MultiplateT Analyzer für die Thrombozytenfunktionstestung. Literaturübersicht

Transcription

1 MultiplateT Analyzer für die Thrombozytenfunktionstestung Literaturübersicht

2 MultiplateT Analyzer Stärkung des Hämostaseportfolios Die Gerinnungsdiagnostik gehört fur Roche Diagnostics zu den wichtigsten Investitionsbereichen der nächsten Jahre. Unser Anspruch ist, unsere Kompetenzen und Konzepte der Klinischen Chemie, Immunologie und POC-Diagnostik hinsichtlich Produktqualität, Prozesseffizienz, Flexibilität und Bedienerfreundlichkeit auch in unserem neuen Gerinnungsportfolio zu etablieren. Neben den Systemen der cobas t Plattform für die Diagnostik der plasmatischen Gerinnung, bietet der Multiplate Analyzer die Möglichkeit einer schnellen Bestimmung der Thrombozytenfunktion. Die Kenntnis der Thrombozytenfunktion von Patienten kann für den behandelnden Arzt ein wichtiger Baustein sein, klinische Entscheidungen zu treffen und umzusetzen, insbesondere in der Kardiologie, Chirurgie und Intensivmedizin. Mit Übernahme der Verum Diagnostica GmbH stehtroche Diagnostics mit dem Multiplate Analyzer eine innovative und überzeugende Lösung für die Thrombozytenfunktionstestung zur Verfügung. Die hohe medizinische Wertigkeit des Systems resultiert aus seiner überzeugenden Prädiktivität für Thrombosen und Blutungen. Das ermöglicht eine patientenindividuelle aggregationshemmende Medikation und die Stratifizierung eines perioperativen Blutungsrisikos. Insbesondere durch den hohen Standardisierungsgrad des Messverfahrens kann der Multiplate Analyzer im Bereich der primären Hämostase neue Maßstäbe bei der Patientenversorgung setzen. Das System ist daher für Roche eine ideale Ergänzung, die Vision, Innovationen mit bewahrtem medizinischem und diagnostischem Know-how zu kombinieren, in das neue Gerinnungsportfolio zu übertragen.

3 Überzeugende Prädiktivität MultiplateT Analyzer Medizinischer Fokus Mehr als 200 Publikationen mit dem Multiplate Analyzer in Medline Konsensuspapier der Arbeitsgruppe für high on treatment platelet reactivity mit der Multiplate- Methode Richtlinien mit Empfehlungen zur Thrombozyten- Funktionstestung bei koronaren Bypass- Operationen und koronaren Interventionen für Patienten unter Clopidogrel-Therapie MultiplateT Analyzer Hohe Prädiktivität für eine individuell angepasste, aggregationshemmende Therapie Ca. 20 % der Patienten unter Clopidogrel- Therapie zeigen keine ausreichende Medikamentenwirkung (low-responder) Low-responder, die mittels der Multiplate- Testung detektiert wurden, haben ein 5 10-fach höheres Risiko für ischämische Ereignisse Eine Umstellung auf neuere und potentere Medikamente hat Vor- und Nachteile; sie ist mit fach höheren Kosten verbunden Patienten, die überdurchschnittlich gut auf Clopidogrel ansprechen (high-responder), weisen ein 2,6-fach höheres Risiko für schwere Blutungen auf Viele Arbeitsgruppen berichten über die erfolgreiche Umsetzung von Multiplate- Ergebnissen in eine individuell angepasste, aggregationshemmende Therapie Zuverlässige Ergebnisse Einfaches, standardisiertes und schnelles Testverfahren aus Vollblut Umfangreiches, CE-markiertes Parametermenü 5 Kanäle für hohen Durchsatz Hohe Sensitivität Parallelbestimmung (2 Sensorenpaare) zur Qualitätskontrolle Hohe Prädiktivität zur Stratifizierung des Blutungsrisikos Patienten, bei denen mittels Multiplate- Analyse eine Thrombozytenfunktionsstörung festgestellt wurde, haben ein erhöhtes Risiko für intra- und postoperative Blutungskomplikationen und/oder Transfusionsbedarf Der Einsatz des Multiplate-Analyzers kann das Management von Blutungskomplikationen im Rahmen chirurgischer Eingriffe verbessern

