This commentary discusses particularities of application of the EuroFlow standardization of flow cytometric analyses on three different flow cytometers. The EuroFlow consortium developed a fully standardized approach for flow cytometric immunophenotyping of hematological malignancies and primary immunodeficiencies. Standardized instrument setup is an essential part of EuroFlow standardization. Initially, the EuroFlow Consortium developed and optimized a step-by-step standard operating procedure (SOP) to setup 8-color BD FACSCanto II flow cytometer (Canto), with the later inclusion of Navios (Beckman Coulter) and BD FACSLyric (Lyric). Those SOPs were developed to enable standardized and fully comparable fluorescence measurements in the three flow cytometers. In Canto and Navios, mean fluorescence intensity (MFI) of a reference peak of Rainbow beads calibration particles is used to set up photomultiplier (PMT) voltages for each detector channel in individual instruments to reach the same MFI across distinct instruments. In turn, a new feature of Lyric instruments allows to share collection of attributes that are used to place the positive population at the same position among instruments in the form of assays, as one of its components integrated in the Cytometer Setup and Tracking (CS&T) module. The EuroFlow Lyric assays thus allow for standardized acquisition of 8-color EuroFlow panels on Lyric without the need to setup the PMT voltages on the individual instruments manually. In summary, the standardized instrument setup developed by EuroFlow enables cross-platform inter- and intra-laboratory standardization of flow cytometric measurements. This commentary provides a perspective on the modifications of the standardized EuroFlow instrument setup of Canto, Navios and Lyric instruments that are described in detail in individual instrument-specfic SOPs available at the EuroFlow website.

2nd Department of Medicine University Hospital of Schleswig Holstein Campus Kiel Kiel Germany

Cancer Research Center CB16 12 00400 Instituto de Salud Carlos 3 Madrid Spain

CLIP Department of Paediatric Hematology and Oncology 2nd Faculty of Medicine Charles University and University Hospital Motol Prague Czech Republic

Department 3 of Internal Medicine University Medical Center Rostock Rostock Germany

Department of Immunohematology and Blood Transfusion Leiden University Medical Center Leiden the Netherlands

Department of Immunology Erasmus MC University Medical Center Rotterdam Rotterdam the Netherlands

Department of Microbiology and Immunology Medical University of Silesia in Katowice Zabrze Poland

FACS Stem cell Laboratory Kantonsspital Aarau Aarau Switzerland

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