A heparin-based method for flow cytometric analysis of microparticles directly from platelet-poor plasma in calcium containing buffer

Abstract

Background: Characterization of circulating microparticles (MPs) is usually performed by flow cytometry. Annexin V, a protein that Ca(2+)-dependently binds to phosphatidylserine, has been used to define entire microparticle (MP) populations, but not all MPs bind AnxV. Recent reports have correlated AnxV negative MPs to clinical parameters in systemic diseases, which emphasize the importance of including characterization of AnxV-binding. An obstacle in flow cytometric analysis of AnxV-binding to MPs is that plasma may clot when adding the Ca(2+)-containing buffers. We here devise a simple method for comprehensive assessment of circulating MPs directly from platelet-poor plasma with characterization of AnxV-binding and of cellular origin of MPs.

Materials and methods: With 49 samples (20 healthy controls and 29 SLE patients) a flow cytometric method analyzing MPs directly from platelet-poor plasma was developed and compared with an established, more laborious method. The method relies on using heparin to inhibit plasma coagulation induced by Ca(2+) and subsequent incubation with labeling reagents directly in platelet-poor plasma.

Results: In comparison with standard methods the new direct method showed low variability (1-12% in total MP measurement), had higher MP counts (i.e. prevents loss of MPs), was less time consuming (saved 2/3 of sample processing time) and results correlated well with an established method of analysis of washed MPs by flow cytometry (r>0.7; p<0.0001). Additionally heparin was shown not to impact MP counts, longtime storage did not alter MP concentrations, and distinct effects of freezing on MP characteristics were confirmed.

Conclusions: The direct method reduces variability due its simplicity and faster handling, and it saves sample material. It is a convenient, fast, and reproducible method for assessing the population of circulating MPs and correlates well with more cumbersome approaches. These benefits make the method well suited for large studies.