4 Prediction of early coronary stent thrombosis based on Multiplate platelet reactivity High residual platelet reactivity in patients on clopidogrel therapy undergoing percutaneous coronary intervention (PCI) is associated with an increased risk of ischemic events. 1 Sibbing, Braun, Morath et al. (2009) investigated in a prospective trial if platelet reactivity to clopidogrel assessed with multiple electrode platelet aggregometry (MEA, Multiplate ) correlates with the risk of early drug-eluting stent thrombosis. 2 With 1,608 CAD patients enrolled who were scheduled for drug eluting stent PCI, this study is among the largest ones conducted on this topic. The primary end point was definite ST at 30 days. Before PCI, all patients received 600 mg clopidogrel. Blood was obtained directly before PCI and tested with the Multiplate ADPtest more than 2 hours after clopidogrel loading. The upper 20% of patients according to MEA measurements (n = 323) were defined as clopidogrel low responders. Low responders had an approximately 10 times higher risk of definite ST within 30 days compared to normal responders, (2.2% vs. 0.2%; odds ratio [OR]: 9.4; 95% confidence interval [CI]: 3.1 to 28.4; p < ). In Sibbing et al s (2009) study hirudin anticoagulated blood was used for analysis. The optimal cut-off value to predict the occurrence of 30-day stent thrombosis according to ROC analysis was 47 U. This study has been featured in an article in 3 where Sibbing stated: We can t say whether the MEA assay is better than other assays or not, but we are very happy with it, and we are convinced by the results we have seen in this study. It also works out at a reasonable cost for each test. When we started this study, we were not routinely measuring platelet responsiveness in the cath lab, but now we are measuring it with this device. Definite stent thrombosis 30 days post DES-PCI Odds ratio % CI, P < Risk of ST [%] Responders to clopidogrel Low responders to clopidogrel

5 Prediction of coronary stent thrombosis based on Multiplate platelet reactivity: 6 month results High residual platelet reactivity in patients on clopidogrel therapy undergoing percutaneous coronary intervention (PCI) is an independent predictor of early definite stent thrombosis (ST) within 30 days. This association was established by Sibbing, Morath, Braun et al. (2009) using the Multiplate ADPtest to identify low responders resulting in an odds ratio of 9.4; 95% [CI]: 3.1 to 28.4; p < ) for definite stent thrombosis in low responders versus normal responders. 2 Sibbing et al. (2010) also reported the six-month follow-up results of these study patients. 4 The upper 20% of patients according to Multiplate measurements (n = 323) were defined as clopidogrel low responders using a cut-off value of 42 U. Definite stent thrombosis 6 months post PCI Risk of ST [%] Odds ratio % CI, P < Low responders had an approximately 7 times higher risk of definite stent thrombosis within 6 months compared to normal responders, (2.5% vs. 0.4%; OR: 6.5; 95% [CI]: ; p < 0.001) When considering definite and probable stent thrombosis combined, low responders exhibited an approximate 6-fold increased risk of ST upon a 6-month follow-up (4.1% vs. 0.7%; OR: 5.8; 95% [CI]: ; p < ). 0.0 Responders to clopidogrel Low responders to clopidogrel Of note is the observation that the majority of low responders to clopidogrel identified by MEA suffer ischemic events early in the course following PCI, usually within 3 months. Combined definite and probable ST 6 months post PCI Odds ratio % CI, P < Risk of ST [%] Responders to clopidogrel Low responders to clopidogrel

