Calcium-phosphate microprecipitates mimic microparticles when examined with flow cytometry

Authors

  • Michael C. Larson,

    Corresponding author
    1. Department of Biophysics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
    2. Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, Wisconsin 53226
    • Department of Biophysics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA
    Search for more papers by this author
  • Maia R. Luthi,

    1. Department of Biomedical Sciences, Marquette University, Milwaukee, Wisconsin 53233
    Search for more papers by this author
  • Neil Hogg,

    1. Department of Biophysics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
    Search for more papers by this author
  • Cheryl A. Hillery

    1. Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, Wisconsin 53226
    2. Department of Pediatrics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
    Search for more papers by this author

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. © 2012 International Society for Advancement of Cytometry

Ancillary