Aim
To determine if forward (FSC) or side scatter (SSC) is the better sizing parameter for enumeration of circulating microvesicles (MV) by flow cytometry.
Method A FACS Canto flow cytometer (BD Biosciences, San Jose, CA, USA) was optimised for MV analysis using either FSC or SSC by calibration with Megamix or Megamix-Plus SSC beads (Biocytex, Marseille, France), respectively. A series of 74 platelet free plasma samples from patients with type II diabetes were analysed for MV expressing phosphatidylserine, CD41, CD235, CD31/CD42b, CD105 and CD62e. Events were acquired for 120s on low flow rate and a known amount of CountBright counting beads (Molecular Probes, Eugene, OR, USA) were included in each sample to enable calculation of absolute MV [MV/ul = (MV count/bead count) x (total # beads/test volume)]. Non parametric tests were used to determine differences and correlations between FSC and SSC results. A p-value <0.05 was considered significant.
Results The Canto was able to adequately resolve 0.5 um beads by FSC and 0.16 um beads by SSC, but there were significantly more background events using SSC compared to FSC (3113, 2098-23860 vs 470, 404-3994; p=0.008). In general, sample analysis by SSC resulted in significantly higher numbers of MV (p<0.0001) but was nonetheless well correlated to enumeration by FSC for all MV subtypes (ρ=0.62-0.89, p<0.000001). However, there were significantly less counting beads enumerated when SSC was used (730, 595-826 vs 766,649-882; p=0.000001) and this was negatively correlated to the number of events in the MV gate (ρ=-0.366; p=0.001).
Conclusion Although SSC may provide better resolution of small MV; high background negatively affects counting bead enumeration and overall MP calculations. Strategies to reduce SSC background would have to be employed in order to reliably use this technique.