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How to make usage of the standardized EuroFlow 8-color protocols possible for instruments of different manufacturers.

  • Nováková, Michaela1
  • Glier, Hana2
  • Brdičková, Naděžda1
  • Vlková, Marcela3
  • Santos, Ana Helena4
  • Lima, Margarida4
  • Roussel, Mikael5
  • Flores-Montero, Juan6
  • Szczepanski, Tomasz7
  • Böttcher, Sebastian8
  • van der Velden, Vincent H J9
  • Fernandez, Paula2
  • Mejstříková, Ester1
  • Burgos, Leire10
  • Paiva, Bruno10
  • van Dongen, Jacques J M11
  • Orfao, Alberto6
  • Kalina, Tomáš12
  • 1 CLIP - Childhood Leukaemia Investigation Prague, Department of Paediatric Haematology and Oncology, Second Faculty of Medicine, Charles University and University Hospital Motol, Prague, Czech Republic. , (Czechia)
  • 2 Kantonsspital Aarau, Aarau, Switzerland. , (Switzerland)
  • 3 Department of Clinical Immunology and Allergology, St. Anne's University Hospital in Brno, Czech Republic. , (Czechia)
  • 4 Laboratory of Flow Cytometry, Department of Hematology, Hospital de Santo António, Centro Hospitalar do Porto, Unidade Multidisciplinar de Investigação Biomédica (UMIB), Porto, Portugal. , (Portugal)
  • 5 Laboratoire d'Hématologie, Pôle Cellules et Tissus, CHU, INSERM UMR 1236, Rennes, France. , (France)
  • 6 Cancer Research Centre (IBMCC, USAL-CSIC), Institute for Biomedical Research of Salamanca (IBSAL), Department of Medicine and Cytometry Service (NUCLEUS Research Support Platform), University of Salamanca (USAL), Salamanca and Centro de Investigación Biomédica en Red de Cáncer, Instituto Carlos III, Madrid, Spain. , (Spain)
  • 7 Department of Pediatric Hematology and Oncology, Zabrze Medical University of Silesia, Katowice, Poland. , (Poland)
  • 8 University of Rostock, Division of Internal Medicine, Hematology laboratory, Medical Clinic III, Hematology, Oncology, and Palliative Medicine, Rostock, Germany. , (Germany)
  • 9 Department of Immunology, Laboratory for Medical Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands. , (Netherlands)
  • 10 Clínica Universidad de Navarra, Centro de Investigación Medica aplicada (CIMA), IDISNAm, Pamplona, Spain. , (Spain)
  • 11 Department of Immunohematology and Blood Transfusion (IHB), Leiden University Medical Center (LUMC), Leiden, The Netherlands. , (Netherlands)
  • 12 CLIP - Childhood Leukaemia Investigation Prague, Department of Paediatric Haematology and Oncology, Second Faculty of Medicine, Charles University and University Hospital Motol, Prague, Czech Republic. Electronic address: [email protected]. , (Czechia)
Published Article
Journal of immunological methods
Publication Date
Dec 01, 2019
DOI: 10.1016/j.jim.2017.11.007
PMID: 29154914


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. Copyright © 2017. Published by Elsevier B.V.

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