferred into standard
cartridges, and time necessary to occlude a microscopic aperture in
a membrane coated with collagen and ADP was measured. The
results are reported as closure time (s). As this technology does not
measure platelet aggregation directly but rather assesses the time
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demonstrated.3,5 However, no method of quantification of platelet
function inhibition by clopidogrel has consensually been
recommended.
Although light transmission aggregometry (LTA), which
measures luminosity as aggregation occurs in adenosine diphosphate (ADP)-stimulated platelet-rich plasma (PRP), is considered
by many as the current gold standard in platelet function testing,
the technique demands specific knowledge and skills, requires
specialized equipments, and is labour-intensive.6 Fast and easyto-use point-of-care methodologies are now widely available and
often employed to assess platelet response to clopidogrel.
However, little is known about the comparability or interchangeability of these tests.
Hence, we performed this study to evaluate the comparability of
four major platelet function assays in assessing platelet inhibition
provided by clopidogrel in patients with stable CAD requiring elective angiography.
M. Lordkipanidzé et al.
2879
Assessing inhibition of platelet function
although statistically significant, was small and resulted in almost
superimposable platelet aggregation profiles obtained at baseline
and post-clopidogrel administration (Figure 1F).
Sample size and statistical analysis
Inter-test correlations
Sample size was not calculated a priori to correlate platelet function
results from different assays at the time of diagnostic coronary angiography. Post hoc calculations showed that the study had a power of
80%, with a two-sided a-value of 0.05, to detect a correlation coefficient of at least 0.25 between any of the paired platelet aggregation
data sets obtained from the different platelet function assays (PASS
2002, NCSS 2004 statistical software, Kaysville, UT, USA).
Normally distributed continuous variables are presented as mean
(standard deviation), non-normally distributed continuous variables
as median (inter-quartile range), and categorical variables as frequencies (percentages). Variables were analysed for a normal distribution
with the Kolmogorov– Smirnov test. Continuous variables were compared using the paired t-test or the signed-rank test for comparison of
paired samples (the Wilcoxon test), and categorical variables were
compared using the x2 or Fisher exact test, when applicable. To
account for the randomization group, partial correlations between
results obtained with the various assays were calculated. Agreement
between assays to evaluate the inhibition of platelet aggregation
from baseline (%inhibition) was assessed through the Bland – Altman
agreement analysis. This analysis specifically measures bias, which can
be defined as a systematic error responsible for either under- or overestimation of a value, and sets limits agreement, similar to confidence
intervals, which indicate the range of under- or overestimation of one
reading in comparison with the other.10 Agreement among assays to
identify patients with insufficient platelet aggregation, as recommended
in current American guidelines, was assessed through the k statistic.
A two-sided P-value of ,0.05 was considered significant. Analyses
were performed with SPSS 14.0 for Windows (SPSS Institute,
Chicago, IL, USA) and GraphPad Prism 5 for Windows (GraphPad
Software Inc., San Diego, CA, USA).
Baseline
Before clopidogrel initiation, subjects displayed important variability in platelet function (Figure 1). Table 1 and Figure 2A show
Spearman’s coefficients of correlation between the various
platelet function assays. When the same assay was employed
using different concentrations of the same agonist, correlation
between results was strong. However, results obtained with
different assays yielded no or low correlation. Thus, results
obtained through LTA and WBA were associated poorly among
themselves (0.2 , r , 0.4), and point-of-care assays, namely
VerifyNoww P2Y12 and PFA-100w, lacked correlation with any of
the assays studied (r , 0.2).
Results
Patients
Of the 116 patients studied, 92 (79.3%) were male. Mean age was
60.2 + 9.0 years (range from 38 to 80 years). Eight (6.9%) were
diabetic, 75 (64.7%) had hypertension, 100 (86.2%) suffered from
dyslipidaemia, and 28 (24.1%) were active smokers. After diagnostic angiography, PCI was performed in 33 patients (28.4%). The
remaining 83 patients were classified as either presenting nonsignificant stenosis (n ¼ 31) or stenoses not requiring or amenable
to PCI (n ¼ 52).
Platelet aggregation studies
All patients underwent the analysis of platelet aggregation by LTA,
WBA, and PFA-100w. However, owing to irregularities in cartridge
supply, VerifyNoww P2Y12 assay was performed in only 72 subjects.
