Preanalytical requirements for flow cytometric evaluation of platelet activation: choice of anticoagulant

Accurate assessment of in vivo or in vitro platelet activation requires opt imal preanalytical conditions to prevent artefactual in vitro activation of the platelets. The choice of anticoagulant is one of the critical preanaly tical conditions as anticoagulants exert different effects on the activatio n of platelets ex vivo. We tested the effectiveness of Diatube-H (also know n as CTAD; sodium citrate, theophylline, adenosine and dipyridamole) and ci trate vacutainer tubes in preventing artefactual activation of platelets an d preserving functional reserve. Platelet surface expression of the CD62P ( reflecting alpha granule release), CD63 (reflecting lysosomal release) and modulation of normal platelet membrane glycoproteins CD41a and CD42b, were measured in whole blood and in isolated platelets immediately after collect ion and at 6, 24 and 48 h after venipuncture. Samples taken into Diatube-H showed less spontaneous platelet activation than did those taken into citra te. To measure in vitro platelet functional reserve, thrombin was added as agonist to blood stored for varying periods up to 48 h. Although Diatube-H suppressed in vitro platelet activation for up to 4 h, in samples kept for 6-24 h before thrombin addition, the inhibitory effect was lost and platele ts responded fully to agonist activation. Hence, Diatube-H preserved platel ets and allowed for measurement of in vivo platelet activation as well as t hrombin-induced in vitro platelet activation after 6-24 h, in both whole bl ood and isolated platelets.