Development of a time-resolved immunofluorometric assay for quantifying platelet-derived microparticles in human plasma

Platelet-derived microparticles (PMPs) are considered a marker of platelet activation. They vary considerably in size, and flow cytometry, the predominant method used to assay PMPs, is only detecting larger PMPs (> 0.1 mu m). We describe here a method that quantifies the amount of PMP-located GPIIb antigen in detergent-treated platelet-free plasma (PPP) by means of a one-step time-resolved immunofluorometric assay (TR-IFMA). This assay uses a streptavidin-coated microwell plate and two different monoclonal antibodies to GPIIb (CD41), one conjugated to biotin and the other labeled with europium ion. A wide linear range standard curve with low background and a high sensitivity was obtained. Pre-assay ultracentrifugation or filtration of PPP extensively reduced the fluorometric signal, indicating that the GPIIb antigen is mainly particle-located. A strong correlation between the amount of GPIIb and PMP as detected by flow cytometry was found. Consequently, the assay can be used to study PMP-related phenomena and, in contrast to flow cytometry, can be used on frozen samples and is independent of PMP size. (c) 2005 Elsevier Ltd. All rights reserved.