Standardization of Flow Cytometric Immunophenotyping for Hematological Malignancies: The FranceFlow Group Experience

Abstract

Flow cytometry is broadly used for the identification, characterization, and monitoring of hematological malignancies. However, the use of clinical flow cytometry is restricted by its lack of reproducibility across multiple centers. Since 2006, the EuroFlow consortium has been developing a standardized procedure detailing the whole process from instrument settings to data analysis. The FranceFlow group was created in 2010 with the intention to educate participating centers in France about the standardized instrument setting protocol (SOP) developed by the EuroFlow consortium and to organise several rounds of quality controls (QCs) in order to evaluate the feasibility of its application and its results. Here, we report the 5 year experience of the FranceFlow group and the results of the seven QCs of 23 instruments, involving up to 19 centers, in France and in Belgium. The FranceFlow group demonstrates that both the distribution and applicability of the SOP have been successful. Intercenter reproducibility was evaluated using both normal and pathological blood samples. Coefficients of variation (CVs) across the centers were <7% for the percentages of cell subsets and <30% for the median fluorescence intensities (MFIs) of the markers tested. Intracenter reproducibility provided similar results with CVs of <3% for the percentages of the majority of cell subsets, and CVs of <20% for the MFI values for the majority of markers. Altogether, the FranceFlow group show that the 19 participating labs might be considered as one unique laboratory with 23 identical flow cytometers able to reproduce identical results. Therefore, SOP significantly improves reproducibility of clinical flow in hematology and opens new avenues by providing a robust companion diagnostic tool for clinical trials in hematology.

Applicability and reproducibility of acute myeloid leukaemia stem cell assessment in a multi‐centre setting

“…Sophisticated 8–10 colour flow cytometry is at the basis of the diagnosis, characterisation and monitoring of haematological malignancies. Correct implementation of the technique and standardisation in its applications is of high importance and several guidelines to achieve this have recently been published 22,23 . In this study, we demonstrate that harmonisation between flow‐cytometers is required for comparable results.…”

“…Correct implementation of the technique and standardisation in its applications is of high importance and several guidelines to achieve this have recently been published. 22,23 In this study, we demonstrate that harmonisation between flow-cytometers is required for comparable results. Here, the use of BD's FC beads or setup according to Euroflow protocol was adequate to result in comparable measurements as percentages of lymphocytes, blasts, CD34 + blasts, CD34 + CD38 dim and CD34 + CD38fractions were decidedly comparable among all institutes (data not shown).…”

Automated identification of leukocyte subsets improves standardization of database-guided expert-supervised diagnostic orientation in acute leukemia: a EuroFlow study

“…Previous experience within EuroFlow demonstrated that subsequent training of centers with higher percentage of checks/alarms show decrease number of alarms. Strong compliance to the instrument setup and sample processing SOPs are thereby of utmost importance for appropriate use of both the AGI tools and database-guided diagnostic orientation toward the most appropriate subsequent AL panel, and should be verified with already existing quality controls and the local average number of checks [24][25][26][27]. Of note, the use of validated alternative reagents, like the OneFlow ALOT and the Cytognos ALOT kits, resulted in highly comparable results.…”

MiSet RFC Standards: Defining a Universal Minimum Set of Standards Required for Reproducibility and Rigor in Research Flow Cytometry Experiments

“…For example, the EuroFLOW consortium has developed a standardized procedure detailing the entire process from instrument settings to data analysis, including tools to process large data files in diagnostic laboratories (58,59). EuroFLOW standards are commonly considered a robust approach (61,62). These included issues related to operator/center expertise and training, panel design, data analysis and interpretation of results.…”

“…These included issues related to operator/center expertise and training, panel design, data analysis and interpretation of results. EuroFLOW standards are commonly considered a robust approach (61,62). Minimum standards have also been defined in the setting of flow cytometry SRLs, providing guidance for best practices in instrumentation, training, experimental design, biosafety standards, and sample handling (11,20,21).…”