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Calcium‐phosphate microprecipitates mimic microparticles when examined with flow cytometry
Author(s) -
Larson Michael C.,
Luthi Maia R.,
Hogg Neil,
Hillery Cheryl A.
Publication year - 2013
Publication title -
cytometry part a
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.316
H-Index - 90
eISSN - 1552-4930
pISSN - 1552-4922
DOI - 10.1002/cyto.a.22222
Subject(s) - flow cytometry , calcium , annexin , phosphatidylserine , chemistry , phosphate , chelation , cytometry , fluorescence , biophysics , biochemistry , microbiology and biotechnology , biology , phospholipid , physics , organic chemistry , quantum mechanics , membrane
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. © 2012 International Society for Advancement of Cytometry