6 Further support for variable anti-platelet drug efficacy and the clinical relevance of Multiplate testing Müller-Schunk, Linn, Peters, Spannagl et al. conducted the first outcome related clinical trial with the Multiplate analyzer, published in 2008 in AJNR neurologic patients scheduled for supra-aortic stent placement were enrolled. The aim was to determine platelet inhibition after clopidogrel treatment using the Multiplate analyzer and correlate the results with the clinical outcome. Adverse ischemic events were registered in 10% of patients and a statistically significant correlation of clopidogrel non-response and adverse events has been reported. In a prospective study from Siller-Matula, Christ, Lang et al. (2009) CAD patients scheduled for PCI were enrolled and assessed for clopidogrel response in Multiplate and VASP assays. The rate of stent thrombosis was recorded during a 6-month follow-up. Positive predictive value for the prediction of stent thrombosis and outcome related sensitivity and specificity was determined to be superior in Multiplate testing. The authors conclude that the assessment of the antiplatelet effect of clopidogrel by platelet aggregometry [with Multiplate] was more predictive for stent thrombosis than the VASP assay. Adverse events in supra-aortic stent placement 5 Total # of adverse events Responders to clopidogrel 5.0 Non-responders to clopidogrel Sensitivity and specificity for definite ST 6 Sensitivity Specificity 100% 100% 100% 86% 80% 60% 40% 37% 20% 0% Multiplate cut-off 54 U VASP cut-off 42% PRI

7 Tailoring antiplatelet therapy with Multiplate and the potential to reduce the incidence of adverse ischemic events after PCI Various medical centers use the Multiplate analyzer to assess and monitor platelet function in patients receiving antiplatelet drug therapy, like Aspirin or clopidogrel, to minimize the risk of on-treatment complications. High residual platelet reactivity (HPR) in patients on clopidogrel therapy undergoing percutaneous coronary intervention (PCI) is associated with increased risk of ischemic events (e.g. stent thrombosis). Prasugrel and ticagrelor are novel potent ADP-receptor inhibiting drugs that act more consistently and significantly reduce the risk of ischemic events compared to clopidogrel therapy. However, these drugs are also associated with a risk of increased major bleeding and other side effects that negatively impact compliance and patient outcomes. Furthermore, the treatment of patients with prasugrel or ticagrelor is up to x more expensive compared to clopidogrel. Twenty-four hours before PCI, all patients received either 300 mg (patients with angina pectoris) or 600 mg (patients with acute coronary syndrome) loading dose of clopidogrel. In the tailored group, platelet function was tested with the Multiplate ADPtest 24 hours after clopidogrel loading and at indicated time points (MEA). Patients with HPR (>46 U) received an additional 600 mg loading daily dose and 150 mg daily dose of clopidogrel for 30 days. Thereafter the 75 mg daily dose was resumed for the remainder of the study. Patients in the non-tailored group received a standard daily dose of 75 mg clopidogrel for 180 days after PCI. During 6 months of follow-up, no ischemic events occurred in the Multiplate tailored patient group. However, 5.3% of patients undergoing uniform standard dose clopidogrel therapy suffered severe cardiac and cerebovascular complications such as cardiac death or myocardial infarction (Fig. 1). Several studies support that routine tailoring of antiplatelet therapy using Multiplate testing has the potential to significantly reduce the incidence of major adverse ischemic complications. A study of Hazarbasanov, Velchev, Finkov et al. (2012) 7 aimed to analyze the clinical impact of a platelet function-guided antiplatelet therapy compared to standard dose clopidogrel treatment in patients after PCI. This prospective, randomized, open-label, interventional study enrolled 192 patients. The primary end point was the incidence of major adverse cardiac and cerebovascular events such as cardiac death, myocardial infarction, stent thrombosis or ischemic stroke at six months (180 days).

