Calcium-phosphate microprecipitates mimic microparticles when examined with flow cytometry. Cytometry A 2013 Feb;83(2):242-50
Date
11/06/2012Pubmed ID
23125136Pubmed Central ID
PMC3615643DOI
10.1002/cyto.a.22222Scopus ID
2-s2.0-84873873127 (requires institutional sign-in at Scopus site) 35 CitationsAbstract
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.
Author List
Larson MC, Luthi MR, Hogg N, Hillery CAAuthor
Neil Hogg PhD Senior Associate Dean, Professor in the Biophysics department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
Annexin A5Artifacts
Calcium Phosphates
Cell-Derived Microparticles
Chemical Precipitation
Flow Cytometry
Fluorescent Antibody Technique
Humans
Light
Particle Size
Phosphatidylserines
Protein Binding
Reference Standards
Scattering, Radiation
