Inserm, Institut national de la santé et de la recherche médicale
Faculté de pharmacie, Aix Marseille Université

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

Robert S, Poncelet P, Lacroix R, Arnaud L, Giraudo L, Hauchard A, Sampol J and Dignat-George F. J Thromb Haemost. 2009 ;7:190-7. IF= 6,3


BACKGROUND : 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. OBJECTIVES : 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.

METHODS : 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.

RESULTS : 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))].

CONCLUSIONS : The present strategy provides a new option for PMP count standardization and thus opens the way for multicenter studies.