All platelet function assays were able to discern a statistically
significant difference in platelet function following clopidogrel
intake, however to varying degrees (Figure 1). With most assays,
clopidogrel ingestion resulted in a major inhibitory shift in platelet
aggregation profiles, although some overlap between baseline and
post-clopidogrel values was notable (Figure 1A –E). On the other
hand, the prolongation of closure time found with PFA-100w,
After clopidogrel administration
Following clopidogrel administration, wider inter-subject variability
was present (Figure 1). As at baseline, the use of different concentrations of an agonist with the same assay resulted in highly correlated results (Table 2 and Figure 2B). The association between most
analysed pairs of assays improved slightly compared with what was
observed at baseline, but remained poor.
Agreement among assays in measuring
clopidogrel-induced platelet inhibition
The agreement between clopidogrel-induced platelet inhibition
results among platelet function assays was studied by the Bland–
Altman analysis of agreement. As ADP-induced LTA is the generally accepted gold standard in platelet function testing, we first
compared platelet inhibition reported by both concentration of
ADP with this methodology (Figure 3). The inhibition of platelet
aggregation induced by ADP 5 and 20 mM showed a slight bias
of 4.5% [95% confidence interval (95% CI) 1.3– 7.7, P ¼ 0.006 by
paired t-test], indicating greater inhibition when 5 mM of ADP
was used. However, the limits of agreement varied from 229
(95% CI from 234 to 225) to 38% (95% CI 34– 42), indicating
that the two ADP concentrations may significantly disagree in
certain individuals (Figure 3).
Since the ADP concentration most commonly used in the literature is 20 mM, the extent of platelet inhibition measured by WBA
and VerifyNoww P2Y12 was compared with that obtained with
20 mM ADP-induced LTA. Because the PFA-100w assay results
cannot be converted into %inhibition, the assay has been excluded
from agreement analysis.
The inhibition of platelet aggregation was overestimated by 13%
[95% CI 2.9 –24.0, P ¼ 0.01, limits of agreement 297 (95% CI
2112 to 282) to 124% (95% CI 109–139)] when 5 mM
ADP-induced WBA was used and underestimated by 11% [95%
CI 220.7 to 21.8, P ¼ 0.02, limits of agreement 2110 (95% CI
2123 to 297) to 87% (95% CI 74–100)] when 20 mM
ADP-induced WBA was used in comparison with 20 mM
ADP-induced LTA (Figure 4A and B). When platelet inhibition
assessed by the VerifyNoww P2Y12 assay was compared with
20 mM ADP-induced LTA, VerifyNoww P2Y12 overestimated
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required for clot formation, platelet inhibition cannot be calculated.
Thus, the impact of clopidogrel administration is reported as the absolute prolongation of closure time (s) from baseline.
2880
M. Lordkipanidzé et al.
Table 1 Correlation (95% confidence interval) between platelet function tests at baseline
Platelet
function test
LTA ADP 20 mM
LTA ADP 5 mM
0.766a (0.679–0.832)
WBA ADP 5 mM
WBA ADP 20 mM
PFA-100w
VerifyNoww P2Y12
...............................................................................................................................................................................
LTA ADP
20 mM
0.229a (0.049–0.394)
a
0.249 (0.070–0.412)
WBA ADP
5 mM
0.326a (0.153–0.479)
0.102 (20.081 to 0.279)
0.167 (20.067 to 0.383)
0.309a (0.135–0.465)
0.076 (20.107 to 0.254)
0.134 (20.100 to 0.354)
0.732a (0.635–0.806)
20.197a (20.366 to 20.016)
0.052 (20.181 to 0.280)
0.036 (20.147 to 0.216)
0.006 (20.225 to 0.237)
WBA ADP
20 mM
PFA-100w
a
20.020 (20.212 to 0.250)
Correlation is significant at P , 0.05.
inhibition by a mean of 6.3% (95% CI 21.6–14.2%, P ¼ 0.117), with
wide limits of agreement [254.4 (95% CI 264 to 244) to 67.0%
(95% CI 57–77), Figure 4C], indicating poor accord between assays.
Agreement among assays to identify
patients with insufficient platelet
inhibition
Current guidelines by ACC/AHA/SCAI without any supporting evidence state that adjusting the clopidogrel dose may be considered
if ,50% inhibition of platelet aggregation is detected on clopidogrel therapy.3 Results were reanalysed seeking to investigate
whether clinical conduct in such a context would be influenced
by the platelet function-testing methodology chosen. Insufficient
platelet inhibition (,50%) was found in 55% of patients by 5 mM
ADP-induced LTA, 66% of patients by 20 mM ADP-induced LTA,
71% of patients by 5 mM ADP-induced WBA, 47% of patients by
20 mM ADP-induced WBA, and 61% of patients by VerifyNoww
P2Y12. Overall, agreement was poor, as assessed by the k statistic
and presented in Table 3. Although WBA failed to select the same
patients as candidates for intensified therapy as either LTA or
VerifyNoww P2Y12, the latter showed fair agreement with LTA,
with 20 mM of ADP yielding better results. The only results displaying strong agreement were the different ADP concentrations
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Figure 1 Platelet function data obtained from various platelet function assays, before and after clopidogrel intake. (A) Light transmission
aggregometry stimulated with 5 mM of ADP. (B) Light transmission aggregometry stimulated with 20 mM of ADP. (C) VerifyNoww P2Y12.
