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

Abstract

Accurate assessment of in vivo or in vitro platelet activation requires optimal preanalytical conditions to prevent artefactual in vitro activation of the platelets. The choice of anticoagulant is one of the critical preanalytical conditions as anticoagulants exert different effects on the activation of platelets ex vivo. We tested the effectiveness of Diatube-H (also known as CTAD; sodium citrate, theophylline, adenosine and dipyridamole) and citrate vacutainer tubes in preventing artefactual activation of platelets and 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 collection and at 6, 24 and 48 h after venipuncture. Samples taken into Diatube-H showed less spontaneous platelet activation than did those taken into citrate. 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 platelets responded fully to agonist activation. Hence, Diatube-H preserved platelets and allowed for measurement of in vivo platelet activation as well as thrombin-induced in vitro platelet activation after 6-24 h, in both whole blood and isolated platelets.

Interaction with damaged vessel wall in vivo in humans induces platelets to express CD40L resulting in endothelial activation with no effect of aspirin intake

“…Blood was immediately added to tubes kept in crushed ice containing a mixture of sodium citrate, theophylline, adenosine, and dipyridamole (CTAD) (Becton Dickinson, Franklin Lakes, NJ) in PBS (31). After each pipetting, any residual blood at the skin-wound site was carefully removed with filter paper.…”

Preanalytical Recommendations of the ‘Groupe d’Etude sur l’Hémostase et la Thrombose’ (GEHT) for Venous Blood Testing in Hemostasis Laboratories

“…This mixture is well suited for monitoring heparin therapy either by APTT or by anti-Xa assays [25,26] since it reduces heparin neutralization by platelet factor IV very significantly. Recently, the use of the CTAD has been proposed for flow-cytometric analysis of platelets [27][28][29][30]. On the other hand, its use is proscribed for any type of platelet function testing as it inhibits platelet function.…”

Platelet activation and hypercoagulability following treatment with porcine factor VIII (HYATE:C)

“…With informed consent, blood for platelet studies was drawn as previously described [8] into Diatube-H Vacutainer tubes (Becton Dickinson, Rutherford, NJ) or into 0.129 M citrate Vacutainer tubes for plasma hypercoagulability studies. For the studies of e ect in vivo, samples were drawn prior to and at intervals (10 min, 1 hr, and 2 hr) following administration of PFVIII (HYATE:C, Ipsen Ltd (Speywood Biopharm Ltd), Wrexham, U.K.).…”

“…Samples were then incubated at 37°C for 30 min and stained with uorochrome-labeled antibodies as above. Flow cytometry was performed as previously described [8,13]. Microparticles were distinguished from intact normal platelets as described by Abrams et al [14] and were de®ned as the proportion of all GPIb-or GPIIbIIIa-positive cells smaller than 0.8 l in size based on forward scatter pro®le; they showed reduced uorescence intensity for GPIb or GPIIbIIIa compared to intact platelets.…”