Assessment of in vitro-generated platelet microparticles using a modified flow cytometric strategy

Quantification of platelet microparticles (PMPs) may be a useful marker for the detection of in vivo platelet activation. Optimisation of flow cytomet ric methods for detection and quantification of PMPs has not been systemica lly evaluated. This study reports the optimisation of flow cytometric proce dures for the detection of PMPs, the determination of limits of size detect ion using microbeads, and the characterisation of PMP generation by in vitr o activation of platelets using collagen and adenosine 5' diphosphate (ADP) . Fluorescent and plain microbeads proved useful for defining the limits of the flow cytometer in detecting PMPs. A systematic calibration of the forw ard scatter (FS) threshold parameter (size) of the flow cytometer using mic robeads allowed for the detection of very small particles (down to 0.1 mum diameter). PMPs generated in vitro using ADP and collagen were reliably det ected by flow cytometry using monoclonal antibodies (MAb) directed towards platelet surface membrane glycoproteins (Gp). The PMP events were detected in the FS low (i.e., small size events) and fluorescence (FL) high (i.e., p latelet Gp MAb-labelled events) region. PMPs of different size profiles wer e observed for each of the agonists. Flow cytometry can be used as a tool i n the assessment of PMPs. As detection of particles of this type is at the limit of resolution of flow cytometers, careful attention is required with the choice of platelet-specific MAb, isotype control, and optimisation of p rocedure setup and performance. (C) 2001 Elsevier Science Ltd. All rights r eserved.