Platelet Function analysis using Multiple Electrode Aggregometry (Multiplate )

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1 Platelet Function analysis using Multiple Electrode Aggregometry (Multiplate )

2 Multiplate platelet function analysis in whole blood based on impedance aggregometry

3 Multiplate instrument 5 channels for parallel tests easy to use Windows XP based software automatic analysis and documentation duplicate sensor for internal quality control electronic pipetting

4 applications Multiplate is already one of the most widely used platelet function tests

5 Performing the test put the test cell into the measuring position attach the sensor cable pipette 300 µl of saline µl of blood* allow 3 minutes for warming and equilibration add the activator after 6 minutes: print the results discard the test cell * usually hirudin or heparin blood

6 Multiplate reagents comprehensive line of dedicated reagents

7 Multiplate tests 1/2 activation inhibition 1 release of arachidonic acid COLtest Collagen TXA 2 ASPItest Arachidonic Acid Aspirin NSAID TXA 2 ArA 1 COX TRAPtest TRAP ADPtest ADP resting platelet Clopidogrel Prasugrel Cangrelor PGE1 ADPtest HS (ADP + PGE1) platelet activation GpIIb /IIIa receptor exposure degranulation GpIIb/IIIa antagonists: Reopro (abciximab) Aggrastat (Tirofiban) Integrillin (Eptifibatid) activated platelet

8 Multiplate tests 2/2 test activation sensitivity not sensitive for ASPItest arachidonic acid: is converted to TXA2 by platelet-own cyclooxygenase aspirin, IIb/IIIa antagonists clopidogrel, vwf ADPtest ADP: binds onto platelet ADP receptors clopidogrel, IIb/IIIa antagonists aspirin, vwf ADPtest HS ADP + prostaglandin E1 (Prostaglandin is a natural inhibitor and enhances the sensitivity of the assay for clopidogrel) clopidogrel, IIb/IIIa antagonists aspirin, vwf TRAPtest TRAP-6 (thrombin receptor activating peptide): TRAP-6 is a potent agonist which mimicks the platelet-activating action of thrombin IIb/IIIa antagonists vwf, aspirin, clopidogrel (weak effect on TRAPtest) COLtest collagen: collagen activates platelet and triggers a release of arachidonic acid from the platelet membrane, which is converted to TXA2 by the Cyclooxygenase aspirin, IIb/IIIa antagonists clopidogrel, vwf RISTOtest Ristocetin: vwf dependent platelet activation via the GpIb receptor Bernard-Soulier syndrome, severe vwd, aspirin mild vwd

9 multiple electrode aggregometry = MEA - one Multiplate test cell incorporates two independent sensor units. - the increase of impedance due to the attachment of platelets to the electrodes is detected for each sensor unit separately and transformed to arbitrary aggregation units (AU) that are plotted against time. - the duplicate sensors serve as an internal control test during each measurement Pearson s correlation coefficient of single measurements of the curves assessed by the two electrode pairs and the difference of the two AUCs is calculated. The result is flagged if the values are outside of the acceptance range (correlation coefficient <0.98, difference to the mean curve >20%). D. Sibbing et al, Thromb Haemost Jan;99(1):121-6.

10 blood sample for Multiplate analysis - citrated blood is the typical sample anticoagulant used for platelet function analysis - citrate complexes approx. 98% of the free calcium in the sample - however calcium is an important second messenger of platelet activation - therefore citrate has the potential to disturb platelet function tests - for Multiplate analysis a tight attachment of platelets onto the impedance sensor is mandatory, which enhances the effect of calcium depletion on this method - therefore the analysis of blood anticoagulated by hirudin or heparin is recommended - hirudin blood collection tubes are commercially available from the manufacturer of Multiplate Since hirudin and other direct acting thrombin inhibitors do not reduce the concentration of divalent cations in plasma, these can be considered to provide a more physiological environment than the use of sodium citrate. On this basis, we would agree with the recommendation by Dynabyte that the use of hirudin or another direct acting thrombin inhibitor is preferable for measurements of platelet aggregation performed by MEA (multiple eletrode aggregometry = Multiplate). A. Johnson, Thromb Haemost Jun;99(6):

11 Why study platelet function in whole blood rather than in PRP? In PRP not all platelets may be present, the most active platelets and larger platelets may have been lost during centrifugation In PRP platelets are studied in isolation which is NOT their natural situation In whole blood other blood cells are also present - erythrocytes (a potential source of ADP and ATP and a means of removing adenosine) and leucocytes which also influence platelet aggregation. S. Heptinstal et al

12 examples TRAPtest ASPItest ADPtest no platelet inhibition 113 U 102 U 89 U 17 U 100 mg aspirin qd 139 U 134 U 75 mg clopidogrel qd 98 U 89 U 31 U 100 mg aspirin + 75 mg clopidogrel qd 88 U 8 U 17 U tirofiban (Aggrastat i.v.) 7 U 3 U 3 U

13 A.B. *1971 very weak aggregation in all tests due to Glanzman Thrombasthenia

14 near-patient application of Multiplate university clinic Frankurt / Main Hannover medical school Essen university clinic

15 Multiplate in interventional cardiology / neuroradiology

16 2 patients before and after aggrastat loading dose Aggrastat Aggrastat case report by Dr. med. Stefanie Müller-Schunk, Neuroradiologie, Klinikum der Universität München

17 2 patients before and after aspirin 500 mg i.v. aspirin 500 mg i.v. aspirin 500 mg i.v. 50 min after aspirin application case reports by Dr. med. Stefanie Müller-Schunk, Neuroradiologie, Klinikum der Universität München

18 increase of clopidogrel dose Nonresponse Response clopidogrel 75mg clopidogrel 2x75mg

19 change from clopidogrel to prasugrel Stent thrombosis AU AU 0 6 min Clopidogrel 150mg/d F. Krötz, Kardiologie, Klinikum der Universität München 0 6 min Prasugrel 10mg/d

20 Platelet Function Analysis Market simple whole-bloodbased 1-2 different tests available single chanel Multiplate complicated low standardisation used in research and reference labs cartridge-based platelet function tests easy-to-use multi-channel several tests available open system optical aggregometry flow cytometry

21 Summary Multiplate is one of the newer platelet function tests. The system has a high sensitivity for anti-platelet drugs (Aspirin, Clopidogrel, Prasugrel, IIbIIIa antagonists). It is widely used in laboratory and near-patient settings. Studies have shown a good predictivity towards thromboembolism in platelet non-responsiveness (10 fold enhanced risk for stent thrombosis in a study including 1608 patients following PCI). Studies have shown a predictivity for transfusion requirements during / after cardiac surgery. Further applications include the assessment of platelet disorders, drug interactions and animal models.

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