Centrally Determined Standardization of Flow Cytometry Methods Reduces Interlaboratory Variation in a Prospective Multicenter Study

Abstract

Objectives:Flow cytometry (FC) aids in characterization of cellular and molecular factors involved in pathologic immune responses. Although FC has potential to facilitate early drug development in inflammatory bowel disease, interlaboratory variability limits its use in multicenter trials. Standardization of methods may address this limitation. We compared variability in FC-aided quantitation of T-cell responses across international laboratories using three analytical strategies.Methods:Peripheral blood mononuclear cells (PBMCs) were isolated from three healthy donors, stimulated with phorbol 12-myristate 13-acetate and ionomycin at a central laboratory, fixed, frozen, and shipped to seven international laboratories. Permeabilization and staining was performed in triplicate at each laboratory using a common protocol and centrally provided reagents. Gating was performed using local gating with a local strategy (LGLS), local gating with a central strategy (LGCS), and central gating (CG). Median cell percentages were calculated across triplicates and donors, and reported for each condition and strategy. The coefficient of variation (CV) was calculated across laboratories. Between-strategy comparisons were made using a two-way analysis of variance adjusting for donor.Results:Mean interlaboratory CV ranged from 1.8 to 102.1% depending on cell population and gating strategy (LGLS, 4.4–102.1% LGCS, 10.9–65.6% CG, 1.8–20.9%). Mean interlaboratory CV differed significantly across strategies and was consistently lower with CG.Conclusions:Central gating was the only strategy with mean CVs consistently lower than 25%, which is a proposed standard for pharmacodynamic and exploratory biomarker assays.

Multicentre Harmonisation of a Six-Colour Flow Cytometry Panel for Naïve/Memory T Cell Immunomonitoring

“…In Figure 5(a), bars represent median values among the three donor-specific CVs (obtained on the three replicates for each cPBMC (n = 3) and each WB (n = 3) specimen. As expected [30], the median CV was lower for abundant populations (i.e., CD3+ cells within lymphocytes, and CD4+ or CD8+ cells within CD3+ T cells), and higher for lessrepresented and poorly resolved subsets, such as CD4+ TD cells. In particular, acceptable precision values (CV below 0.20) were obtained for 8/15 vs. 12/15 parameters in cPBMC and for 6/15 vs. 13/15 parameters in WB experiments, in local vs. centralised analysis, respectively.…”

Validation of CyTOF Against Flow Cytometry for Immunological Studies and Monitoring of Human Cancer Clinical Trials

“…The data analysis of CyTOF is perhaps the most challenging part of the workflow. With cytometry data in general, manual gating is the one of the main contributor to inter-laboratory variations (31). An optimally designed panel, with a well-matched biomarkers and metals-tags as mentioned above, will cause less trouble gating and resolving positive events.…”

Cyt‐Geist: Current and Future Challenges in Cytometry: Reports of the CYTO 2018 Conference Workshops

“…Improving reproducibility of analysis starts with the use of standardized gating protocols, encoded in SOPs, gating templates and the use of biological or unstained controls . Instrument‐associated variability can be addressed through inter‐instrument alignment and monitoring .…”

“…However, these options are not scalable to larger studies, due to the time required to analyze each sample. This leads on to the growing use of specialist software tools to reduce subjectivity in reporting . Automated FCM analysis approaches have reached a demonstrated level of maturity in clinical trials and in the FDA‐approval process .…”

“…In this approach, a limited number of regional facilities with appropriated aligned procedures and standardized instruments are used to process and analyze samples. Data analysis and reporting is then conducted via a central controlling facility . Although currently rarely used by the participants, in future automation could be introduced at all stages of sample preparation (implementation of robotics).…”