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A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood
Published on: February 8, 2016
Standardizing Flow Cytometry Immunophenotyping Analysis from the Human ImmunoPhenotyping Consortium
Greg Finak1, Marc Langweiler2, Maria Jaimes3
1Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, 98109, WA.
Insights
Standardizing immunophenotyping with pre-configured reagent panels and automated gating significantly reduces cross-laboratory variability. This approach enhances data comparability across studies and centers for immune cell analysis.
Area of Science:
- Immunology
- Biotechnology
- Data Science
Background:
- Standardization of immunophenotyping is crucial for reliable cross-study and cross-center data comparison.
- Variability in reagents, sample handling, instrument setup, and data analysis impacts data consistency.
Purpose of the Study:
- To evaluate standardized, pre-configured immunophenotyping panels and automated gating for reducing cross-center variability.
- To compare the performance of centralized manual gating versus site-specific manual and automated gating.
Main Methods:
- Development and use of five standardized, eight-color immunophenotyping panels in lyophilized, 96-well plates.
- Coordinated analysis across nine laboratories using standardized operating procedures (SOPs).
- Comparison of manual gating (site-specific and central) with automated gating algorithms.
Main Results:
- Within-site variability was consistently low across all experiments.
- Cross-site variability was reduced with centralized manual gating compared to site-specific analysis.
- Automated gating demonstrated performance comparable to central manual analysis, with minimal bias and variability.
Conclusions:
- Standardized staining, data collection, and automated gating effectively reduce variability in immunophenotyping.
- Automated methods can streamline analysis and improve the power of immunophenotyping studies.
- Implementation of these standardized approaches enhances data reliability for immune cell subset identification.
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.
