Standardization of 8-color flow cytometry across different flow cytometer instruments: A feasibility study in...

The EuroFlow Consortium developed a fully standardized flow cytometric approach from instrument settings, through antibody panel, reagents and sample preparation protocols, to data acquisition and analysis. The Swiss Cytometry Society (SCS) promoted a study to evaluate the feasibility of using such standardized measurements of 8-color data across two different flow cytometry platforms - Becton Dickinson (BD) FACSCanto II and Beckman Coulter (BC) Navios, aiming at increasing reproducibility and inter-laboratory comparability of immunophenotypic data in clinical laboratories in Switzerland. The study was performed in two phases, i.e. a learning phase (round 1) and an analytical phase (rounds 2 and 3) consisting of a total of three rounds. Overall, 10 laboratories using BD FACSCanto II (n=6) or BC Navios (n=4) flow cytometers participated. Each laboratory measured peripheral blood samples from healthy donors stained with a uniform antibody panel of reagents - EuroFlow Lymphoid Screening Tube (LST) - applying the EuroFlow standardized protocols for instrument setup and sample preparation (www.EuroFlow.org). All data files were analyzed centrally and median fluorescence intensity (MedFI) values for individual markers on defined lymphocyte subsets were recorded; variability from reference MedFI values was assessed using performance scores. Data troubleshooting and discussion of the results with the participants followed after each round at SCS meetings. The results of the learning phase demonstrated that standardized instrument setup and data acquisition are feasible in routine clinical laboratories without previous experience with EuroFlow. During the analytical phase, highly comparable data were obtained at the different laboratories using either BD FACSCanto II or BC Navios. The coefficient of variation of MedFI for 7 of 11 markers performed repeatedly below 30%. In the last study round, 89% of participants scored over 90% MedFI values within the acceptance criteria (P-score), in line with the results of the EuroFlow quality assessment rounds performed by the EuroFlow expert laboratories(Kalina et al., 2015). Central analysis of data allowed identification of deviations from the standardized procedures and technical issues (e.g. failure to perform correct instrument setup and improper compensation). In summary, here we show that inter-laboratory cross-platform standardization of 8-color flow cytometric measurements in clinical laboratories is feasible and allows for fully comparable MedFI results across BD FACSCanto II and BC Navios instruments. However, adherence to standardized protocols is crucial. Thus, training of the laboratory personnel in the EuroFlow standardized procedures is highly recommended to prevent errors in instrument setup and sample preparation.