Regular Article Flow cytometric detection of endothelial microparticles (EMP): Effects of centrifugation and storage alter with the phenotype studied ☆ Sabrina H. van Ierssel a, ⁎, Emeline M. Van Craenenbroeck b , Viviane M. Conraads b , Viggo F. Van Tendeloo c , Christiaan J. Vrints b , Philippe G. Jorens a , Vicky Y. Hoymans b a Department of Critical Care Medicine, Antwerp University Hospital (UZA), University of Antwerp, Edegem, Belgium b Laboratory of Cellular and Molecular Cardiology, Antwerp University Hospital (UZA), University of Antwerp, Edegem, Belgium c Centre for Cellular Therapy and Regenerative Medicine, Antwerp University Hospital (UZA), University of Antwerp, Edegem, Belgium abstract article info Article history: Received 5 October 2009 Received in revised form 17 December 2009 Accepted 30 December 2009 Available online 1 February 2010 Keywords: Centrifugation detection endothelial microparticles flow cytometry phenotype storage Introduction: Endothelial microparticles (EMP) are released into the circulation in case of endothelial disturbance, and are therefore increasingly investigated as a biomarker reflecting disease activity. Numerous pre-analytic methods have been proposed for their flow cytometric enumeration, but standardization is still lacking. In this study we evaluated the influence of centrifugation and storage conditions on EMP quantification. Materials and Methods: Platelet-poor plasma (PPP) from 10 healthy volunteers was prepared by centrifugation at 1 550 g for 20 minutes twice. A first aliquot of PPP was analyzed immediately, a second after storage at 4 °C for 7 hours. A third and fourth aliquot were snap-frozen and stored at -80 °C for 7 and 28 days. A final aliquot was further centrifuged at 10 000 g for 10 minutes and analyzed immediately. EMP were defined as CD31+CD42b-, CD62E+, CD144+ or CD144+CD105+ particles, smaller than 1.0 μm. Results: High speed centrifugation led to a significant loss of CD31+CD42b- EMP (p = 0.004). A good correlation between PPP and high speed centrifuged PPP was only found for CD144+ EMP (Kendall tau b = 0.611, p = 0.025). Storage at 4 °C did not affect EMP quantification. However, freezing at -80 °C increased CD31+CD42b- and CD62E+ EMP counts, and lowered CD144+ EMP (p b 0.05). Nevertheless, the agreement among the different storage conditions was relatively good (Kendall coefficient of concordance N 0.487; p b 0.05). Conclusion: The flow cytometric detection of EMP varies with the centrifugation protocol and the storage method used, and these changes also depend on the phenotype studied. The results of this study caution against comparing study results gathered with different EMP laboratory protocols. © 2010 Elsevier Ltd. All rights reserved. Introduction Endothelial microparticles (EMP) are small membrane vesicles, b 1.0 μm diameter, released from activated, damaged and apoptotic endothelial cells [1]. EMP can be characterized by the presence of endothelial-specific surface antigens. These membrane markers vary with the microparticle (MP) generating process: CD31+, CD105+ and Annexin V+ EMP are generated mainly during apoptosis; while CD62E, CD54 and CD106 expression are increased on EMP released upon activation [2,3]. To date, growing evidence indicates that EMP are more than simple markers of the endothelial status [1,4]. EMP likely have an important function in various physiological processes, including the modulation of inflammation, coagulation and vascular function [1,4]. As such, there is now growing interest in the detection of EMP as a novel biomarker of endothelial disturbance. Circulating EMP are commonly measured in plasma samples by flow cytometry, due to its ability to perform multiparametric analysis of a high number of events in a very short time period [5,6].A generally accepted uniform flow cytometry protocol to enumerate EMP is lacking. Moreover, reliable EMP enumeration poses a technical challenge, as their size is near the detection limit of currently available flow cytometers, resulting in different instrument settings [5,7]. In their search for the superior marker in a particular disease, laboratories have used diverse combinations of antibodies for the detection of EMP [5,6]. Finally, there is no inter-laboratory agreement on the pre-analytic preparation of plasma samples [5,6]. In fact the anticoagulant, needle bore size, centrifugation technique, storage time and temperature have been shown to affect the flow cytometric Thrombosis Research 125 (2010) 332–339 Abbreviations: ACD, acid citrate dextrose; EMP, endothelial microparticles; FITC, fluorescein isothiocyanate; MP, microparticle; PBS, phosphate buffered saline; PE, phycoerythrin; PPP, platelet poor plasma. ☆ The results were partially presented as an abstract on the 5th European meeting on Vascular Biology and Medicine, Marseille 14th-17th September 2009. ⁎ Corresponding author. Antwerp University Hospital, Wilrijkstraat 10, 2650 Edegem. Tel.: +32 3 8215248; fax: +32 3 8284882. E-mail address: sabrina.vanierssel@ua.ac.be (S.H. van Ierssel). 0049-3848/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2009.12.019 Contents lists available at ScienceDirect Thrombosis Research journal homepage: www.elsevier.com/locate/thromres