, 2004, Shah et al., 2008, Shet, 2008 and Dignat-George et al., 2009) but without a systematic analysis of individual parameters. The present investigation was undertaken to fill this gap in the literature by evaluating factors that affect MV analysis in terms of venous sample collection, anticoagulants, isolation techniques, staining methods and storage and cytometer settings. An important feature of the present approach was to assess all of these parameters on blood from diverse groups of healthy and diseased individuals so that findings may be generalized. CP-868596 cell line A paramount finding of this study is the impact of anticoagulants on MV recovery.

Counts of platelet and endothelial MV were substantially lower in blood collected in citrate or EDTA than in blood collected in protease inhibitors, either H&S or heparin. This effect of anticoagulants was interpreted by Shah et al. (2008) as arising from microvesiculation in vitro with protease inhibitor anticoagulants. However, results of the present study provide an alternative conclusion, first because

endothelial MV, which Selumetinib supplier cannot be generated in blood in vitro, were effectively removed with chelation of whole blood. Therefore, the difference in MV counts obtained in calcium chelating anticoagulants compared to protease inhibiting anticoagulants reflects loss with chelation rather than gain with protease inhibitors. This conclusion is verified by the finding that adding any anticoagulant to platelet-free plasma prepared without an anticoagulant had no effect on MV counts, which were congruent with those obtained from whole blood anticoagulated by protease inhibition. Chelation-induced association of the MV with platelets is adequate to account for this phenomenon, as it can be recapitulated with PRP prepared from blood collected

in protease inhibiting anticoagulants. Because the degree of loss with chelation is unpredictable, with relative proportions of annexin-V positive and negative platelet MV not falling in predictable register, all prior work on MV from blood anticoagulated by citrate, ACD and Methocarbamol EDTA (Jy et al., 2004) may need reevaluation. That said, our MV counts from citrated plasma lie within the lower group of the wide range among published studies (Yuana et al., 2011). Platelet MV counts remained constant when either H&S or heparin anticoagulated blood was maintained for up to 60 min at 33 °C, and for 30 min at room temperature, but thereafter increased. The temperature effect is commensurate with the sensitivity of platelet shape change as blood cools (Tablin et al., 2000). Counts of endothelial MV did not change over time at either temperature, to indicate that the increase reflected release of MV from the platelets. There is growing and compelling evidence that flow cytometry resolves only the largest membrane vesicles, which comprise a near-negligible portion of the total (Koch et al., 1966, Foladori et al., 2008, Zwicker et al., 2009, Lacroix et al., 2010, Yuana et al.

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