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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?
Author(s) -
ROBERT S.,
PONCELET P.,
LACROIX R.,
ARNAUD L.,
GIRAUDO L.,
HAUCHARD A.,
SAMPOL J.,
DIGNATGEORGE F.
Publication year - 2009
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/j.1538-7836.2008.03200.x
Subject(s) - reproducibility , quartile , coefficient of variation , medicine , microparticle , flow cytometry , bead , protocol (science) , biomedical engineering , nuclear medicine , chromatography , pathology , chemistry , materials science , immunology , biology , confidence interval , astrobiology , alternative medicine , composite material
Summary. 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.