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Analysis of synthetic and biological microparticles on several flow cytometric platforms
Citation:
Fisher, N., M. Mooberry, M. S. CARRAWAY, C. BAILEY, R. Kasthuri, B. Udis, R. Zucker, AND N. Key. Analysis of synthetic and biological microparticles on several flow cytometric platforms. Presented at International Society for Thrombosis and Hemostasis, Liverpool, UK, June 27 - 30, 2012.
Impact/Purpose:
The emerging field of MP analysis has great potential to improve our understanding of pathologic processes and to aid in discovery of novel biomarkers in disease. The particular application of MP detection is an important consideration when interpreting results and selecting an instrument for MP work.
Description:
Microparticles (MPs) are membrane vesicles (0.1 to 1 urn) released from cells upon activation. The limit of detection ofmost standard flow cytometers is just below 1 urn. Recent advances enable detection of particles lower than 0.5 urn, Synthetic. beads are used to define size ranges and to standardize the MP gate. Because beads differ in refractive index (RI) from biological MPs, the interpretation of MP scatter in relation to beads on various cytometers varies with the particular method used to amplify scatter signals. MPs in plasma and MPs derived from platelets activated with ionophore were characterized and enumerated using counting beads, standard reference beads (Megamix" , Biocytex), and a cocktail of 1000, 800,500, and 300 nm beads (BD) spiked with 200 nm Bangs reference beads. We compared flow cytometerswith forward scatter (FSC) photomultiplier tube (PMT) amplification of scatter (BD FACS Canto), FSC diode (Stratedigm SI000), and wide-angle diode detection (Beckman Gallios). Although FSC PMT detector improved resolution and decreased background noise, the primary scatter signal that detected differences in size of polystyrene microparticles below 300 nm was SSC. Our data illustrate the differences in size gating between bead and biological MPs, which is influenced by the method of scatter detection. Increasing RI ofthe particles significantly increased the signal intensity, as predicted by MiePlot analysis. While some instruments had excellent detection of highly refractive particles, others were more sensitive for the measurement of biological MPs, which have a much lower RI. The emerging field of MP analysis has great potential to improve our understanding of pathologic processes and to aid in discovery of novel biomarkers in disease. The particular application of MP detection is an important consideration when interpreting results and selecting an instrument for MP work. This abstract does not reflect EPA policy.