Assessment of In Vitro-Generated Platelet Microparticles Using a Modified Flow Cytometric Strategy

Abstract

Quantification of platelet microparticles (PMPs) may be a useful marker for the detection of in vivo platelet activation. Optimisation of flow cytometric methods for detection and quantification of PMPs has not been systemically evaluated. This study reports the optimisation of flow cytometric procedures for the detection of PMPs, the determination of limits of size detection using microbeads, and the characterisation of PMP generation by in vitro 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 forward scatter (FS) threshold parameter (size) of the flow cytometer using microbeads allowed for the detection of very small particles (down to 0.1 μm diameter). PMPs generated in vitro using ADP and collagen were reliably detected 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., platelet Gp MAb-labelled events) region. PMPs of different size profiles were observed for each of the agonists. Flow cytometry can be used as a tool in 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 procedure setup and performance.