PB1069 - A standardized approach in enumerating microparticles in the Framingham Heart Study using quantitative flow cytometry

Background: Background
Platelet-derived-microparticles (PDMPs) and erythrocyte-derived-microparticles (Erythro-MPs) represent the majority of microparticles detectable in human plasma and are considered as prothrombotic. Flow cytometry allows for the rapid measurement of microparticles, and numerous efforts have been made to standardize the enumeration of PDMPs using flow cytometry.

Aims: To enumerate MPs using a standardized quantitative flow cytometry-based assay

Methods: Citrated whole blood samples were collected from Framingham Heart Study participants (n=279). We used two commercially available microparticle calibration beads with a diameter ranging from 0.16–0.50 µm (from Stago and Spherotech) to optimize the BD Symphony A1 flow cytometer equipped with a microparticle module. To monitor the performance of the SSC-triggered MP parameter, an optimal SSC MP separation index (S.I.) (between bead equivalent size range of 0.16-0.20 µm) of ~4 was set. The electronic noise of each fluorescence channel was monitored, and negative events were set at 2.5 standard deviations away from the electronic noise. The standardized quantitative flow cytometry panel consisted of the lineage markers CD61 FITC (PDMP), CD235a BV786 (Erythro-MPs), and CD31 APC-Cy7 (endothelial-derived MPs).

Results: The S.I. for the SSC-triggered MP threshold was 4.350.14, and the electronic noise for the fluorescence channel used to detect was 17.3 for PDMPs, 16.5 for erythro-MPs and 14.9 for endothelial-derived MPs. Using the standardized SSC trigger threshold, PDMPs were reproducibly detectable at an absolute fluorescence intensity of 6956 MEFL, Erythrocyte derived MPs were detectable at 1017 MEBV786 and endothelial-derived MPs at 1526 MEAPCY7.

Conclusion(s): The reporting of instrument performance and microparticle trigger threshold monitoring may be essential in the reproducibility and reporting of large-scale MP data. The use of calibration beads and reporting on the sensitivity of detecting microparticles may provide a useful tool in assessing the quality of acquired flow cytometry-derived MP data in large population-based studies.