Platelet Function analysis using Multiple Electrode Aggregometry (Multiplate ® )

► Multiplate ® platelet function analysis in whole blood based on impedance aggregometry

► 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

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

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 ► Performing the test

► Multiplate ® reagents comprehensive line of dedicated reagents

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

► Multiplate tests 2/2 testactivationsensitivitynot sensitive for ASPItestarachidonic acid: is converted to TXA2 by platelet-own cyclooxygenase aspirin, IIb/IIIa antagonists clopidogrel, vWF ADPtestADP: binds onto platelet ADP receptorsclopidogrel, IIb/IIIa antagonists aspirin, vWF ADPtest HSADP + prostaglandin E1 (Prostaglandin is a natural inhibitor and enhances the sensitivity of the assay for clopidogrel) clopidogrel, IIb/IIIa antagonists aspirin, vWF TRAPtestTRAP-6 (thrombin receptor activating peptide): TRAP-6 is a potent agonist which mimicks the platelet-activating action of thrombin IIb/IIIa antagonistsvWF, aspirin, clopidogrel (weak effect on TRAPtest) COLtestcollagen: 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 RISTOtestRistocetin: vWF dependent platelet activation via the GpIb receptor Bernard-Soulier syndrome, severe vWD, aspirin mild vWD

► multiple electrode aggregometry = MEA test 1+2 -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 -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 20%). D. Sibbing et al, Thromb Haemost Jan;99(1):121-6.

► 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):

 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 ► Why study platelet function in whole blood rather than in PRP?

TRAPtest ASPItestADPtest no platelet inhibition 100 mg aspirin qd 75 mg clopidogrel qd 100 mg aspirin + 75 mg clopidogrel qd tirofiban (Aggrastat ® i.v.) 17 U 134 U139 U 98 U89 U 31 U 8 U 88 U 17 U 113 U102 U 89 U 7 U3 U ► examples

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

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

► Multiplate in interventional cardiology / neuroradiology

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

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

clopidogrel 2x75mg clopidogrel 75mg Non- response Response ► increase of clopidogrel dose

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

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

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. ► Summary