Quantification and Characterization of Platelet Microparticles

Blood (10 pL) preferably anticoagulated with EDTA/CTAD is incubated for 5 min at 4°C with FITC-isotype control (2l) and PE-isotype control (2l) or with FITC-CD42a (2l) and PE-CD62P (2l). Samples are diluted to 1 mL with HBSS-BSA containing LDS-751 (as above).

Unlabeled polystyrene spheres 1.09 m in diameter (at 1 X 10"6 from the stock solution supplied Sigma-Aldrich) and EDTA/CTAD at 0.25 of the concentration used for preventing blood coagulation are added and analyzed immediately by flow cytometry.

Fig. 7. Analysis of platelet microparticles and platelets. The blood sample was stained with fluorescein isothiocyanate (FITC)-conjugated CD42a and phycoerythrin (PE)-conjugated CD62P, and events were displayed in a plot of side light scatter (logarithmic scale, ordinate) and forward light scatter (logarithmic scale, abscissa; dot plot A). To assess CD62P expression and platelet and platelet microparticle numbers, one gate (A) was set to encompass platelets and putative platelet microparticles and another (B) was set to encompass just the 1.09-m-diameter polystyrene beads (dot plot A). The events from A and B were counted and displayed in frequency histograms of forward light scatter (logarithmic scale); those from B are not illustrated in plot B, and those from A are shown in plot C. The value obtained for the mean forward light scatter signal of the 1.09-m-diameter beads (from B) was used to set the position of the cursor (in plot B) used to divide the events from A into putative microparticles (C) and platelets (D). Plot B shows the distribution of normal platelets, and plot D shows the distribution of aged blood (48 h), in which high numbers of microparticles have formed. The gated events in regions C and D are displayed in plots of PE-CD62P fluorescence (FL2; logarithmic scale, ordinate) versus FITC-CD42a fluorescence (FL1; logarithmic scale, abscissa; plots E and F, respectively). CD42a+ events were considered microparticles (plot E) and platelets (plot F), and the percentages of these that were also CD62P+ and their mean fluorescence intensity were recorded.

Labeled samples are first displayed in a plot of SSC (logarithmic scale) versus forward light scatter (logarithmic scale) (Fig. 7). Events with the light scatter characteristics of platelets and putative platelet microparticles are displayed in a

Fig. 8. The flow cytometric analysis of platelet leukocyte aggregates (PLAs) in whole blood. The cells in blood are analyzed for LDS-751 expression, and leukocytes positive for this are gated by A in histogram A to exclude red cells and platelets. These events are then backgated to a histogram of forward scatter and side scatter (histogram B). Amorphous regions B, C, and D are set around the lymphocytes, monocytes, and neutrophils, respectively. These are then analyzed in a dual-fluorescence histogram of PE-CD62P and FITC-CD42a. Events in regions B-D that are positive for CD42a are considered to be platelet leukocyte conjugates. Single positive events are considered to be platelet-free leukocytes. The relative percentage of platelet leukocyte conjugates may then be determined. The use of CD62P allows the identification of activated platelets bound to the leukocytes. Quantum Red-conjugated CD3, CD14, and CD16 may also be used to identify lymphocytes, monocytes, and neutrophils, respectively.

Fig. 8. The flow cytometric analysis of platelet leukocyte aggregates (PLAs) in whole blood. The cells in blood are analyzed for LDS-751 expression, and leukocytes positive for this are gated by A in histogram A to exclude red cells and platelets. These events are then backgated to a histogram of forward scatter and side scatter (histogram B). Amorphous regions B, C, and D are set around the lymphocytes, monocytes, and neutrophils, respectively. These are then analyzed in a dual-fluorescence histogram of PE-CD62P and FITC-CD42a. Events in regions B-D that are positive for CD42a are considered to be platelet leukocyte conjugates. Single positive events are considered to be platelet-free leukocytes. The relative percentage of platelet leukocyte conjugates may then be determined. The use of CD62P allows the identification of activated platelets bound to the leukocytes. Quantum Red-conjugated CD3, CD14, and CD16 may also be used to identify lymphocytes, monocytes, and neutrophils, respectively.

frequency histogram of forward light scatter. Those with forward light scatter signals greater than that of 1.09-m-diameter polystyrene beads are considered platelets, whereas those with lesser signals are considered putative micro-particles. The gated events (i.e., platelets and putative platelet microparticles) are displayed in plots of PE-CD62P fluorescence versus FITC-CD42a fluorescence; CD42a+ events are considered platelets or platelet microparticles, and PE-CD62P fluorescence data are recorded.

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