Standardization of platelet‐derived microparticle enumeration by flow cytometry with calibrated beads: results of the International Society on Thrombosis and Haemostasis SSC Collaborative workshop

Microparticles (MPs) are sub-micrometer sized vesicles released from cell membranes in response to activation or apoptosis [1]. MPs originating from several cell sources have been described in human plasma. Among them, plateletderivedMPs (PMPs) are believed to account for themajority of circulating MPs in healthy subjects [2]. Their levels are increased in several prothrombotic and inflammatory disorders [1]. In these clinical settings, PMP counts may be useful for identifying patients at risk for vascular disorders and for monitoring response to treatment [3]. However, their clinical use is not fully established, because standardized methodologies for PMP counting are lacking. A previous International Society on Thrombosis and Haemostasis (ISTH) Vascular Biology Subcommittee survey indicated that approximately 75% of laboratories use flow cytometry (FCM) to enumerate MPs in clinical samples. However, a wide variety of preanalytic variables and analytic variables have been reported in the literature, resulting in a wide range of PMP values in platelet-free plasma (PFP) of healthy subjects (100–4000 PMPs lL). This lack of consensus stresses the need for standardization [4]. Three ISTH Scientific and Standardization Subcommittees (SSC Vascular Biology, DIC, and Haemostasis &Malignancy) have initiated a project aimed at standardizing the enumeration of cellularMPs by FCM.Afirstcollaborative workshopwas set up, to: first, establish the resolution and the level of background noise of the flow cytometers currently used in laboratories with respect to the strategy requirements; and second, define the interinstrument reproducibility of PMP enumeration in human plasma. This strategy was based on the use of fluorescent calibrated sub-micrometer beads (Megamix beads; BioCytex, Marseille, France), which allow the window of MP analysis to be reproducibly set [5]. The study included 40 laboratories accounting for 59 flow cytometers, and was performed in two stages (Fig. S1): in stage A, participating laboratories received Megamix beads and were asked to set up the FCM protocol and to validate an instrument protocol adapted from a previously described method [5]. On the basis of forward scatter (FS)/FS channeling (FSC) resolution and background characteristics, stage A results led to acceptance or rejection of the tested instruments, with some time being allowed for technical intervention in order to improve any deficient performance. In stage B, selected laboratories received PFP samples prepared as frozen aliquots by the core laboratory, and were asked to analyze them with the previously validated instrument(s), common reagents, and the FCM protocol established in stage A. A detailed description of the methodology is available in the Supporting Information (Data S1). The purpose of this initial phase was to check whether the instrument to be used to enumerate PMPs demonstrated the required performance with a blend of fluorescent beads with well-known sizes and relative amounts. The instruments were validated on the basis of their capacity to discriminate between 0.5-lm and 0.9-lm Megamix beads using the FS/FSC parameter, as well as their background noise. Instruments detecting < 0.1% of fluorescent bead events among total events were rejected, because such a level of background may impede the electronics functions and induce amajor loss of events owing to coincidences and electronic aborts (Fig. S2A,B). Analysis of the results demonstrated that instruments were heterogeneous with respect to FS/FSC resolution and background noise. Furthermore, the level of performance could vary over time (Fig. S2C). Some of the parameters affecting FS/FSC resolution were identified with Megamix beads. Among them, FS/ FSC gain, FS/FSCmode, neutral density (intensity scavenging) Correspondence: Francoise Dignat-George, UMR-S608, 27 Bd Jean Moulin, 13005 Marseille, France. Tel.: +33 491 385600; fax: +33 491 385602. E-mail: francoise.dignat-george@univmed.fr Journal of Thrombosis and Haemostasis, 8: 2571–2574 DOI: 10.1111/j.1538-7836.2010.04047.x