(D) Whole-blood aggregometry stimulated with 5 mM of ADP. (E) Whole-blood aggregometry stimulated with 20 mM of ADP.
(F ) PFA-100w Line represents the median, and whiskers the inter-quartile range. ADP, adenosine diphosphate.
2881
Assessing inhibition of platelet function
Table 2 Correlation (95% confidence interval) between platelet function tests after clopidogrel administration
Platelet
function
test
LTA ADP 20 mM
LTA ADP
5 mM
LTA ADP
20 mM
WBA ADP
5 mM
WBA ADP
20 mM
0.902a (0.862–0.931)
WBA ADP 5 mM
WBA ADP 20 mM
PFA-100w
VerifyNoww P2Y12
...............................................................................................................................................................................
0.255a (0.077–0.417)
0.307a (0.133–0.463)
20.270a (20.438 to 20.102)
0.370a (0.152–0.554)
0.291a (0.112–0.449)
0.382a (0.215–0.527)
20.274a (20.434 to 20.097)
0.496a (0.299–0.652)
0.881a (0.833–0.916)
20.139 (20.313 to 0.044)
0.187 (20.046 to 0.401)
20.150 (20.322 to 0.033)
0.293a (0.066–0.491)
PFA-100w
20.334a (20.525 to 20.111)
a
Correlation is significant at P , 0.05.
used in LTA, thus suggesting that ADP concentration plays a minor
role in quantifying platelet response to clopidogrel when assessed
optically.
Discussion
Ability to identify patients with insufficient platelet inhibition by
clopidogrel using different platelet function assays varies greatly
according to the platelet function assay used, and correlation
between the tests is poor. Although all assays studied were
found sensitive to clopidogrel therapy, none could distinguish categorically patients who were treated with clopidogrel from those
who were not. Comparability between assays was low, both at
baseline and after clopidogrel intake, as demonstrated by poor
agreement through the Bland– Altman analysis of clopidogrelinduced platelet inhibition. Recommendation to increase clopidogrel dosing if insufficient platelet inhibition by clopidogrel is
demonstrated, as suggested in the current ACC/AHA/SCAI
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Figure 2 Partial correlation (controlling for the randomization group) between aggregation results obtained by the various platelet function
assays. (A) Correlation between tests at baseline. (B) Correlation between tests after clopidogrel ingestion. ADP, adenosine diphosphate; LTA,
light transmission aggregometry; WBA, whole-blood aggregometry.
2882
bition assessed by 5 and 20 mM ADP-induced LTA. Solid red
line represents bias (4.5%), dashed blue lines represent 95% confidence interval (from 1.3 – 7.7), dotted green lines represent
limits of agreement (229 to 38), and dashed grey line represents
95% confidence intervals of limits of agreement. ADP, adenosine
diphosphate; LTA, light transmission aggregometry.
guidelines, would benefit from a revision to specify platelet function assay selection, as the current available assays are clearly
not interchangeable.
A number of studies have looked at the correlation between
various assays in defining platelet response to clopidogrel.11 – 13
Although the use of correlation to report on the association
between two measurements is widespread, it is often inappropriate as it does not imply agreement between methods, nor does
it evaluate bias.14 To weigh one method against another, or to
assess how closely two measurements are related, the Bland–
Altman agreement analysis was used in this study to overcome
the shortcomings of correlation. Thus, this study expands on our
current understanding of how each assay measures up against
the rest.
