EFFECT OF DIFFERENT IN VITRO ANTICOAGULANTS ON LABORATORY MEASUREMENT OF PLATELET FUNCTION

Light transmission aggregometry (LTA) is commonly applied in the diagnosis of platelet bleeding disorders and in the research setting to monitor platelet function in the evaluation and development of antiplatelet agents. The guidelines for the standardisation of LTA for the study of platelet aggregation (PA) published in 2013 by the SSC-ISTH were largely based on the consensus of experts, because methodological studies directly comparing different procedures were lacking. We tested the cogency of some SSC-ISTH recommendations: 1) buffered citrate (109 or 129 mM) should be used as anticoagulant to help keep the pH stable; 2) after centrifugation, platelet-rich plasma (PRP) should sit at room temperature for 15 min before testing; 3) LTA studies should be completed within a maximum of 4h after blood sampling. Blood from 10 healthy volunteers was collected into buffered and non-buffered citrate (109 and 129 mM). PA was measured by LTA in PRP stimulated by ADP (2 μM) immediately after preparation, 15 min thereafter (=1h after blood sampling), 2, 3 and 4h after blood sampling. Plasma pH and platelet count were measured at the same time points as LTA. PA changed within the first 15 minutes after PRP preparation, then remained stable up to 2h after blood sampling, and decreased thereafter; 2) PA tended to be higher in 109 mM than in 129 mM citrate; 3) there was no difference in PA between buffered and non-buffered citrate; 4) pH in buffered samples was lower than in non-buffered samples, but increased in all samples to pH 8 at 4h; 5) platelet count in non-buffered citrate PRP was lower than in buffered citrate and decreased over time in all samples. The loss of platelets was inhibited by addition of prostaglandin E1 (PGE1, 2 μM) to whole blood upon collection. We conclude that PRP should rest for 15 min before testing; buffering of citrate does not affect PA significantly but does affect platelet count: therefore, it is uncertain whether the use of buffered citrate is preferable over non-buffered citrate; LTA studies should be completed within 2h rather than 4h after blood sampling.