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Standardizing Flow Cytometry Immunophenotyping Analysis from the Human ImmunoPhenotyping Consortium.


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.

SUBMITTER: Finak G 

PROVIDER: S-EPMC4748244 | biostudies-other | 2016 Feb

REPOSITORIES: biostudies-other

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Standardizing Flow Cytometry Immunophenotyping Analysis from the Human ImmunoPhenotyping Consortium.

Finak Greg G   Langweiler Marc M   Jaimes Maria M   Malek Mehrnoush M   Taghiyar Jafar J   Korin Yael Y   Raddassi Khadir K   Devine Lesley L   Obermoser Gerlinde G   Pekalski Marcin L ML   Pontikos Nikolas N   Diaz Alain A   Heck Susanne S   Villanova Federica F   Terrazzini Nadia N   Kern Florian F   Qian Yu Y   Stanton Rick R   Wang Kui K   Brandes Aaron A   Ramey John J   Aghaeepour Nima N   Mosmann Tim T   Scheuermann Richard H RH   Reed Elaine E   Palucka Karolina K   Pascual Virginia V   Blomberg Bonnie B BB   Nestle Frank F   Nussenblatt Robert B RB   Brinkman Ryan Remy RR   Gottardo Raphael R   Maecker Holden H   McCoy J Philip JP  

Scientific reports 20160210


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 laboratorie  ...[more]

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