8 Hazarbasanov et al. s (2012) study demonstrated that a treatment strategy incorporating the Multiplate analyzer to assess a tailored dose of clopidogrel may reduce the incidence of ischemic events in patients after PCI. Furthermore, a study by Christ, Francesconi et al. (2011) 8 of 507 patients undergoing PCI has shown that routine tailoring of antiplatelet therapy using Multiplate testing helps prevent early definite stent thrombosis in compliant patients. In this study patients undergoing PCI received a personalized antiplatelet treatment of either clopidogrel or prasugrel depending on the clinical status and platelet reactivity test result. STEMI patients received first line prasugrel, while all other patients received clopidogrel and were tested for their platelet response. Patients with a low response to the anti-platelet treatment (ADPtest > 50 U) were either reloaded with clopidogrel (patients with prior cerebrovascular events) or with prasugrel (all other patients). Christ et al. (2011) reported a very low incidence of stent thrombosis in this patient cohort treated with a personalized anti-platelet therapy. Another study by Sibbing, Mayer, Bernlocher et al. (2012) 9 aimed to investigate whether Multiplate tailored antiplatelet therapy with prasugrel in patients displaying HPR on clopidogrel treatment reduces the incidence of stent thrombosis after PCI. Before PCI, all patients received a loading dose of 600 mg clopidogrel. In the study design one group of patients received a standard clopidogrel treatment whereas another cohort of patients were switched from clopidogrel to prasugrel based on Multiplate testing. The primary outcome was the incidence of stent thrombosis (ST) within 30 days after PCI. Survival analysis 180 days post PCI 7 MACCE free survival [%] Fig % Log-rank P = % Multiplate tailored Standard clopidogrel treatment N = 97 treatment N = 97 Stent thrombosis incidence 30 days post PCI Odds ratio (adj) % Cl, P = Stent thrombosis was 4-fold higher in the patient group without antiplatelet therapy adjustment compared to the patient group with Multiplate-tailored treatment (Fig. 2). Routine platelet function testing for guidance of antiplatelet treatment markedly reduced risk of stent thrombosis in patients with HPR while on clopidogrel therapy. Incidence of ST [%] Fig Treatment adjustment N = 143 Standard clopidogrel treatment N = 428

9 Multiplate analysis: Prediction of bleeding and transfusion in coronary artery surgery Reece et al. (2011) investigated the Multiplate analyzer for determining platelet aggregation in 44 patients during coronary surgery. 10 The results demonstrated that platelet aggregation was reduced during and after cardiopulmonary bypass surgery (CPB) as compared to baseline values with evidence of slight recovery in platelet function post chest closure. Patients receiving transfusion products displayed lower levels of ADP and TRAP induced platelet aggregation than patients not receiving transfusions. It was concluded that the Multiplate analyzer can detect platelet dysfunction in the setting of CPB. Platelet aggregation and transfusion in patients undergoing cardiac surgery 10 Transfusion 100 No transfusion P = Rahe-Meyer et al. (2008) showed in a study of 100 surgical patients 11 that in the case of possible Aspirin ingestion, the Multiplate system is a better predictor of platelet related coagulopathy or transfusion than patient self-reporting. Ranucci et al. (2011) aimed to determine if a preoperative test of platelet function could determine postoperative risk of excessive bleeding and transfusion requirements. 12 They tested 87 patients who discontinued thienopyridine treatment no more than 7 days prior to surgery. Platelet aggregation [U] 50 P = Multivariable linear regression analysis confirmed the Multiplate ADPtest (p = 0.007) as independently associated with postoperative bleeding. The accuracy of prediction was good with an area under the curve of 0.71, p= Using the Youden index the best cut-off value for the ADP test was determined to be 31 U with an associated sensitivity of 72%, specificity 66%, negative predictive value 92%, and positive predictive value 29%. 0 ADP TRAP Platelet dysfunction and bleeding in cardiac surgery 12 Patients with excessive bleeding The incidence of excessive bleeding was significantly higher in patients with ADPtest results below the cutpoint (31 U) in comparison to patients with ADPtest results higher than the cutpoint (29% vs 8%) Cut-off value 31 U Sens = 72% Spec = 66% NPV =92% PPV = 29% 10.0 Number of patients [n] ADP - induced platelet aggregation [U]

10 Bleeding risk and platelet function testing with Multiplate analysis in PCI High residual platelet reactivity in patients on clopidogrel therapy has been associated with increased risk of thrombotic events in PCI; however, it is also recognized that enhanced response to clopidogrel carries an associated risk of increased major bleeding. Both conditions significantly increase mortality. TIMI Major bleeds Odds ratio % CI, P = The association of enhanced platelet response to clopidogrel treatment in PCI as evidenced by low levels of platelet reactivity measured with the Multiplate analyzer and its association with bleeding was first elucidated by Sibbing, Schulz, Braun, Morath et al. (2010). 13 Sibbing s group evaluated 2,533 CAD patients scheduled for drug-eluting stent PCI. All patients received a clopidogrel loading dose of 600mg. Blood was obtained directly before PCI, more than 2 hours after clopidogrel loading and tested with the Multiplate ADPtest. ROC analysis identified the optimal cut-off of 19 U to identify enhanced responders. The incidence of major bleeding was significantly higher in enhanced clopidogrel responders (n = 975) as compared with the remaining patients (n = 1,558) [21 (2.2%) vs. 13 (0.8%); OR 2.6, 95% CI ; P = 0.005]. Risk of in-hospital major bleeding [%] Remaining patients Enhanced responders

