Synapse Research Institute developed a flow cytometry-based whole blood platelet activation test (WB-PACT) which measures platelet activation in response to a specific agonist. The test is optimized for (1) the GPVI agonist collagen related peptide (CRP), (2) the PAR-1 agonist thrombin receptor activating peptide (TRAP) and (3) the P2Y12 receptor agonist ADP. Simultaneously, the activation of GPIIb/IIIa and the expression of P-selectin on the platelet surface is quantified, correlating with the aggregation and granule release potential of platelets, respectively.
Test strips consisting of a specific agonist and antibodies against GPIb (platelet marker), activated GPIIb/IIIa and P-selectin can be prepared in advance and stored at -20°C. The reaction is initiated by the addition of 5 µl diluted whole blood. Flow cytometry was used to discriminate platelets from other cells by gating on the GPIb positive cells and to quantify the density of the activation markers.
The PACT has many advantages:
(1) flow cytometry measures within limited time the specific characteristics of a large number of individual platelets;
(2) platelets are analyzed in their physiological milieu of whole blood;
(3) platelet function can be measured without pretreatment of the blood, allowing measuring both the baseline activation state of circulating platelets and their reactivity in response to various agonists without the influence of artefactual in vitro activation by pre-conditioning of blood;
(4) the technique permits to investigate the aggregation potential as well as the granule release capacity of platelets;
(5) only minimal amounts of blood (5 µl) are needed.
The WB-PACT was studied in relation to several haemostatic complications, including secondary thrombotic events, bleeding disorders, inflammation, platelet function during surgery and platelet function in thrombocytopenia.
Huskens D, Sang Y, Konings J, van der Vorm L, de Laat B, Kelchtermans H, Roest M. Standardization and reference ranges for whole blood platelet function measurements using a flow cytometric platelet activation test. PloS One. 2018; 13(2):e0192079.
Eising HP, Roest M, de Groot PG, Huskens D, Konings J, Urbanus RT, de Laat B, Remijn JA. High prevalence of reduced thrombin generation and/or decreased platelet response in women with unexplained heavy menstrual bleeding. Int J Lab Hematol. 2018
Batman B, van Bladel ER, van Hamersveld M, Pasker-de Jong PCM, Korporaal SJA, Urbanus RT, Roest M, Boven LA, Fijnheer R. Agonist-induced platelet reactivity correlates with bleeding in haemato-oncological patients. Vox Sang. 2017;112(8):773-779.
Kicken CH, Roest M, Henskens YM, de Laat B, Huskens D. Application of an optimized flow cytometry-based quantification of Platelet Activation PACT): Monitoring platelet activation in platelet concentrates. PLoS One. 2017;12(2):e0172265.
Leunissen TC, Wisman PP, van Holten TC, de Groot PG, Korporaal SJ, Koekman AC, Moll FL, Teraa M, Verhaar MC, de Borst GJ, Urbanus RT, Roest M. The effect of P2Y12 inhibition on platelet activation assessed with aggregation- and flow cytometry-based assays. Platelets. 2017; 28(6):567-575.
Leunissen TC, Gijsberts CM, Wisman PP, Huisman A, Berg MT, Asselbergs FW, Hoefer IE, Pasterkamp G, Moll FL, de Borst GJ, Roest M. Lower platelet reactivity is associated with presentation of unstable coronary artery disease. Int J Angiol. 2016;25(4):210-218.
Van Poucke S, Zhang Z, Roest M, Vukicevic M, Beran M, Lauwereins B, Zheng MH, Henskens Y, Lancé M, Marcus A. Normalization methods in time series of platelet function assays: A SQUIRE compliant study. Medicine (Baltimore). 2016 ;95(28):e4188.
Biedermann JS, Cannegieter SC, Roest M, van der Meer FJ, Reitsma PH, Kruip MJ, Lijfering WM. Platelet reactivity in patients with venous thrombosis who use rosuvastatin: a randomized controlled clinical trial. J Thromb Haemost. 2016;14(7):1404-1409.
Bikker A, Bouman E, Sebastian S, Korporaal SJ, Urbanus RT, Fijnheer R, Boven LA, Roest M. Functional recovery of stored platelets after transfusion. Transfusion. 2016;56(5):1030-1037
Wisman PP, Teraa M, de Borst GJ, Verhaar MC, Roest M, Moll FL. Baseline platelet activation and reactivity in patients with critical limb ischemia. PLoS One. 2015;10(7):e0131356.
van Geffen JP, Kleinegris MC, Verdoold R, Baaten CC, Cosemans JM, Clemetson KJ, Ten Cate H, Roest M, de Laat B, Heemskerk JW. Normal platelet activation profile in patients with peripheral arterial disease on aspirin. Thromb Res. 2015;135(3):513-20.