Blood platelet activation evaluated by flow cytometry: optimised methods for clinical studies

A variety of flow cytometry techniques are in use to evaluate in vivo blood platelet activation. We have in this study further developed and optimised these methods to be suitable for use in clinical studies, By preloading th e Monovette(R) EDTA vacuum blood sampling tubes with 1/8 vol 4% (w/v) paraf ormaldehyde (PFA), we were able to assess platelet CD62P (P-selectin) expre ssion in whole blood with less than 0.2% activated platelets. No washing or neutralising steps were required to remove excess fixative, Both basal and agonist-stimulated CD62P expression were stable for at least 48 h after sa mpling, The standard curve was linear from 1.9 (basal) to 8.1.10(3) (TRAP-s timulated) molecules of equivalent soluble fluorochrome units (MESF) in phy coerythrein-conjugated anti-CD62P labelled whole blood samples, These assay conditions were also well suited for assessment of platelet expression of CD41, CD42a, CD61 and CD63. The preanalytic storage period was extended fro m 10 min to at least 2h for platelet PAC-1 and fibrinogen binding analysis by preloading Monovette(R) citrate tubes with 8/10vol buffer. With PFA prel oading, blood sampled into citrated tubes could be analysed for fractions o f microparticles and platelet-platelet aggregates as well as for aggregate size.