11 Multiplate analyzer in heparin-induced thrombocytopenia (HIT) and von Willebrand disease (VWD) The Multiplate analyzer has been widely used to evaluate patient responses to platelet aggregation inhibiting drugs. Moreover, its role as a reliable diagnostic tool for platelet-related disorders is becoming more obvious. Heparin-induced thrombocytopenia (HIT) is a severe complication of anticoagulant therapy with heparin. Some patients develop abnormal antibodies of the IgG class against heparin when it is bound to a platelet protein called platelet factor 4 (PF4). This antigen-antibody complex then binds to the FcγIIa receptor on the platelet surface and leads to platelet activation and consequent formation of blood clots. Additionally, the platelet count may fall in an immune-mediated thrombocytopenia. The diagnosis of HIT is still very challenging as the functional assays are complex, long-lasting and require qualified medical staff. The 14C-serotonin release assay (SRA) is considered to be the gold standard for HIT diagnosis but is not suitable for routine use because of the difficulties mentioned above. Morel-Kopp et al. (2012) 14 conducted a large study to validate Multiplate analysis as a suitable diagnostic tool for functional assessment of HIT. Morel-Kopp et al. s (2012) study 14 demonstrated, that the Multiplate analyzer is not only an easy-to-use and rapid test system, but its high sensitivity and specificity is comparable to those of the accepted gold standard serotonin release assay. Thus, Multiplate analysis should be considered as a confirmatory assay for platelet activating HIT antibodies. Another study by Valarche et al. (2011) 15 compared the MEA analyzer with the classic diagnostic method LTA (light transmission aggregometry) to evaluate the sensitivity of MEA for von Willebrand disease (VWD). It was shown that MEA was as sensitive as LTA in detecting VWD, with 76% correlation between the two methods. Thus, MEA is a reliable tool in association with other standard test systems, for the evaluation of VWD. Correlation between the functional SRA and MEA for HIT diagnosis 14 Sensitivity Specificity PPV 100 They tested 181 patient samples positive for Heparin-PF4 antibodies via MEA and SRA tests to assess the diagnosis of true HIT. Using differrent criteria for SRA-positivity (serotonin release >20% with 0.1 IU/ml heparin and <20% release with 100 IU/ml heparin/threshold of >50% serotonin release), Multiplate analysis showed a high sensitivity and specificity for HIT. Correlation [%] Strict SRA positivity Modified SRA positivity

12 Evaluation of paediatric reference values for normal platelet function based on Multiplate impedance aggregometry Hereditary platelet disorders are rare but can lead to bleeding syndromes appearing during infancy or early childhood. Patients with platelet dysfunction disorders possess a higher risk of bleeding complications during complex surgeries or percutaneous injuries. Evaluation of reference diagnostic parameters is necessary for the comparison between normal and disordered platelet function. The study of Halimeh, de Angelis, Sander et al. (2010) 16 evaluated paediatric reference values for the Multiplate system in infants and children from different age groups ( months, years, 5-9 years and 10- to <18 years). Platelet aggregation was evaluated using the ADPtest (ADP, 6.5 um), COLtest (collagen 3.2 ug), TRAPtest (thrombin receptoractivating peptide 6; TRAP-6, 32 um), ASPItest (arachidonic acid 0.5 mm) and RISTOtest (ristocetin 0.77 mg/ml). Infants aged months displayed significantly lower aggregation values in TRAPtest and ASPItest compared with the older age groups. Therefore adult reference values can provide a valid guidance for the evaluation of platelet function of children at age > 1 year. Aggegometry values in healthy infants, children and adoloscents months years 5-9 years 10 - < 18 years Platelet aggregation [AUC; U] ** ** 20 0 ADPtest COLtest TRAPtest ASPItest RISTOtest ** P < 0.001

