Calcium-phosphate microprecipitates mimic microparticles when examined with flow cytometry.

ABSTRACT

There are increased levels of circulating microparticles (MPs) in several disease states. Flow cytometry is a common method to examine MPs, but their small size necessitates the use of markers to distinguish specifically MPs from artifact. Annexin V, which binds phosphatidylserine, is a commonly used marker for MP detection. Annexin V requires millimolar calcium ion for optimum binding. Ca(++) can precipitate with phosphate in phosphate-buffered saline (PBS). Calcium-phosphate microprecipitates were formed by titrating Ca(++) into PBS and examined using flow cytometry. Calcium-phosphate microprecipitates were compared with MPs derived from aged donor blood units. Microprecipitates were ∼0.7-0.9 µm in diameter compared with standard beads of known size. The microprecipitates disappeared with the addition of Ca(++) chelator. When we added fluorescently labeled antibodies to microprecipitates, the median fluorescent signal increased with increasing Ca(++) concentration regardless of specificity of the antibody. When repeated with a biological sample, there was an apparent increase in the fluorescent signal that returned to baseline after Ca(++) chelation. The flow cytometry signal of calcium-phosphate microprecipitates overlaps with the MP signal. Since Ca(++) is essential for annexin V binding, it is essential to avoid artifacts from calcium-phosphate microprecipitates when using any buffer or biological fluid containing phosphate. This also highlights the potential utility of flow cytometry for the analysis of crystals in biological fluids.