A cytofluorometric method to quantify membrane antigens on individual alveolar macrophages

An immunofluorescence method for assaying membrane-bound antigens on individual alveolar macrophages (AM) collected by bronchoalveolar lavage (BAL) is described. Cells were labelled with FITC-conjugated anti-HLA-DR antibodies in a single (direct) step. Quantification of the fluorescence was performed by computer-assisted cytophotometry. Alveolar macrophages, especially when obtained from cigarette smokers, exhibited an autofluorescence which interfered with the measurement of specific fluorescence. The specific fluorescence was calculated by determination of total fluorescence in the wave-length optimal for FITC and the non-specific fluorescence in a different wavelength during a second measurement. Specificity and reproducibility testing confirmed the reliability of the method.