Flow cytometric quantitation of immunofluorescence intensity: Problems and perspectives

Authors

  • Jan W. Gratama,

    Corresponding author
    1. Department of Clinical and Tumor Immunology, Daniel den Hoed Kliniek, Rotterdam, The Netherlands
    • Department of Clinical and Tumor Immunology, Daniel den Hoed Cancer Center, P.O. Box 5201, 3008 AE Rotterdam, The Netherlands
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  • Jean-Luc D'hautcourt,

    1. Hôpital de Warquignies, Boussu, Belgium
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  • Frank Mandy,

    1. National Laboratory for Analytical Cytology, Health Protection Branch, Health Canada, Ottawa, Canada
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  • Gregor Rothe,

    1. Institut für Klinische Chemie und Laboratoriumsmedizin, Klinikum der Universität Regensburg, Regensburg, Germany
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  • David Barnett,

    1. Department of Haematology, Royal Hallamshire Hospital, Sheffield, England
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  • George Janossy,

    1. Department of Clinical Immunology, Royal Free Hospital School of Medicine, London, England
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  • Stefano Papa,

    1. Istituto di Scienze Morfologiche, Università di Urbino, Urbino, Italy
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  • Gerd Schmitz,

    1. Institut für Klinische Chemie und Laboratoriumsmedizin, Klinikum der Universität Regensburg, Regensburg, Germany
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  • Rodica Lenkei,

    1. CALAB Research, S:t. Görans Sjukhus, Stockholm, Sweden
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  • The European Working Group on Clinical Cell Analysis

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    • The European Working Group on Clinical Cell Analysis (EWGCCA) is a collaborative initiative of scientists active in the field of clinical cell analysis from 13 European countries. The goal of this working group, which is open to scientists active in this field, is the evaluation, refinement, and standardization of new analytical techniques for clinical cell analysis. The group is divided into subsections according to the different tasks and types of analytical techniques, i.e., flow and image cytometry or molecular biology. The core members of the EWGCCA are G. Schmitz (Regensburg, Germany), coordinator; B. Autran (Paris, France); B. Brando (Milan, Italy); J.L. D'hautcourt (Boussu, Belgium); J.W. Gratama (Rotterdam, The Netherlands); A. Huber (Aarau, Switzerland); G. Janossy (London, England); H.E. Johnsen (Copenhagen, Denmark); J. Kappelmayer (Debrecen, Hungary); R. Lenkei (Stockholm, Sweden); A. Orfao (Salamanca, Spain); S. Papa (Urbino, Italy); M. Papamichail (Athens, Greece); G. Valet (Martinsried, Germany); T. Tötterman (Uppsala, Sweden); G. Rothe (Regensburg, Germany); and B. Zupanska (Warsaw, Poland).


Abstract

Quantitation of immunofluorescence intensity serves to estimate the number of defined molecules expressed on or in cells. Clinical applications of this diagnostic tool are increasing, e.g., aberrant expression of various antigens (Ag) by leukemic blasts or lymphoma cells, intensity of CD38 expression by CD8+ T-lymphocytes to monitor activation status, and intensity of CD62P to detect platelet activation. In this report we discuss the quality-control measures required for quantitation of fluorescence intensity, and we review seven concepts that have been developed to quantify fluorescence intensity during the past 15 years. Initial work addressed the conversion of logarithmic channel numbers into units of relative fluorescence. The design and use of calibration beads labeled with predefined amounts of dye allowed instrument-independent expression of fluorescence intensity in units of molecules of equivalent soluble fluorochrome (MESF). This method was refined by the combined use of such standards with monoclonal antibodies (mAb) conjugated 1:1 with phycoerythrin (PE), allowing translation of fluorescence intensity into numbers of antibodies bound per cell. Alternatively, the use of 1:1 PE-conjugated mAb under the assumption that CD4+ lymphocytes reproducibly bind 50,000 CD4 mAb molecules was proposed to convert units of relative fluorescence intensity into units of antibodies bound per cell. The use of antibody-binding capacity as a surrogate marker for quantification of Ag expression was addressed more directly by the development of antibody-binding standards. The quantitative indirect immunofluorescence assay is based on beads labeled with various amounts of CD5 mAb that calibrate the binding of the secondary antibody in units of antibody-binding capacity. Alternatively, goat anti-mouse–labeled calibration beads have been developed. Published results obtained with the latter calibrators showed an unexpected inaccuracy. The different ways in which calibrators and cells under study bind mAb (i.e., Fab mediated versus Fc mediated) may have contributed to this variation. Recently, the use of stabilized cell populations expressing Ag in a specified range of concentrations has been proposed as an Ag-specific calibration system of mAb binding. We identify several issues on the level of instrumentation, reagents, and cells under study that should be solved to allow standardization of quantitative assessments of immunofluorescence intensity. Cytometry 33: 166–178, 1998. © 1998 Wiley-Liss, Inc.

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