Bio‐maleimide as a generic stain for detection and quantitation of microparticles

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Abstract
Microparticles (MP) are small fragments of cytoplasm shed from a cell surface and their role in the pathophysiology of disease is being extensively investigated. A novel staining technique for quantifying total MP in peripheral blood was evaluated in this study. Evaluation of Bodipy-maleimide (or bio-maleimide) as a stain for quantifying total MP in peripheral blood by flow cytometry. Samples were obtained from 10 healthy donors after informed consent. Plasma was prepared by sequential centrifugation at 1500 g followed by 13,000 g and stained with Annexin V and bio-maleimide. Enumeration beads were added after 15 min of incubation with the stain and samples analyzed on a FACS Canto flow cytometer. Detection and quantification of MP by bio-maleimide staining was comparable with that by Annexin V. The total mean MP level with bio-maleimide staining was 34 +/- 19.7/microl (range of 11.6-68.1/microl) and with Annexin V staining it was 38.9 +/- 29.8/microl (range of 10.6 to 112.9/microl). There was no significant difference using a paired t-test and methods were comparable using a Bland-Altman plot. Bio-maleimide is a useful and inexpensive stain to measure total MP levels in peripheral blood by flow cytometry. This technique could be employed to study thrombotic risks in a variety of disease states.