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

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. © 2019 International Society for Advancement of Cytometry.

Flow cytometric quantification of all phases of the cell cycle and apoptosis in a two-color fluorescence plot.

An optimal technology for cell cycle analysis would allow the concomitant measurement of apoptosis, G0, G1, S, G2 and M phases in combination with cell surface phenotyping. We have developed an easy method in flow cytometry allowing this discrimination in an only two-color fluorescent plot. It is based on the concomitant use of 7-amino-actinomycin D and the antibodies anti-Ki67 and anti-phospho(Ser10)-histone H3, both conjugated to Alexa Fluor®488 to discriminate G0 and M phases, respectively. The method is particularly valuable in a clinical setting as verified in our laboratory by analyzing human leukemic cells from marrow samples or after exposure to cell cycle modifiers.

All-trans retinoic acid prevents apoptosis of human marrow CD34+ cells deprived of haematopoietic growth factors.

The regulation of apoptosis plays a key role in haematopoiesis. It has been demonstrated that haematopoietic progenitor cells progressively undergo apoptotic cell death in the absence of appropriate growth factors. We studied the effects of pharmacological doses of all-transretinoic acid (ATRA) on the apoptosis of human adult marrow CD34+ progenitor cells cultured for 7 d in a serum-free medium. We quantified CD34+ cells, clonogenic progenitors and 5 week colony-forming cells (CFC) before and after ATRA exposure. Moreover, we defined the apoptotic status of the CD34+ cell fraction by analysis of phosphatidylserine externalization (using annexin V), the relative membrane permeability to 7-aminoactinomycin D (7AAD) and the mitochondrial membrane potential [using 3,3'-dihexyloxacarbocyanine iodide, DiOC6(3)]. In the drastic experimental conditions used, a decrease in viable CD34+ cells, granulocyte-macrophage colony-forming units (CFU-GM), erythroid burst-forming units (BFU-E) and 5 week CFC were observed. Exposure to ATRA partially prevented the decrease in viable CD34+, without a concomitant effect on the clonogenic and more immature progenitors. ATRA-treated CD34+ cells displayed changes in apoptotic status compared with control cultures, particularly in lower annexin V-binding. These results were confirmed using 7AAD and DiOC6(3). Our results demonstrate that ATRA exerts a protective effect on CD34+ cells exposed to such apoptotic stress.