LTA is considered by many as the gold standard in platelet function testing.6 In addition to having been used extensively in the last
50 years, it remains to this day the most widely used assay in the
assessment of platelet response to clopidogrel, and it targets, with
the use of ADP as the agonist, the platelet activation pathway
inhibited by clopidogrel, namely the purinergic pathway. Even
though the assay has become better automated, it remains timeand labour-intensive, requires technical expertise, and thus is
restricted to specialized laboratories. Its major drawback
however is the lack of standardization, which renders the assay
results hard to compare between research teams.15 As this study
is a single-centre study and all analyses were performed by a
single highly trained and experienced technician, the latter limitation of LTA could be avoided, thus minimizing the variability of
LTA and resulting in more precise measurements. Predisposing
conditions that can create disparities in results between laboratories include different blood specimen temperatures, choice of
anticoagulant agent, varying delays of testing from blood collection,
adjustment of platelet count to a pre-specified level in the PRP
specimen, varying ADP concentrations, and quantification of platelet aggregation at maximal levels vs. at the end of the experiment.15,16 Although each of these variables affects the outcome
of aggregometry measurement and interpretation, LTA remains
among the leading platelet function testing tools to evaluate platelet response to clopidogrel.
Although WBA is seldom used in the literature to evaluate
platelet response to clopidogrel (like LTA, it remains restricted
to specialized laboratories), the poor correlation found between
WBA and LTA results in the current study is consistent with a
recent report, which described a similar correlation (r ¼ 0.257)
between 20 mM ADP-induced LTA and WBA in 27 patients scheduled to undergo PCI.17 Similarly, in 17 healthy volunteers, 20 mM
ADP-induced LTA and WBA disagreed significantly in identifying
patients with .50% inhibition of platelet aggregation.18 In comparison with LTA, aggregometry measured in whole blood requires
less manipulation of the specimen, thus making the assay less prone
to artefactual platelet aggregation during preparatory steps.19,20 It
also offers the advantage of a more physiologically relevant
milieu for platelets, said to be more sensitive to the antiplatelet
effect of clopidogrel.21,22 However, the presence of other blood
constituents alters platelet aggregation profiles and produces less
consistent results than those obtained in PRP.23 Erythrocytes and
leukocytes have been shown to actively regulate the availability
of ADP in blood, and higher doses of ADP are believed to be
necessary to elicit platelet aggregation in whole blood.21,24 This
particularity may explain the important variability detected in
whole blood clopidogrel-induced platelet inhibition in our study
and the subsequent lack of agreement with LTA results obtained
with the same agonist concentrations.
In an effort to make platelet function testing widely available
outside of specialized laboratories, several point-of-care assays
have been commercialized. The VerifyNoww P2Y12 assay was
developed and Food and Drug Administration-approved to specifically evaluate the effect of P2Y12 receptor blockade on platelet
aggregation. This technology offered the novelty of isolating the
effect of ADP stimulation on the P2Y12 receptor from that of
the P2Y1 receptor, by incorporating the effects of PGE1 in addition
to ADP.25 This design aimed at limiting the variability of response
to clopidogrel by removing the contribution of the P2Y1 ADP
receptor, which is unaffected by clopidogrel administration.
However, our results and others published recently detected
significant variability in platelet aggregation measured with this
device, with an important overlap between pre- and postclopidogrel values.26 In comparing these results with those
obtained through LTA, we found weak-to-moderate correlation,
which contrasts with recent reports describing a much stronger
correlation (0.73 , r , 0.86) between these methodologies.11,12
Moreover, we found that agreement between these methodologies in selecting patients with insufficient platelet inhibition
was low, concordant with a recent report on 1267 patients
admitted for acute coronary syndrome.13 We further compared
the VerifyNoww P2Y12 device results with those obtained
through WBA, as the VerifyNoww P2Y12 device also requires a
whole-blood specimen. It is worth mentioning that the disadvantages of whole-blood specimens apply to the VerifyNoww P2Y12
technology as well. The association between the VerifyNoww
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Figure 3 Bland – Altman agreement analysis in platelet inhi-
M. Lordkipanidzé et al.
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Assessing inhibition of platelet function
P2Y12 results and WBA was not improved in comparison with
LTA. It also resulted in a complete lack of agreement in selecting
patients with insufficient clopidogrel-induced platelet inhibition.
The PFA-100w is another point-of-care device intended for the
detection of platelet dysfunctions. However, our results are in
agreement with several reports describing its inability to monitor
clopidogrel therapy.27 – 30 As described previously, although a statistical difference could be detected, clopidogrel ingestion did not
result in a major inhibitory shift of platelet aggregation. Furthermore, the PFA-100w results lacked meaningful correlation with
all other platelet function assays studied, in accordance with
previous studies.28,30 As the assay cannot quantify the inhibition
of platelet aggregation by clopidogrel from baseline, it fails to highlight patient populations requiring intensified therapy and, as such,
is inadequate to evaluate the effect of clopidogrel.
