The influence of pre-analytical conditions on platelet-derived microparticles.

Background: Microparticles (MP) have recently become a focus of both research and clinical investigations. As pre-analytical conditions frequently remain unpublished, further studies are needed to analyze their impact on MP release.

Methods: This prospective study investigated the effect of sequential storage under three different sets of conditions (fresh; storage at 4 degrees C for 24 hours, SC1; storage at -70 degrees C for 24 hours, SC2) and agitation on platelet-derived MP (PMP) in 11 healthy blood donors (6 male, 5 female). PMP were quantified using flow cytometry (FCM) for analysis of all events positive for both CD41a-PE and Annexin-V-FITC. Newly developed calibration beads for FCM (size of 0.3 - 0.9 microm) were applied for FCM. For functional testing a phospholipid-dependent clotting assay (XACT) was used.

Results: PMP concentration increased 1.7-fold in platelet-poor plasma (PPP) under SC1 and further increased 1.6-fold (p < 0.001) under SC2 (p = 0.005). Overall, samples of SC2 had a 5.5-fold increased count of large PMP (0.5 - 0.9 microm) compared to baseline. Results in samples of SC2 ranged from 40.1 seconds to 80.3 seconds but on average the CT was also shortened compared to the CT for SC1 and fresh samples. Additional agitation before PPP preparation reduced the PMP concentration by around 50% (p = 0.025). 135% more small PMP were detected with recently developed calibration beads. Compared to CT (XACT) flow cytometry using Megamix Plus calibration beads is able to reveal significant differences between the analyzed preanalytical conditions.

Conclusions: Fresh blood samples should be used for standardizing PMP analysis. Calibration beads for FCM (size of 0.3 - 0.9 microm) have shown to be a reliable tool for PMP quantification especially for PMP of smaller sizes up to 300 nm. Agitation of blood samples before PMP analysis should be avoided. The application of XACT is limited for the analysis of preanalytical conditions.