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Centrally Determined Standardization of Flow Cytometry Methods Reduces Interlaboratory Variation in a Prospective
Liset Westera1, Tanja van Viegen2, Jenny Jeyarajah3
1Tytgat Institute for Liver and Intestinal Research, Amsterdam, Netherlands.
Clinical and Translational Gastroenterology
|November 3, 2017
Summary
Standardizing flow cytometry (FC) methods, specifically central gating, significantly reduces interlaboratory variability in T-cell response analysis for inflammatory bowel disease drug development. This improves assay reliability for multicenter trials.
Area of Science:
- Immunology
- Biotechnology
- Pharmacology
Background:
- Flow cytometry (FC) is crucial for analyzing immune responses in diseases like inflammatory bowel disease (IBD).
- Interlaboratory variability in FC methods hinders its application in multicenter drug development trials for IBD.
- Standardization of FC protocols is necessary to overcome these limitations.
Purpose of the Study:
- To compare the variability of FC-based T-cell response quantitation across international laboratories.
- To evaluate three different analytical strategies for gating in FC analysis.
- To identify a standardized method for reliable FC data in multicenter IBD studies.
Main Methods:
- Peripheral blood mononuclear cells (PBMCs) from healthy donors were stimulated and distributed to seven international labs.
- Standardized reagents and protocols were used for permeabilization and staining.
- Three gating strategies were compared: local gating with local strategy (LGLS), local gating with central strategy (LGCS), and central gating (CG).
Main Results:
- Interlaboratory coefficients of variation (CVs) varied widely (1.8% to 102.1%) depending on the cell population and gating strategy.
- Central gating (CG) demonstrated consistently lower mean interlaboratory CVs compared to LGLS and LGCS.
- Mean CVs were significantly lower across strategies, with CG achieving the lowest variability.
Conclusions:
- Central gating was the only strategy yielding mean CVs below 25%, a benchmark for pharmacodynamic and exploratory biomarker assays.
- Implementing central gating in FC analysis can enhance reproducibility for multicenter IBD drug development.
- Standardization through central gating is vital for reliable FC data in clinical research.
