1Department of Clinical Immunology, Royal Free Hospital School of Medicine, London
2UK NEQAS for Leucocyte Immunophenotyping, Haematology Department, Royal Hallamshire Hospital, Sheffield
Current clinical interest in the quantification of antigens has heightened the requirement to standardize immunofluorescent (IF) analysis. This is because the use of different flow cytometers and the plethora of monoclonal antibodies conjugated to different fluorochromes causes difficulties in the interlaboratory comparisons of the mean fluorescence intensity (MFI) obtained by flow cytometry. We compared two commercial methods currently abailable for antigen quantitation: the quantitative indirect immunofluorescent test (QIFI) and Quantum Simply Cellular (QSC) beads. We have also introduced a new concept for quantitative antigen analysis termed: stabilized cellular immunofluorescent analysis (SCIFA). To achieve this, stabilized cell preparations were produced with a long shelf life (>200 days) in order to serve as cellular controls for quantitative flow cytometry. These, together with fresh normal blood were used to determine the precision and reproducibility of the AIFI and QSC methods for a wide range of different CD antibodies. The study revealed tha the QIFI assay had greater precision and reproducibility than the QSC system. The latter was greatly influenced by the variable conditions of antibody incubation with the QSC beads which affected the antibody binding capacity (ABC) value. In addition, the QIFI system can be used to control the QSC system. Importantly, the study assigned ABC values for a variety of antigens on lymphocytes, monocytes, and granulocytes for the stabilized samples. These reference values facilitated the construction of calibration curves for stabilized cellular immunofluorescence analysis (SCIFA). Such values can be used as internal cellular controls, or calibrators, for either the QIFI or QSC methods. Such and approach will facilitate the standardization of flow cytometric ABC determination, used when examining aberrant antigenic expression in leukaemic samples, e.g., CD45, CD10, CD5, CD20, and CD34, and also activation antigens monitored in immune disorders such as HIV infection (e.g., HLA-DR, and CD38). The cell populations produced are available for inter-laboratory comparative studies through the Biomed-2 Concerted Action programme and the UK NEQAS scheme.