Standardizing Flow Cytometry Immunophenotyping Analysis from the Human ImmunoPhenotyping Consortium

Greg Finak, Marc Langweiler, Maria Jaimes, Mehrnoush Malek, Jafar Taghiyar, Yael Korin, Khadir Raddassi, Lesley Devine, Gerlinde Obermoser, Marcin L. Pekalski, Nikolas Pontikos, Alain Diaz, Susanne Heck, Federica Villanova, Nadia Terrazzini, Florian Kern, Yu Qian, Rick Stanton, Kui Wang, Aaron BrandesJohn Ramey, Nima Aghaeepour, Tim Mosmann, Richard H. Scheuermann, Elaine Reed, Karolina Palucka, Virginia Pascual, Bonnie B. Blomberg, Frank Nestle, Robert B. Nussenblatt, Ryan Remy Brinkman, Raphael Gottardo, Holden Maecker, J. Philip McCoy

Research output: Contribution to journalArticlepeer-review

149 Scopus citations


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.

Original languageEnglish (US)
Article number20686
JournalScientific reports
StatePublished - Feb 10 2016

ASJC Scopus subject areas

  • General


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