MULTIPARAMETER FLOW CYTOMETRIC ANALYSIS OF INFLAMMATORY CELLS CONTAINED IN BRONCHOALVEOLAR LAVAGE FLUID

Quantitative analysis of surface molecule expression on viable alveola r macrophages (AM) by use of flow cytometry is hampered by non-specifi c antibody binding to various AM Fc(IgG) receptors as well as extensiv e and heterogeneous autofluorescence of this cell type. The following approaches were undertaken to circumvent these obstacles. Fc(IgG) rece ptors were blocked by excess human immunoglobulin. The use of a long w ave-emitting dye (phycoerythrin/cyanine-5 tandem conjugate) permitted avoidance of the peak (green) AM autofluorescence range. Moreover, a c ell-by-cell compensation for the remaining red autofluorescence backgr ound was employed. This was based on two facts: (i) strict correlation between green (F488/530) and red autofluorescence (F488/660) for all AM populations investigated; and (ii) neglectable overlap of the antib ody-associated red fluorescence into the 530 nm autofluorescence detec tion wavelength. A fraction of the green autofluorescence (F488/530; c hannel 1) was then substracted from the red fluorescence (F488/660; ch annel 2) on a cell-by-cell basis using standard two colour fluorescenc e compensation circuits. The validity of this FACS technique was confi rmed by comparison with immunocytochemical staining and a reverse rese tting method. On AM lavaged from carcinoma-bearing but otherwise disea se-free human lungs, the pattern of surface antigen expression was ass essed with a panel of monoclonal antibodies. When applying to complex mixtures of bronchoalveolar lavage cells, the autofluorescence was emp loyed to separate AM from granulocytes and lymphocytes. In conclusion, the presently described FACS technique allows quantitative immunostai ning of surface molecules on AM, even when present in low copy numbers on highly autofluorescent cells originating from smokers.