Standardization of immunological assays, including flow cytometry, in terms of reagents, sample handling, instrument setup, and data analysis, is essential for successful cross-study and cross-center analysis in order to mitigate the effects of technical variability in assay results. The Human Immunology Project and the Federation of Clinical Immunology Societies (FOCIS) have partnered to develop five standardized, lyophilized, eight-color staining reagent panels (termed lyoplates) for this purpose. In collaboration with the FlowCAP consortium, standardized samples (Cytotrol control cells) were distributed to nine participating centers and analyzed by flow cytometry using the lyoplate reagents and SOP's to minimize experimental variability. Data from two of these panels (T-cell and B-cell) were entered into the FlowCAP-III challenge, where participants analyzed the data using automated gating methods for comparison against cell population statistics for major T and B-cell subsets as defined by a consensus manual gating scheme. This evaluation showed that several automated gating algorithms could successfully recapitulate centralized manual gating statistics for T-cell and B-cell subsets with little statistical bias, and with within-center and between-center variability as low or lower than centralized manual gating. These results demonstrate that automated gating algorithms are ready for use in performing reproducible analyses and comparisons of immunological data.
- Copyright © 2013 by The American Association of Immunologists, Inc.