Standardization of platelet-derived microparticle counting using calibrated beads and a Cytomics FC500 routine flow cytometer: a first step towards multicenter studies?

Abstract

Platelet microparticles (PMPs) have proved useful to identify patients with vascular risk. However, PMP counting, which is currently done by flow cytometry (FCM), needs to be standardized. The objectives were (i) to standardize FCM settings for PMP counts on a routine instrument (Cytomics FC500) using size-calibrated fluorescent beads; (ii) to determine intra-instrument and inter-instrument reproducibility; and (iii) to establish PMP values in healthy subjects. Using a blend of size-calibrated fluorescent beads (0.5 and 0.9 mum) in a fixed numerical ratio (Megamix), we gated PMPs in a restricted size window. To test intra-instrument and inter-instrument reproducibility, annexin V and CD41 coexpression were used to count PMPs in frozen aliquots of the same platelet-free plasma (PFP) over 4 months and in PFP from 10 healthy subjects on three independent flow cytometers. This calibrated-bead strategy allowed full long-term control of the FCM-based microparticle protocol and reproducible PMP counts over time [coefficient of variation (CV) < 10%]. Optimal settings were easily transferred from one instrument to another, using Megamix as a stable template. Similar PMP counts (CV < 12%) were obtained using the three instruments. With such a standardized FCM protocol, PMP values were established in healthy subjects (n = 60) with significantly higher levels in women than in men [median (1st quartile to 3rd quartile): 1775 microL(-1) (1014-3039 microL(-1)) vs. 656 microL(-1) (407-962 microL(-1))]. The present strategy provides a new option for PMP count standardization and thus opens the way for multicenter studies.