1. Assessing Platelet Reactivity with Point-of care Systems Christian Valina Herz-Zentrum Bad Krozingen Oral Antiplatelet Therapy After PCI: Optimizing Outcomes in Clinical Practice

2. Assessing Platelet Reactivity with Point-of care Systems Platelet-function tests What kind of POC-tests are available? Differences in test results

3. Assessing Platelet Reactivity with Point-of care Systems Platelet-function tests What kind of POC-tests are available? Differences in test results

4. Platelet-function tests Test principleTests Cessation of bleeding from a standardized wound in vivo Bleeding time AggregometryLight transmission aggregometry (LTA) Multiplate ® WBA VerifyNow ® Platelet counting method (e.g. Ichor-plateletworks ® ) Thrombovision T ® guide Flow cytometryDetection of activation markers ex vivo Ex vivo stimulation and detection of activation markers VASP phosphorylation Shear dependent platelet function within whole blood PFA-100 ® IMPACT ® -CPA Placor PRT-7000 ® Global haemostasis testsThrombelastography (TEG ® ) Thrombelastometry (ROTEM ® ) Haemostasis Analysis System ® Thromboxane measurementsSerum thromboxane B 2 Harrison, Hämostaseologie 2009; 29: 25

5. Platelet function testing: Some Problems No definitive screening test Methodological variability within each technique Labor intensive Costly Time consuming Requirement of fair degree of expertise and experience to perform and interpret Often requirement of additional expensive specialist tests Lack of a standardized definition of suboptimal platelet response

6. Assessing Platelet Reactivity with Point-of care Systems Platelet-function tests What kind of POC-Devices are available? Differences in test results

7. Point-of-care-testing: What should they can? Bed-side testing No sample preparation Ready-to-use Easy to use Rapid read out Short Turn-around-Time (TAT)

8. Point-of-care-testing: Test Principles POC-SystemTest principle VerifyNow ® Multiplate ® WBA Aggregometry PFA-100 ® IMPACT ® -CPA Placor PRT-7000 ® Shear dependent platelet function within whole blood Thrombelastography (TEG ® ) Thrombelastometry (ROTEM ® ) Haemostasis Analysis System ® Global haemostasis tests, monitoring of rate and quality of clot formation Ichor-Plateletworks ® Platelet counting pre- and postactivation HemoStatus ® Platelet procoagulant activity

9. VerifyNow ® Whole blood (citrated) without further sample preparation No need for time-consuming centrifugation steps Turbidimetric based optical detection of platelet aggregation stimulated by a specific agonist Disposable cartridges contain fibrinogen-coated beads and platelet activator (3 cartridges available: aspirin, P2Y 12, GPIIb/IIIa)

10. VerifyNow ® Technology

11. Results Screen VerifyNow P2Y12 Assay PRU result is ‘P2Y12-mediated platelet aggregation’ via adenosine diphosphate (ADP) pathway VerifyNow ® P2Y12 results can then be reported as % platelet inhibition Base result is ‘Maximal platelet aggregation’ via Thrombin Receptor Activating Peptide (TRAP) pathway which is independent of aspirin and clopidogrel Print

12. Correlation of VerifyNow ® with LTA

13. Multiplate ® system (“multiple platelet function analyzer”) Whole blood (hirudinized) Multiple electrodes in the disposable test cell (4 electrodes form 2 independent sensor units), multiple channels of the instrument (5). Detects the effects of the platelet inhibitors Aspirin ®, clopidogrel and GpIIb/IIIa antagonists, and also the newer direct ADP receptor antagonists. No luminescence available, low shear system, does not simulate normal haemostasis. Continuously records platelet aggregation. Increase of impedance by the attachment of Platelets onto the Multiplate ® sensors is transformed to arbitrary aggregation units (AU) and plotted against time. Three parameters: –Area under the aggregation curve (AUC). –Aggregation is the height of the curve. V –Velocity is the maximum slope of the curve.

15. Platelet Function Assay 100 (PFA-100) Analyzer device and disposable cartridge Whole blood Measurment of time to occlusion of aperture in mebrane coated with collagen/ADP or collagen/epinephrin under high shear stress conditions Cannot distinguish thienopyridene effect 160µm - 40mbar Flow direction

16. Thromboelastography (TEG) Haemoscope's TEG® 5000 system Small sample of blood (typically 0.36 ml), is placed into a gently rotating cuvette (imitates venous flow and activate coagulation) => Sensor shaft inserted into the sample => a clot forms between the cup and the sensor. Speed and strength of clot formation is measured. Patterns of changes in strength and elasticity in the clot provide information. Four values that represent clot formation are determined by this test: –the R value (or reaction time): represents the speed of clot formation –the K value: end or R until the clot reaches 20mm –the angle and –the MA (maximum amplitude). clot strength Coagulation Index (CI) (or overall assessment of coagulability).

18. Assessing Platelet Reactivity with Point-of care Systems Platelet-function tests: Advantages of POC-Systems Platelet-function tests: What kind of POC-tests are available? Differences in test results

19. Differences in test results: Are they measuring the same? Low shear systems (e.g. Multiplate) does not simulate normal haemostasis Different stimulation reagents and dosages Is „baseline aggregation“ really „baseline“? „Units“: –% platelet inhibition or P2Y12 reaction units (VerifyNow) –Arbitrary aggregation units (Multiplate) –Closure time (PFA-100) –Maximum aggregation, residual aggregation, … (LTA) –Platelet reacitivity index (VASP-P) Cut-off values

21. Assessing Platelet Reactivity with Point-of care Systems Platelet-function tests: Advantages of POC-Systems Platelet-function tests: What kind of POC-tests are available? Differences in test results Cut-off values

22. VerifyNow: Cut off levels for „low response“ Trenk, 2008; TRIGGER(enrolling)> 208 PRU Godino, 2009> 213 PRU Price et al., 2008≥ 235 PRU ARMYDA-PRO, GRAVITAS (enrolling)≥ 240 PRU Paniccia et al., 2007 ≥ 264 PRU

23. Assessment of On-Clopidogrel Platelet Reactivity by LTA and VerifyNow 17.6 ± 16.2 % n=307 161.6 ± 90.5 n=307 LTA VerifyNow

24. VerifyNow ® PRU n=307 r 2 =0.441 Assessment of On-Clopidogrel Platelet Reactivity Correlation Between LTA and VerifyNow

25. AUC: 0.833 Sensitivity for PRU 208: 0.53 Specificity for PRU 208: 0.88 Assessment of On-Clopidogrel Platelet Reactivity LTA versus VerifyNow by ROC Analysis

26. Cut off levels for „low response“ LTA:> 62% (Angiolillo, max. aggregation, ROC) TEG:> 50% (5mM ADP) >70% (10mM, ADP) VASP platelet reactivity index:>48% …..