Dataset Information

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-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Publications

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]

PMID: 26861911

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Project description:This 40-color flow cytometry-based panel was developed for in-depth immunophenotyping of the major cell subsets present in human peripheral blood. Sample availability can often be limited, especially in cases of clinical trial material, when multiple types of testing are required from a single sample or timepoint. Maximizing the amount of information that can be obtained from a single sample not only provides more in-depth characterization of the immune system but also serves to address the issue of limited sample availability. The panel presented here identifies CD4 T cells, CD8 T cells, regulatory T cells, γδ T cells, NKT-like cells, B cells, NK cells, monocytes and dendritic cells. For each specific cell type, the panel includes markers for further characterization by including a selection of activation and differentiation markers, as well as chemokine receptors. Moreover, the combination of multiple markers in one tube might lead to the discovery of new immune phenotypes and their relevance in certain diseases. Of note, this panel was designed to include only surface markers to avoid the need for fixation and permeabilization steps. The panel can be used for studies aimed at characterizing the immune response in the context of infectious or autoimmune diseases, monitoring cancer patients on immuno- or chemotherapy, and discovery of unique and targetable biomarkers. Different from all previously published OMIPs, this panel was developed using a full spectrum flow cytometer, a technology that has allowed the effective use of 40 fluorescent markers in a single panel. The panel was developed using cryopreserved human peripheral blood mononuclear cells (PBMC) from healthy adults (Table 1). Although we have not tested the panel on fresh PBMCs or whole blood, it is anticipated that the panel could be used in those sample preparations without further optimization. @ 2020 Cytek Biosciences, Inc. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.

Dataset Information

Standardizing Flow Cytometry Immunophenotyping Analysis from the Human ImmunoPhenotyping Consortium.

Publications

Standardizing Flow Cytometry Immunophenotyping Analysis from the Human ImmunoPhenotyping Consortium.

Similar Datasets

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