13 Multiple electrode aggregometry (MEA) is a reliable test device for diagnostic assessment of Glanzmann thrombasthenia Glanzmann thrombasthenia is a rare hereditary disease which can lead to severe mucocutaneous bleeds. Glanzmann thrombasthenia caused by a genetic defect of the GPIIb/IIIa receptor. The platelet GPIIb/IIIa receptor binds to fibrinogen and is the most important principle receptor for platelet aggregation. Quantitative and qualitative defects in GPIIb/IIIa receptors lead to deficient platelet aggregation followed by a tendency for increased bleeding. The detection of Glanzmann thrombasthenia using light transmission aggregometry is time-consuming and weakly standardized. The study of Awidi, Maqablah, Dweik et al. (2009) 17 investigated the suitability of Multiplate analysis using whole blood for the diagnosis of patients with Glanzmann thrombasthenia. Blood samples were taken from patients with Glanzmann thrombasthenia who were not taking platelet function inhibiting medications for at least 7 days. Platelet aggregation in platelet-rich plasma was monitored by LTA (a), whereas platelet aggregation in whole blood was studied by Multiplate (b) after stimulation of platelets with ADP (ADPtest), collagen (COLtest) or ristocetin (RISTOtest). In both LTA and Multiplate a very low level of aggregation was witnessed in response to all examined agonists. Thus, Multiplate analysis provides comparable results to LTA. Awidi et al. s (2009) study demonstrated that MEA performed via the Multiplate analyzer is a fast and standardized method to assess severe platelet function disorders like Glanzmann thrombasthenia in whole blood. Evaluation of platelet aggregation via LTA after addition of specific activators (a) Platelet aggregation [%] Ristocetin ADP Collagen 2 X 10 M 0.19 mg/ml 1.5 mg/ml Evaluation of platelet aggregation via Multiplate after addition of specific activators (b) Platelet aggregation [AUC; U]] ADPtest 6.4 µ M COLtest 3.2 µ g/ml RISTOtest 0.77 mg/ml

14 Cross validation of Multiple Electrode Aggregometry A prospective trial in healthy volunteers At present dual drug therapy with Aspirin and clopidogrel is the standard treatment of patients undergoing percutaneous coronary intervention (PCI). However, there is strong variability in the individual response to these antiplatelet drugs, that carries a high risk of bleeding by high-responders and an increased risk of stent thrombosis by low/non-responders. There are several standard test systems which allow the determination of platelet function in patients undergoing clopidogrel and Aspirin therapy. Multiplate analysis is a fast and easy-to-use platelet function test method that analyzes whole blood. Multiplate PFA-100 VASP assay CPA SC CPA AS Clopidogrel 11.3 n.d Aspirin n.d Tab. 1 according to Siller-Matula et al. (2009) the effect size for clopidogrel and Aspirin measured with different tests as median values. SC surface coverage; AS average size of platelet aggregation. Siller-Matula, Gouya, Wolzt and Jilma (2009) 18 investigated the sensitivity of Multiplate analysis to clopidogrel and Aspirin effects and compared it with other platelet function tests such as the Cone and Platelet Analyzer (CPA), PFA-100 and VASP phosphorylation assay. In this study design each of 9 healthy volunteers received a loading dose of 300 mg Aspirin and 300 mg clopidogrel on the first day, and a standard dose of 100 mg Aspirin and 75 mg clopidogrel on each of the three consecutive days. Blood samples were analyzed at baseline (pre Aspirin and clopidogrel intake), and 2, 4, 6 and 72 hours after drug ingestion. Clopidogrel effect was investigated by means of ADP-induced platelet aggregation, whereas Aspirin effect was measured by means of arachidonic acid-induced platelet aggregation. The effect size for the clopidogrel and Aspirin treatment was calculated as the average of the amplitude between baseline and maximal platelet inhibition at nadir for each patient. The best effect resolution for both Aspirin and clopidogrel according to the greatest signal magnitude was found for Multiplate analysis (Tab. 1).

