Reproducibility of Flow Cytometry Through Standardization: Opportunities and Challenges

Abstract

There is an agreement in the field that interlaboratory reproducibility of flow cytometry measurements as well as the whole studies might be improved by a consensual use of methodological approach. Typically, a consensus is made on a crucial markers needed in the immunostaining panel, sometimes on the particular fluorochrome conjugates and rarely on a complete set of methods for sample preparation. The term "standardization" is used to describe the complete set of methodical steps, while "harmonization" is used for partial agreement on the method. Standardization can provide a platform for improved reproducibility of cytometry results over prolonged periods of time, across different sites and across different instruments. For the purpose of structured discussion, several desired aims are described: common interpretation of the immunophenotype definition of a target subset, accurate quantification, reproducible pattern of a multicolor immunophenotype, and reproducible intensity of all measured parameters. An overview of how standardization was approached by several large consortia is provided: EuroFlow, The ONE Study, Human Immunology Project Consortium (HIPC), and several other groups. Their particular aims and the tools adopted to reach those aims are noted. How those standardization efforts were adopted in the field and how the resulting outcome was evaluated is reviewed. Multiple challenges in the instrument hardware design, instrument setup tools, reagent design, and quality features need to be addressed to achieve optimal standardization. Furthermore, the aims of different studies vary, and thus, the reasonable requirements for standardization differ. A framework of reference for the reasonable outcomes of different approaches is offered. Finally, it is argued that complete standardization is important not only for the reproducibility of measurements but also for education, for quality assessment and for algorithmic data analysis. The different standardized approaches can and in fact should serve as benchmarking reference tools for the development of future flow cytometry studies.

Analytical performance of a 25‐marker spectral cytometry immune monitoring assay in peripheral blood

“…The recent Cytometry Part A Special Issue: Rigor and Reproducibility highlighted the importance of antibody validation, sample preparation and instrument, assay and post‐analysis standardization [21–23]. These practices are indispensable to successful analytical performance or complete validation of high‐dimensional assays.…”

“…It is imperative to address these technicalities and develop a gating scheme that is accurate and relatively robust across individual samples. Multicenter manual gating alone can be a significant source of assay variability [1,23], although others report that biological variability often exceeds technical variability [2].…”

“…Using our 25‐biomarker assay, only the phenotypic e‐MDSC population either failed repeatability in blood (>20% CV) or was incalculable in PBMC due to fewer than 100 cells at the employed acquisition settings. Disease sample benchmarking could potentially improve panel performance [23] as nonhealthy samples typically have expanded MDSC compartments [11]. Immune biomarkers and populations that were sensitive to 24+ h blood storage or PBMC manipulation (or both) include CXCR3 expression on memory CD4 T cells, Th1 or Th17 cells, CD45RA+ Tregs, CD38+ CD4 T cells, classical and nonclassical monocytes, M‐MDSCs, CD15+CD11b+ LDGs, and CD11cloCD123hi pDCs.…”

A Cytometrist's Guide to Coordinating and Performing Effective COVID‐19 Research

“…The key elements for successful translational and clinical science involve standardization (42), harmonization, and method validation. As we are still in the early days of SARS‐CoV2 research, progress in standardization and harmonization is critically needed.…”

Flow Cytometry Contributions for the Diagnosis and Immunopathological Characterization of Primary Immunodeficiency Diseases With Immune Dysregulation

“…Before reviewing the contribution of flow cytometry to the characterization of PIDs with immune dysregulation, we emphasize that in order to perform molecular characterization of inborn errors of immunity in diagnostic laboratories, one needs to become familiar with the flow cytometry guidelines and parameters, which have been previously reported (31, 99–104) They were discussed in detail with focus on technical flow cytometry aspects. For example, flow cytometry parameters of general importance are the determination and validation of flow cytometry positive controls (e.g., fluorescence compensation controls as well as resting and activation controls in the case of inducible molecules), the establishment of appropriate cutoffs (e.g., by defining the 10th percentile of normal controls as a center-specific lower limit of normal), and avoiding misinterpretation of results due to inter-laboratory variability, specificity, and sensitivity, particularly in patients with low peripheral blood lymphocyte counts.…”