Limitations
As this report is a substudy, some limitations are inherent to the
study design. Mainly, the sample size was not determined beforehand, and the analysis was conducted post hoc of the parent
randomized, double-blind, placebo-controlled trial.7 However,
power calculations demonstrated sufficient power to detect
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Figure 4 Bland – Altman analysis of agreement to assess how closely two measurements are related to each other, with limits of agreement
depicting the range of variability in agreement between the two measures. Bias is a measure of a systematic error leading to over- or underestimation of a known value (in this case 20 mM ADP-induced LTA) by alternative measurements made with (A) 5 mM ADP-induced WBA.
Solid red line represents bias (13%), dashed blue lines represent 95% confidence intervals (2.9 – 24.0), dotted green lines represent limits of
agreement (297 to 124), and dashed grey line represents 95% confidence intervals of limits of agreement. (B) 20 mM ADP-induced WBA.
Solid red line represents bias (211%), dashed blue lines represent 95% confidence intervals (220.7 to 21.8), dotted green lines represent
limits of agreement (2110 to 87), and dashed grey line represents 95% confidence intervals of limits of agreement. (C) VerifyNoww P2Y12.
Solid line represents bias (6.3%), dashed blue lines represent 95% confidence intervals (21.6 to 14.2), dotted green lines represent limits
of agreement (254 to 67), and dashed grey line represents 95% confidence intervals of limits of agreement. ADP, adenosine diphosphate;
LTA, light transmission aggregometry; WBA, whole-blood aggregometry.
2884
M. Lordkipanidzé et al.
Table 3 Agreement to identify patients with
insufficient platelet inhibition (<50%) among assays as
assessed by the k statistic
VerifyNoww
P2Y12
Platelet
function
test
LTA
ADP
20 mM
WBA
ADP
5 mM
WBA
ADP
20 mM
LTA ADP
5 mM
0.679a
20.117
0.057
0.295a
20.187a
0.101
0.364a
................................................................................
LTA ADP
20 mM
WBA ADP
5 mM
20.047
0.132
Funding
Fondation de l’Hôpital du Sacré-Cœur de Montréal, Fonds de
Recherche en Cardiologie of Hôpital du Sacré-Cœur de Montréal.
Acknowledgements
It is generally accepted that a k statistic of 0 –0.2 translates into slight agreement,
0.2 –0.4 into fair agreement, 0.4– 0.6 into moderate agreement, 0.6–0.8 into
substantial agreement, and 0.8– 1 into almost perfect agreement.
a
Agreement (k statistic) is significant at P , 0.05.
clinically meaningful correlations between assay results. Although
clopidogrel metabolites were not measured to ensure that patients
took clopidogrel, compliance was verified by pill count and personal interview. The cut-off value of 50% to indicate inadequate
clopidogrel-induced platelet inhibition is arbitrary, and no strong
evidence indicates that this cut-off is a predictor of adverse cardiovascular events. However, this cut-off value is recommended by
the current American guidelines as an indicator of insufficient
platelet inhibition by clopidogrel and has therefore been selected
as most relevant herein. It should be noted that this study was
not designed to evaluate the clinical utility of platelet inhibition
measurements. The small sample size precludes us from stating
that either test is better suited to do so. However, the current
study highlights that the available platelet function assays are not
interchangeable and thus the recommendation to use available
platelet function tests interchangeably to screen non-responders
to clopidogrel is premature.
Several other platelet function assays are available to evaluate
clopidogrel-induced platelet inhibition. Some examples include
the flow cytometric measurement of the vasodilator-stimulated
phosphoprotein (VASP) phosphorylation status, the Thromboelastographw, and Plateletworksw. As these techniques were not evaluated in the current study, their comparability with present platelet
function assays cannot be discussed. However, because the VASP
index is highly P2Y12-specific, it may prove more sensitive to the
evaluation of clopidogrel-induced platelet inhibition.4
Conclusion
At present, no platelet function assay can be acclaimed as optimal
for quantifying the inhibition of platelet aggregation by clopidogrel.
Although most studied assays were sensitive to clopidogrelinduced platelet inhibition, the results showed only weak association among platelet function test results, and agreement
between tests to select patients requiring intensified clopidogrel
therapy was accordingly low. Consequently, before implementing
We wish to acknowledge the technical assistance of our laboratory
technician, Edmond Sia, as well as the assistance of our research
nurse, Chantale Mercure, in the recruitment and blood sampling
of the patients.
M.L. is a recipient of the Fonds de recherche en santé du
Québec PhD training award.
Conflict of interest: none declared.
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