Abstract
Microparticles, which include exosomes, microโvesicles, apoptotic bodies and apoptotic microparticles, are small (0.05 โ 3 ฮผm in diameter), membranous vesicles that can contain DNA, RNA, miRNA, intracellular proteins and express extracellular surface markers from the parental cells. They can be secreted from intracellular multivesicular bodies or released from the surface of blebbing membranes. Circulating microparticles are abundant in the plasma of normal individuals and can be derived from circulating blood cells such as platelets, red blood cells and leukocytes as well as from tissue sources, such as endothelial and placental tissues. Elevated levels of microparticles are associated with various diseases such as thrombosis (platelet microparticles), congestive heart failure (endothelial microparticles), breast cancer patients (leukocyte microparticles) and women with preeclampsia (syncytiotrophoblast microparticles). Although microparticles can be detected by microscopy, enzymeโlinked immunoassays and functional assays, flow cytometry is the preferred method because of the ability to quantitate (fluorescent beadโ or flow rateโbased method) and because of polychromatic capabilities. However, standardization of preโanalytical and analytical modus operandi for isolating, enumerating and fluorescent labeling of microparticles remains a challenge. The primary focus of this article is to review the preliminary steps required to optimally study circulating in vivo microparticles which include: 1) centrifugation speed used, 2) quantitation of microparticles before antibody labeling, 3) levels of fluorescence intensity of antibodyโlabeled microparticles, 4) polychromatic flow cytometric analysis of microparticle subโpopulations and 5) use of polyclonal antibodies designed for Western blotting for flow cytometry. These studies determine a roadmap to develop microparticles as biomarkers for a variety of conditions. ยฉ 2010 International Society for Advancement of Cytometry