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

• Published in: Journal of thrombosis and haemostasis Vol. 7; no. 1; pp. 190 - 197
• Main Authors: ROBERT, S., PONCELET, P., LACROIX, R., ARNAUD, L., GIRAUDO, L., HAUCHARD, A., SAMPOL, J., DIGNAT‐GEORGE, F.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.01.2009
• Subjects: beads; Blood Platelets; Calibration; Cell-Derived Microparticles - pathology; flow cytometry; Flow Cytometry - methods; Flow Cytometry - standards; Humans; megamix; microparticles; Microspheres; Observer Variation; Particle Size; Reference Standards; standardization
• ISSN: 1538-7933; 1538-7836; 1538-7836
• DOI: 10.1111/j.1538-7836.2008.03200.x

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 interinstrument reproducibility; and (iii) to establish PMP values in healthy subjects. Methods: Using a blend of size‐calibrated fluorescent beads (0.5 and 0.9 μm) 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 μL−1 (1014–3039 μL−1) vs. 656 μL−1 (407–962 μL−1)]. Conclusions: The present strategy provides a new option for PMP count standardization and thus opens the way for multicenter studies.