15 Evaluation of Aspirin-induced platelet dysfunction based on Multiplate assessed platelet reactivity Aspirin is a widely used inhibitor of platelet aggregation. Due to its antiplatelet effects, Aspirin intake is associated with increased bleeding risk during and after complex surgery. Jambor, Weber, Gerhardt et al. (2009) 19 showed that multiple electrode aggregometry (MEA) is a reliable test to assess Aspirin-induced platelet dysfunction. In Jambor et al. s (2009) study each of 24 healthy adult volunteers received a single dose of 500 mg of acetylsalicylic acid (Aspirin). Blood samples were collected at several time points after Aspirin intake and analyzed with the Multiplate system. Time point 0 marks the baseline of platelet aggregation in healthy volunteers without Aspirin treatment. Aspirin caused significant platelet inhibition four hours after intake and the inhibitory effect lasted 24 hours by all study participants. Platelet aggregation returned to the baseline range ( U) in 33% of volunteers by 80 hours (day 3) and in 88% of probands by 124 hours (day 5). This study showed that Multiplate analysis is a reliable method for the assessment of Aspirin effect. Platelet aggregation was determined following stimulation by arachidonic acid (ASPItest). ASPItest 0.5 mm Platelet aggregation [AUC; U]] platelet aggragation (AUC; U) Time after Aspirin intake [h] time / h after

16 CLSI guideline adopting Multiplate analysis Since the introduction of the Multiplate analyzer to the market in 2005 the system has seen a great momentum in terms of adoption and medical consensus on the value of Multiplate analysis. Since then over 200 medline listed publications with the use of Multiplate analysis were published, supporting the growing interest and adoption of the Multiplate system in clinical practice. Multiplate analysis is an improvement of traditional whole blood impedance aggregometry, which was introduced in 1979 by Cardinal et al. 20 Multiplate analysis is included in the latest Clinical and Laboratory Standards Institute guideline on platelet function testing (H58-A). 21

17 Cardiology consensus paper supporting best predictivity of Multiplate analysis The addition of ADP receptor antagonists to Aspirin treatment reduces ischemic events in patients with cardiovascular disease. However, recurrent ischemic events during dual antiplatelet therapy, including stent thrombosis, remains a major concern. Platelet function measurements demonstrate a highly variable inhibition of ADP induced platelet function under clopidogrel treatment. High on-treatment platelet reactivity to adenosine diphosphate (ADP) is observed in a significant proportion of clopidogrel-treated individuals. Multiple studies have demonstrated a clear association between high on-treatment platelet reactivity to ADP and the occurrence of adverse events. The document also provides a consensus statement of the definition of high platelet reactivity to ADP on the Multiplate analyzer, defined as an aggregation > 47 U (468 AU*min). This review 1 provides a consensus opinion on the definition of high on-treatment platelet reactivity to ADP based on various methods and proposes how identification of HPR may be used in patient care. Multiplate analysis was included in this consensus document. Table 2 of the document summarized studies linking high ontreatment platelet reactivity to ischemic events. The best prediction of ischemic events was associated with the Multiplate analyser, with an odds ratio of 12.0, while other studies using for example the VerifyNow system showed an odds ratio of only 2-3. Multiplate highest odds ratios in consensus paper. 1 Supporting best predictivity for stent thrombosis Multiplate LTA VerifyNow Platelet-works VASP 14.0 Odds ratio [Risk enhancement] better 0.0 Sibbing et al. (2009) Cuisset et al. (2009) Gurbel et al. (2008) Breet et al. (2010) Marcucci et al. (2009) Breet et al. (2010) Breet et al. (2010) Blindt et al. (2007) Fig.: adapted from Bonello et al. (2010) consensus paper.

18 Guidelines are supporting platelet function testing With the increasing evidence of a poor response to clopidogrel being associated with an increased risk for stent thrombosis and other adverse events, platelet function testing (PFT) has been incorporated into PCI treatment guidelines. PFT is now supported by a 2011 Class IIb recommendation from the American College of Cardiology Foundation/American Heart Association/Society for Cardiovascular Angiography and Interventions Guideline for Percutaneous Coronary Intervention. 22 CLASS IIb 1. Platelet function testing may be considered in patients at high risk for poor clinical outcomes. (Level of Evidence: C) 2. In patients treated with clopidogrel with high platelet reactivity, alternative agents, such as prasugrel or ticagrelor, might be considered. (Level of Evidence: C) 2011 ACCF/AHA/SCAI guideline for PCI Supporting the use of platelet function testing Oral antiplatelet Recommendation Evidence Level agents Aspirin I B P2Y12 Inhibitors I A Clopidogrel I B Prasugrel I B Ticagrelor I B Several anti-platelet agents with level I recommendations Class IIb recommendation 1. Platelet function testing may be considered in patients at high risk for poor clinical outcomes (Level of evidence: C) 2. In patients treated with clopidogrel with high platelet reactivity, alternative agents, such as prasugrel or ticagrelor, might be considered (Level of evidence: C) Relatively careful recommendation for drug selection aided by platelet function testing Tab.: adapted from Levine et al. (2011). 22

