Reproducibility of Flow Cytometry Through Standardization: Opportunities and Challenges
1CLIP-Childhood Leukemia Investigation Prague, Department of Pediatric Hematology and Oncology, 2nd Medical School, Charles University and University Hospital Motol, Prague, Czech Republic.
Insights
Standardizing flow cytometry methods improves interlaboratory reproducibility. This consensus on techniques, from markers to sample preparation, enhances data accuracy and consistency across studies and sites.
Area of Science:
- * Immunology
- * Biomedical Engineering
- * Laboratory Medicine
Background:
- * Interlaboratory reproducibility in flow cytometry is crucial for reliable scientific findings.
- * Current practices often lack consensus on methodological approaches, leading to variability.
- * Standardization and harmonization are proposed as solutions to improve consistency.
Purpose of the Study:
- * To discuss the importance of consensual methodological approaches in flow cytometry.
- * To review existing standardization and harmonization efforts by major consortia.
- * To identify challenges and propose a framework for achieving optimal flow cytometry standardization.
Main Methods:
- * Review of standardization strategies employed by EuroFlow, The ONE Study, and HIPC.
- * Analysis of tools and outcomes of these standardization initiatives.
- * Examination of challenges in instrument design, reagents, and quality control.
Main Results:
- * Standardization efforts aim for common immunophenotype interpretation, accurate quantification, and reproducible multicolor patterns.
- * Various consortia have adopted different tools and approaches to achieve standardization.
- * Challenges remain in instrument hardware, setup, reagents, and quality features.
Conclusions:
- * Complete standardization is vital for measurement reproducibility, education, quality assessment, and algorithmic data analysis.
- * Standardized approaches serve as essential benchmarking tools for future flow cytometry studies.
- * Tailoring standardization requirements to specific study aims is necessary.
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. © 2019 International Society for Advancement of Cytometry.
