Multiple Electrode Aggregometry & Clopidogrel Resistance
Condition: Type 2 Diabetes Mellitus (T2DM); Coronary Artery Disease (CAD); Myocardial Infraction
MeSH terms: Coronary Artery Disease; Myocardial Ischemia; Coronary Disease; Diabetes Mellitus, Type 2
Study type: Observational

Summary: 
      Antiplatelet therapy with aspirin-clopidogrel reduces the risk of cardiovascular episodes
      after percutaneous coronary intervention (PCI) in patients with acute coronary syndromes.
      However, a significant number of patients experience recurrent events while on such therapy.
      The individual response to dual antiplatelet therapy is not uniform, and consistent findings
      across multiple investigations support the association between a lower degree of platelet
      inhibition, high on-treatment platelet reactivity, and the occurrence of atherothrombotic
      events [1, 2]. Particularly in diabetic patients, clopidogrel resistance is more prevalent
      compared with non-diabetics [3,4], which seems to contribute to the increased
      atherothrombotic risk in these patients compared with those without diabetes mellitus (DM)
      [5]. A number of platelet function instruments have now become available that are simple to
      use and can be utilized as point-of-care (POC) instruments in order to monitor antiplatelet
      therapy and potentially assess the risk of a recurrent event [6].
    

Detailed description: 
      The aim of this study is to evaluate the ability of a new POC device, the multiple electrode
      aggregometry (Multiplate, Dynabyte, Munich, Germany) to detect clopidogrel resistance and
      predict the clinical outcome in patients with type 2 diabetes mellitus (T2DM) and coronary
      artery disease (CAD) in comparison with the light transmittance aggregometry (LTA), which is
      considered the gold standard of platelet function testing [7]. 280 patients with T2DM between
      20 and 75 years of age treated with oral and/or parenteral hypoglycaemic therapy for at least
      1 month and angiographically established coronary artery disease on the grounds of a previous
      ST elevation, non-ST elevation acute coronary syndrome or unstable angina treated with
      clopidogrel 75 mg/day in combination with daily per os aspirin therapy for at least 7 days
      after documentation of CAD, will be recruited. Patients' enrollment will take place in the
      Second University Department of Cardiology of the "Attiko" University Hospital in Athens,
      Greece. Baseline assessment will include recording of demographic data, medical history,
      cardiovascular risk factors, hematological parameters, number of diseased vessels, number of
      stented vessels, previous myocardial infarction (MI), previous revascularization, ejection
      fraction and concomitant medications. Patients with renal (creatinine levels > 2.5 mg/dl) or
      hepatic (bilirubin level > 2 mg/dL) insufficiency, malignant disease, use of drugs known to
      affect platelet function, history of bleeding diathesis, platelet count < 100x109/L and a
      hematocrit < 28% will be excluded. The protocol will be approved by the institutional review
      board of "Attiko" University Hospital and all patients will give written informed consent
      before undergoing any study procedure or receiving any study treatment.

      Platelet function measurements will be performed on study enrollment. Patients will be
      identified either as poor or good responders to clopidogrel. The primary end-point will be
      defined as a composite of death from cardiovascular causes, nonfatal myocardial infarction,
      stent thrombosis, ischemic stroke, urgent rehospitalization for acute coronary syndrome
      and/or revascularisation during 1-year follow-up period. Episodes of major bleeding according
      to PLATO criteria will be recorded during follow-up [8].

      The ability of the two methods to detect clopidogrel resistance will be evaluated and the
      potential correlation between response to antiplatelet treatment and clinical outcome will be
      assessed. In parallel, patients with similar characteristics, but treated with prasugrel or
      ticagrelor, will be used as control group of the same sample size.

      Platelet Function Analysis Platelet function analysis will be performed in the Laboratory of
      Haematology and Blood Bank Unit of "Attiko" University Hospital. Blood samples will be
      collected from an antecubital vein 2 to 4 h after antiplatelet therapy intake. The first 2 to
      4 ml of blood will be discarded to avoid spontaneous platelet activation and platelet
      function tests will be performed within two hours of sampling.

      Light transmittance aggregometry (LTA) The whole blood specimen will be collected in 3.8%
      trisodium citrate and centrifuged at 200 g for 10 minutes to obtain platelet-rich plasma
      (PRP). The remaining specimen will be re-centrifuged at 2000 g for 15 minutes to obtain
      platelet-poor plasma (PPP). The platelet count will be adjusted to between 200,000/μl and
      300,000/μl with PPP. Aggregation will be performed using a Biodata-PAP-4 aggregometer
      (Bio/Data Corporation, PA, USA). The 100% line will be set using PPP and a 0% baseline
      established with PRP before addition of the agonist. The agonist used will be ADP 2.0×10-5 M
      (Bio/Data Corporation, PA, USA). Test procedure will be performed as previously described
      [9]. In brief, 0.45 mL PRP is transferred into a cuvette incubated at 37 °C for 3 minutes.
      Then 0.05 mL of the agonist is added into the PRP and the aggregation pattern is allowed to
      generate for 6 minutes. Platelet aggregation was determined as the maximal percent change in
      light transmittance from baseline using PPP as a reference. Cut-off value of > 59%
      aggregation following 20 microM ADP stimulation will be defined as that indicating of high
      on-treatment platelet reactivity [10]. Other tested parameters will be late aggregation and
      the degree of disaggregation.

