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

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    Author
    Alkhatatbeh, MJ; Enjeti, AK; Baqar, S; Ekinci, EI; Liu, D; Thorne, RF; Lincz, LF
    Date
    2018-01
    Source Title
    Journal of Circulating Biomarkers
    Publisher
    Aboutscience Srl
    University of Melbourne Author/s
    Ekinci, Elif; Baqar, Sara
    Affiliation
    Medicine and Radiology
    Medical Education
    Metadata
    Show full item record
    Document Type
    Journal Article
    Citations
    Alkhatatbeh, M. J., Enjeti, A. K., Baqar, S., Ekinci, E. I., Liu, D., Thorne, R. F. & Lincz, L. F. (2018). Strategies for enumeration of circulating microvesicles on a conventional flow cytometer: Counting beads and scatter parameters.. J Circ Biomark, 7, pp.1849454418766966-. https://doi.org/10.1177/1849454418766966.
    Access Status
    Open Access
    URI
    http://hdl.handle.net/11343/253596
    DOI
    10.1177/1849454418766966
    Open Access at PMC
    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5894907
    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.

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