Calcium-phosphate microprecipitates mimic microparticles when examined with flow cytometry

Area of Science:

• Biochemistry
• Analytical Chemistry
• Hematology

Background:

• Circulating microparticles (MPs) are elevated in various disease states.
• Flow cytometry is a key technique for MP analysis, but distinguishing MPs from artifacts is challenging.
• Annexin V, a phosphatidylserine-binding protein, is commonly used to identify MPs, requiring millimolar calcium ion concentrations.

Purpose of the Study:

• To investigate the potential for calcium-phosphate microprecipitates to interfere with microparticle detection via flow cytometry.
• To assess the impact of calcium concentration on the flow cytometry signal of calcium-phosphate microprecipitates.
• To provide recommendations for avoiding artifacts in MP analysis.

Main Methods:

• Calcium-phosphate microprecipitates were generated by titrating calcium ions into phosphate-buffered saline (PBS).
• Microprecipitates and microparticles from aged donor blood were analyzed using flow cytometry.
• The effect of calcium chelators and fluorescently labeled antibodies on microprecipitate signals was examined.

Main Results:

• Calcium-phosphate microprecipitates exhibited flow cytometry signals overlapping with those of microparticles.
• Microprecipitate signals increased with calcium ion concentration and were abolished by calcium chelation.
• Fluorescent signals in biological samples showed an apparent increase with calcium, returning to baseline after chelation.

Conclusions:

• Calcium-phosphate microprecipitates can generate artifacts that mimic microparticle signals in flow cytometry.
• Avoiding phosphate-containing buffers or ensuring adequate calcium chelation is essential when using annexin V for MP detection.
• Flow cytometry may be useful for analyzing crystalline structures in biological fluids.