Strategies for enumeration of circulating microvesicles on a conventional flow cytometer Counting beads and scatter parameters

Mohammad J Alkhatatbeh; Anoop K Enjeti; Sara Baqar; Elif I Ekinci; Dorothy Liu; Rick F Thorne; Lisa F Lincz

doi:10.33393/jcb.2018.2087

Authors

Mohammad J Alkhatatbeh Department of Clinical Pharmacy, Faculty of Pharmacy, Jordan University of Science and Technology, Irbid, Jordan
Anoop K Enjeti Pathology North Hunter, NSW Health Pathology, New South Wales, Australia
Sara Baqar Department of Medicine, Austin Health, The University of Melbourne, Victoria, Australia
Elif I Ekinci Department of Medicine, Austin Health, The University of Melbourne, Victoria, Australia
Dorothy Liu Department of Medicine, Austin Health, The University of Melbourne, Victoria, Australia
Rick F Thorne Faculty of Health and Medicine, University of Newcastle, New South Wales, Australia
Lisa F Lincz Faculty of Health and Medicine, University of Newcastle, New South Wales, Australia

DOI:

https://doi.org/10.33393/jcb.2018.2087

Keywords:

Flow cytometry, absolute counting, microvesicle, microparticles, extracellular vesicles, submicron particles, scatter

Abstract

Enumeration of circulating microvesicles (MVs) by conventional flow cytometry is accomplished by the addition of a known amount of counting beads and calculated from the formula: MV/μl = (MV count/bead count) × final bead concentration. We sought to optimize each variable in the equation by determining the best parameters for detecting ‘MV count’ and examining the effects of different bead preparations and concentrations on the final calculation. Three commercially available bead preparations (TruCount, Flow-Count and CountBright) were tested, and MV detection on a BD FACSCanto was optimized for gating by either forward scatter (FSC) or side scatter (SSC); the results were compared by calculating different subsets of MV on a series of 74 typical patient plasma samples. The relationship between the number of beads added to each test and the number of beads counted by flow cytometry remained linear over a wide range of bead concentrations (R2 ≥ 0.997). However, TruCount beads produced the most consistent (concentration variation = 3.8%) calculated numbers of plasma CD41+/Annexin V+ MV, which were significantly higher from that calculated using either Flow-Count or CountBright (p < 0.001). The FACSCanto was able to resolve 0.5 μm beads by FSC and 0.16 μm beads by SSC, but there were significantly more background events using SSC compared with FSC (3113 vs. 470; p = 0.008). In general, sample analysis by SSC resulted in significantly higher numbers of MV (p < 0.0001) but was well correlated with enumeration by FSC for all MV subtypes (ρ = 0.62–0.89, p < 0.0001). We conclude that all counting beads provided linear results at concentrations ranging from 6 beads/μl to 100 beads/μl, but TruCount was the most consistent. Using SSC to gate MV events produced high background which negatively affected counting bead enumeration and overall MV calculations. Strategies to reduce SSC background should be employed in order to reliably use this technique.