19 PFT has also received a Class IIb recommendation by the Society of Thoracic Surgeons and the Society of Cardiovascular Anesthesiologists. 23 Class IIb. Point-of-care testing for platelet ADP responsiveness might be reasonable to identify clopidogrel nonresponders who are candidates for early operative coronary revascularization and who may not require a preoperative waiting period after clopidogrel discontinuation. (Level of evidence C). The guideline supports the idea of testing platelet reactivity in order to eliminate an unnecessary preoperative waiting period in patients demonstrating no effect from clopidogrel. Class I represents conditions or recommendations for which there is general agreement or evidence, or both, that a procedure is useful or effective, while class IIa and IIb recommendations represent conditions in which opinions diverge, with class IIa carrying a weight of evidence or opinion in favor of the usefulness or effectiveness of a procedure, and class IIb carrying a weight of evidence in which the usefulness or efficacy of the procedure is less well established. 23 Typically as new markers gain more evidence over time the recommendations in guidelines become stronger, which is also expected for platelet function testing. Such guidelines divide their advice into class I, class IIa, class IIb, or class III. References 1 Bonello, L. et al. (2010). J Am Coll Cardiol Sep 14;56(12): Sibbing, D., Braun, S., Morath, T. et al. (2009). Platelet reactivity after clopidogrel treatment assessed with point-of-care analysis and early drug-eluting stent thrombosis. J Am Coll Cardiol;53: Sibbing, D., Morath, T., Braun, S. et al. (2010). Clopidogrel response status assessed with Multiplate point-of-care analysis and the incidence and timing of stent thrombosis. Thromb Haemost:103 (1): Müller-Schunk, S., Linn, J., Peters, N., Spannagl, M., Deisenberg, M., Brückmann, H., Mayer, T.E. (2008). Monitoring of clopidogrel-related platelet inhibition: correlation of nonresponse with clinical outcome in supra-aortic stenting. Am J Neuroradiol. Apr;29(4): Siller-Matula, J., Christ, G., Lang, I.M., Delle-Karth, G., Huber, K., Jilma, B. (2009). Multiple Electrode Aggregometry better predicts stent thrombosis than the VASP assay. J Thromb Haemost. Nov 23. [Epub ahead of print]. 7 Hazarbasanov, D., Velchev, V., Finkov, B., Postadjian, A., Kostov, E., Rifai, N., Aradi, D. (2012). Tailoring clopidogrel dose according to multiple electrode aggregometry decreases the rate of ischemic complications after percutaneous coronary intervention. J Thromb Thrombolysis. Jan 15 (Epub ahead of print). 8 Christ, G., Francesconi, M., Dechant, C., Macura, J., Ruzicka, K., Podczeck-Schweighofer, A. (2011). Routine tailoring of antiplatelet therapy after coronary stent implantation has the potential to eradicate early definite stent thrombosis in compliant patients. Eurointervention, Vol 7;Suppl M May Sibbing, D., Mayer, K., Bernlochner, I. et al. (2012). Platelet function testing guided use of prasugrel in patients with high on-clopidogrel treatment platelet reactivity reduces the risk of early stent thrombosis. J. Am. Coll. Cardiol.;59;E Reece, M. et al. (2011). Near-patient platelet function testing in patients undergoing coronary artery surgery: a pilot study. Anaesthesia, 66, pages Rahe-Meyer, N. et al. (2008). An Evaluation of Cyclooxygenase-1 Inhibition Before Coronary Artery Surgery: Aggregometry Versus Patient Self-Reporting. Anesth Analg;107: Ranucci, M. et al. (2011). 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