Standardizing Flow Cytometry Immunophenotyping Analysis from the Human ImmunoPhenotyping Consortium.

Sci Rep
Authors
Keywords
Abstract

Standardization of immunophenotyping requires careful attention to reagents, sample handling, instrument setup, and data analysis, and is essential for successful cross-study and cross-center comparison of data. Experts developed five standardized, eight-color panels for identification of major immune cell subsets in peripheral blood. These were produced as pre-configured, lyophilized, reagents in 96-well plates. We present the results of a coordinated analysis of samples across nine laboratories using these panels with standardized operating procedures (SOPs). Manual gating was performed by each site and by a central site. Automated gating algorithms were developed and tested by the FlowCAP consortium. Centralized manual gating can reduce cross-center variability, and we sought to determine whether automated methods could streamline and standardize the analysis. Within-site variability was low in all experiments, but cross-site variability was lower when central analysis was performed in comparison with site-specific analysis. It was also lower for clearly defined cell subsets than those based on dim markers and for rare populations. Automated gating was able to match the performance of central manual analysis for all tested panels, exhibiting little to no bias and comparable variability. Standardized staining, data collection, and automated gating can increase power, reduce variability, and streamline analysis for immunophenotyping.

Year of Publication
2016
Journal
Sci Rep
Volume
6
Pages
20686
Date Published
2016 Feb 10
ISSN
2045-2322
URL
DOI
10.1038/srep20686
PubMed ID
26861911
PubMed Central ID
PMC4748244
Links
Grant list
100140 / Wellcome Trust / United Kingdom
5U19AI090019 / AI / NIAID NIH HHS / United States
U19 AI089986 / AI / NIAID NIH HHS / United States
U19 AI090019 / AI / NIAID NIH HHS / United States
Intramural NIH HHS / United States
R01 EB008400 / EB / NIBIB NIH HHS / United States