Standardization of microparticle enumeration across different flow cytometry platforms: results of a multicenter collaborative workshop

Abstract

Summary Background Microparticles are extracellular vesicles resulting from the budding of cellular membranes that have a high potential as emergent biomarkers; however, their clinical relevance is hampered by methodological enumeration concerns and a lack of standardization. Flow cytometry (FCM) remains the most commonly used technique with the best capability to determine the cellular origin of single MPs. However, instruments behave variably depending on which scatter parameter, (Forward (FSC) or Side scatter (SSC)), provides the best resolution to discriminate submicron particles. To overcome this problem, a new approach, based on two sets of selected beads adapted to FSC or SSC optimized instruments, was recently proposed to reproducibly enumerate platelet-derived MP counts among instruments with different optical systems. Objective The objective was to evaluate this strategy in an international workshop that included 44 laboratories accounting for 52 cytometers of 14 types. Methods/Results Using resolution capability and background noise level as criteria to qualify the instruments, the standardization strategy proved to be compatible with 85% (44/52) instruments. All instruments correctly ranked the PMP levels of two platelet-free plasma samples. The inter-laboratory variability of PMP counts was 37% and 28% for each sample. No difference was found between instruments using forward or side scattered light as the relative sizing parameter. Conclusions Despite remaining limitations, this study is the first to demonstrate a real potential of bead-based strategies for standardization of MP enumeration across different FCM platforms. Additional standardization efforts are still mandatory to evaluate MP clinical relevance at a multicenter level.

Detection of platelet vesicles by flow cytometry

“…It is generally recommended to process the blood, to remove platelets and cells and produce cell free plasma, as soon as possible after collection. The ISTH-promulgated protocol of centrifuging blood twice for 15′ at 2500xg is followed in a large fraction of published reports (24–26). Several studies have examined the effect of freezing on EV numbers and size and come to different conclusions as to whether this affects the final results.…”

“…With these caveats about light scatter-based EV detection in mind, extensive efforts to standardize platelet EV measurements using conventional light scatter-based detection have conducted and reported over the past several years, primarily under the auspices of the International Society on Thrombosis and Haemostasis (24–26). In general, these approaches employ uniform polymer beads to standardize a “window of analysis” in the light scatter parameters to facilitate comparison of results over time and across instruments.…”

Changes in plasma circulating microvesicles in patients with HCV‐related cirrhosis after treatment with direct‐acting antivirals

“…The main limitations of this study pertain to the small sample size, notwithstanding the very homogeneously enrolled and longitudinally followed population; the lack of Child's C cirrhotic patients as DAAs regimen is not indicated; the lack of standardised method to analyse circulating MVs; finally, given the relatively small sample size including mostly Child's A patients, the number of clinically relevant complications registered during follow‐up was low and the logistic regression analysis impaired, hence the reason why a composite outcome was used. We acknowledge that the physiopathology of the events recorded is different; however, all the outcomes considered are overall related to cells activation, hence increase vesiculation.…”

“…Prior to the staining, the antibody mixture was centrifuged at 20 000 g for 30 minutes to remove fluorescent particles. The pre‐analytic phase of MVs analysis has previously been reported …”

Scattering the spotlight on microparticles

“…With this goal in mind, the Scientific Standardization Committee on Vascular Biology of the ISTH initiated a collaborative workshop to assess the reproducibility of platelet microparticle measurements derived from a single source across a number of different instruments and centers. In this issue of the Journal of Thrombosis and Haemostasis, Cointe et al report on a study conducted across 17 countries with 52 flow cytometers [10]. Two different sets of proprietary standardization beads (with sizes ranging from 0.1 lm to 0.9 lm) were utilized based on the underlying characteristics of the instruments in order to determine whether microparticle detection was better with side scatter or forward scatter.…”