      Multiple electrode aggregometry (MEA) Platelet aggregation in whole blood will be assessed by
      MEA using an impedance aggregometer (Multiplate, Dynabyte, Munich, Germany). Samples will be
      collected into 3.2% citrate tubes and analyzed within the period of half to two hours after
      blood collection according to manufacturer's instructions. Platelet aggregation will be
      induced by ADP in final concentration 6.5 μM. Each disposable test cell contains two pairs of
      electrodes, thus enabling two simultaneous measurements. Aggregation will reported as area
      under the curve (AUC), an integrated measure of aggregation velocity and maximal aggregation.
      Cut-off value for HRPR by MEA (> 468 arbitrary aggregation units/min in response to ADP by
      Multiplate analyzer) was defined according to the recently published consensus statement on
      the definition of high on-treatment platelet reactivity to adenosine diphosphate [11].

      Statistical Methods

        1. Primary endpoint:

           In this prospective cohort study, the primary end point will be defined as a composite
           of death from cardiovascular causes, nonfatal myocardial infarction, stent thrombosis,
           ischemic stroke, urgent rehospitalisation for acute coronary syndrome and/or
           revascularisation during the 1-year follow-up period. Episodes of major bleeding
           according to PLATO criteria will be recorded during follow-up.

        2. Descriptive statistics:

           They will be presented as means ± SD, medians and interquartile ranges (IQR), or
           percentages when appropriate.

        3. Comparison of baseline data (demographic data, medical history, cardiovascular risk
           factors, hematological parameters, number of diseased vessels, previous myocardial
           infarction, previous revascularisation, ejection fraction and concomitant medications)
           between responders and non-responders to clopidogrel:

           First, we will use the Shapiro-Wilk test to check for the normality of data
           distributions. Continuous variables will be compared by means of the two-sample
           Student's t test (when the distribution is found normal) or by means of the
           non-parametric two-sample Wilcoxon rank-sum (Mann-Whitney) test (when the distribution
           is found non-normal). Categorical variables will be compared using the Pearson's
           chi-squared test.

        4. Agreement of the two methods in detecting resistance to clopidogrel:

           Agreement between "Multiplate" and "ADP-induced LTA" will be determined by kappa
           statistics and the respective p-values. Kappa values of < 0.20 are considered to
           indicate poor agreement, 0.21 to 0.40 indicate fair agreement, 0.41 to 0.60 indicate
           moderate agreement, 0.61 to 0.80 indicate good agreement, and >0.81 indicate very good
           agreement.

        5. Correlation of laboratory resistance with clinical outcome:

           Logistic regression analysis will be used in order to evaluate whether the methods of
           detecting resistance to clopidogrel ("Multiplate" and "ADP-induced LTA") are associated
           with the clinical outcome (end-point). To evaluate whether these associations are
           independent, certain covariates that are well-known risk factors (ie., cardiovascular
           risk factors, number of diseased vessels, previous myocardial infarction, previous
           revascularisation, etc) will be included in the multivariable logistic regression
           models. The crude and the adjusted odds ratios (ORs) and the corresponding 95%
           confidence intervals (CIs) will be presented.

        6. Level of significance:

           For hypothesis testing, a probability level of < 0.05 will be considered as
           statistically significant. All statistical tests will be two-sided.

        7. Statistical software:

           Stata software will be used for all statistical analyses (Stata Corp., College Station,
           TX, USA).

        8. Sample size calculation:

      For the study sample size calculation, we hypothesize that clopidogrel resistance CR(+) is
      more prevalent among T2DM patients compared with non-DM patients [12, 13]; at the same time,
      the DM group of patients with clopidogrel resistance suffer an increased likelihood of
      adverse cardiovascular events [14]. It has been shown that 13% of the general population of
      T2DM patients suffer a major adverse cardiovascular event (MACE) [including death secondary
      to cardiovascular cause, ST-segment elevation myocardial infarction (STEMI), non-ST-segment
      elevation myocardial infarction (NSTEMI) and unstable angina (UA) and stroke]; this number
      increases to 38% among T2DM patients with clopidogrel resistance who remain on clopidogrel
      treatment [15]. The sample size needed to detect this difference in adverse event rates -
      between CR(+) and CR(-) diabetic patients - with a two-sided a risk of 5% and a b risk of 20%
      (power of 80%) will have to be 101 patients [40 in CR(+) group and 61 in CR(-) group]. If we
      assume an attrition rate of 10%, the sample size should be further increased to 111 patients
      [44 in CR(+) group and 67 in CR(-) group]. Finally, taking under consideration that
      clopidogrel resistance among T2DM patients can be estimated to reach approximately 40%
      [16-18], the initial sample of all diabetic patients should be 2.5 times larger, or 278
      patients (111 clopidogrel-resistant and 167 clopidogrel-sensitive). The statistical software
      G*Power 3.1.3 (Kiel, Germany) was used for sample size calculation.
    

Observational model: Other
Time perspective: Prospective
Sampling method: Non-Probability Sample
Study population: 
        type 2 diabetes mellitus (T2DM) and coronary artery disease (CAD)patients who will be
        detected for clopidogrel resistance
      
Keywords: light transmittance aggregometry (LTA),multiple electrode aggregometry, clopidogrel-resistance