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Analysis of synthetic and biological microparticles on several flow cytometric platforms***
Citation:
Fisher, N., M. Mooberry, M. S. CARRAWAY, R. Kasthuri, B. Udis, R. Zzucker, AND N. Key. Analysis of synthetic and biological microparticles on several flow cytometric platforms***. Presented at International Society for Advancement of Cytometry Meeting, Leipzig, GERMANY, June 23, 2012.
Impact/Purpose:
The emerging field of MP analysis is in its infancy, and has great potential for understanding pathophysiology of disease processes and for development as a biomarker in diagnosing or monitoring treatment. The particular application of the MP detection is an important consideration when interpreting results and selecting an instrument for MP work
Description:
Biological microparticles (MPs) are potentially important biomarkers for thrombosis, cancer, glomerulonephritis and other disease states. These MPs are generally accepted to be membrane vesicles extruded following cellular activation. While human blood cells range from 10-15 microns and platelets range from 1-3 microns, MPs are defined as membrane-containing particles smaller than 1 micron. They are detected in human plasma following centrifugation to remove nucleated cells, erythrocytes and platelets. Untilrecently, the limit of detection of common analytical flow cytometers was not much below 1micron. Asknowledge of the clinical relevance of MPs has emerged, we have seen advances in instrumentation that enable detection of these small particles even below 500 nm based on light scatter. Typically, investigators have used reference beads to define size ranges and to standardize the scatter gating of the MPs. These beads of known size differ considerably in refractive index from biological MPs. This difference in refractive index affects the interpretation of MP scatter with regard to the reference beads on various cytometers, which varies based on the particular method of amplifying scatter signals. We compared instruments with FS PMT amplification of scatter (Becton Dickinson LSR II and FACS Canto) and FS Diode (Stratedigm S1000) and wide-angle diode detection (Beckman Gallios). We measured forward and side scatter of MPs in platelet poor plasma using both standard reference beads (Megamix™ from Biocytex) and a cocktail of 1000, 800, 500 and 300 nm beads (Becton Dickinson) spiked with 200 om Bangs reference beads in addition to beads from Spherotech and Thermo. Although the forward scatter PMT detector improved the resolution and decreased the noise of the system, the primary scatter signal that detected differences in size of polystyrene microparticles below 300 om was the side scatter signal on all the machines that could detect them. Our data illustrate the differences in size gating between bead and biological microparticles, which is influenced by the method of scatter detection. Increasing refractive index of the particles significantly increased the signal intensity, as predicted by MiePlot analysis. While some instruments showed excellent detection of highly refractive particles, others were more sensitive for the measurement of biological MPs, which have a much lower refractive index. The emerging field of MP analysis is in its infancy, and has great potential for understanding pathophysiology of disease processes and for development as a biomarker in diagnosing or monitoring treatment. The particular application of the MP detection is an important consideration when interpreting results and selecting an instrument for MP work