Summary. Background: Microparticles (MP) are small vesicles of 0.1-1 lm, released in response to activation or apoptosis. Over the past decade, they received an increasing interest both as biomarkers and biovectors in coagulation, inflammation and cancer. Clinical studies were conducted to assess their contribution to the identification of patients at cardiovascular risk. However, among the limitation of such studies, pre-analytical steps remains an important source of variability and artifacts in MP analysis. Objectives: Because data from the literature are insufficient to establish recommendations, the objective of the present study was to assess the impact of various pre-analytical parameters on MP measurement. These parameters included the type of collection tube, phlebotomy conditions, transportation practices, centrifugation steps and freezing. Methods: MP were assessed by three methods: flow cytometry using a standardized approach, a thrombin generation test (Calibrated Automated Thrombogram Ò ) and a procoagulant phospholipid-dependent clotting time assay (STA Ò -Procoag-PPL). Results: The main results show that the three major preanalytical parameters which impact on MP-related data are the delay before the first centrifugation, agitation of the tubes during transportation and the centrifugation protocol. Conclusions: Based on both this work and literature data, we propose a new protocol that needs to be validated on a larger scale before being applied for multicenter studies.

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Objective-Cellular microparticles (MP) are promising biomarkers in many pathological situations. Although flow cytometry (FCM) is widely used for their measurement, it has raised controversies because the smallest MP size falls below the detection limit of standard FCM (sd-FCM). Following recent technological improvements leading to high sensitivity FCM (hs-FCM), our objectives were (1) to evaluate the potential of hs-FCM for extended MP detection, (2) to set up a standardized protocol for MP enumeration, and (3) to compare MP counts obtained with both sensitivity levels. Methods and Results-Compared with sd-FCM, hs-FCM displayed improved forward scatter resolution and lower background noise, allowing us to discriminate previously undetectable small MP in plasma samples. Using fluorescent beads with appropriate sizes (0.1/0.3/0.5/0.9 m) and relative amounts, a new standardized hs-FCM MP protocol was set up and provided reproducible MP counts. Applied to coronary patient samples, it resulted into 8-to 20-fold increases in MP counts as compared with sd-FCM. Interestingly, the ratio between small and large MP varied according to clinical status but also depending on MP subset, suggesting access to new biological information. 3-5 Circulating MP are elevated in many prothrombotic and inflammatory disorders; cardiovascular, autoimmune, and infectious diseases; and cancer. 4,[6][7][8][9][10] Although MP counts may provide useful diagnostic/prognostic information, assessment of their pathophysiological relevance is hampered by methodological concerns. Flow cytometry (FCM) is the most commonly used technique, 11 allowing both enumeration and characterization of MP cellular origin with high throughput. The forward scatter (FS) parameter is generally used to define the analysis gate for MP. Unfortunately, because of their limited FS sensitivity, standard flow cytometers measure only a small part of the MP population. Recently, high-sensitivity flow cytometers with significantly improved light scatter detection became available. As we first reported, these instruments resolve a previously undetectable subpopulation of small-size MPs. Conclusion-Recent12 Among the technical improvements found in high sensitivity FCM (hs-FCM), a new option of the Beckman Coulter Gallios called W2 increases FS resolution by amplifying the FS signals collected at large angles. 13 Whether this option can be used in a standardized manner, to improve the detection of MPs derived from platelets (PMP), erythrocytes (Ery-MP), leukocytes (Leu-MP), and endothelial cells (EMP), remains an open question. Our objectives were then (1) to evaluate the impact of high-sensitivity flow cytometer technical improvement on the detection of small-size MP, (2) to set up a new standardized protocol based on calibrated microbeads for MP enumeration, and (3) to compare MP counts obtained at both sensitivity levels of FCM. MethodsBlood and plasma samples, MP preparation and flow cytometric analysis, size-conditioned MP filtration, intra-and inter-instrument reproducibi...

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.