A critical component of the EuroFlow standardization of leukemia/lymphoma immunophenotyping is instrument setup. Initially, the EuroFlow consortium developed a step-by-step standard operating protocol for instrument setup of ≥8-color flow cytometers that were available in 2006, when the EuroFlow activities started. Currently, there are 14 instruments from 9 manufacturers capable of 3-laser excitation and ≥8 color measurements. The specific adaptations required in the instrument set-up to enable them to acquire the standardized 8-color EuroFlow protocols are described here. Overall, all 14 instruments can be fitted with similar violet, blue and red lasers for simultaneous measurements of ≥8 fluorescent dyes. Since individual instruments differ both on their dynamic range (scale) and emission filters, it is not accurate to simply recalculate the target values to different scale, but adjustment of PMT voltages to a given emission filter and fluorochrome, is essential. For this purpose, EuroFlow has developed an approach using Type IIB (spectrally matching) particles to set-up standardized and fully comparable fluorescence measurements, in instruments from different manufacturers, as demonstrated here for the FACSCanto II, and Navios and MACSQuant flow cytometers. Data acquired after such adjustment on any of the tested cytometry platforms could be fully superimposed and therefore analyzed together. The proposed approach can be used to derive target values for any combination of spectrally distinct fluorochromes and any distinct emission filter of any new flow cytometry platform, which enables the measurement of the 8-color EuroFlow panels in a standardized way, by creating superimposable datafiles.

Cancer Research Centre Salamanca and Centro de Investigación Biomédica en Red de Cáncer Instituto Carlos 3 Madrid Spain

Clínica Universidad de Navarra Centro de Investigación Medica aplicada IDISNAm Pamplona Spain

CLIP Childhood Leukaemia Investigation Prague Department of Paediatric Haematology and Oncology 2nd Faculty of Medicine Charles University and University Hospital Motol Prague Czech Republic

Department of Clinical Immunology and Allergology St Anne's University Hospital in Brno Czech Republic

Department of Immunohematology and Blood Transfusion Leiden The Netherlands

Department of Immunology Laboratory for Medical Immunology Erasmus MC University Medical Center Rotterdam Rotterdam The Netherlands

Department of Pediatric Hematology and Oncology Zabrze Medical University of Silesia Katowice Poland

Kantonsspital Aarau Aarau Switzerland

Laboratoire d'Hématologie Pôle Cellules et Tissus CHU INSERM UMR 1236 Rennes France

Laboratory of Flow Cytometry Department of Hematology Hospital de Santo António Centro Hospitalar do Porto Unidade Multidisciplinar de Investigação Biomédica Porto Portugal

University of Rostock Division of Internal Medicine Hematology laboratory Medical Clinic 3 Hematology Oncology and Palliative Medicine Rostock Germany

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EuroFlow-Based Flowcytometric Diagnostic Screening and Classification of Primary Immunodeficiencies of the Lymphoid System