Flow cytometric characterization of cell populations in bronchoalveolar lavage and bronchial brushings from patients with chronic obstructive pulmonary disease

Background: Chronic obstructive pulmonary disease (COPD) is a serious, chronic inflammatory disease of the airway associated with cigarette smoking. Leucocytes are involved in the inflammatory process in the airways in COPD. There is a need for accurate characterization of cellular populations in bronchoalveolar lavage (BAL) due to variation in the predominant cell types reported, which were investigated mostly with manual counting techniques. Methods: Bronchial brushings and BAL were obtained from human subjects undergoing fiber optic bronchoscopy. Flow cytometry was applied to identify various cell types. Quenching of autofluorescence of BAL-derived alveolar macrophages was achieved with eta-octyl beta-D-galactopyranoside and crystal violet. Comparisons of cell counts obtained with flow cytometric and manual counting methods were performed. Results: Correlation analysis showed that manual cell counting methods overestimated the percentage of macrophages when compared with flow cytometric methods (R-2 = 0.54). There was also a small tendency by manual counting to underestimate the percentage of lymphocytes and neutrophils. Using flow cytometry, the percentage and absolute numbers of alveolar macrophages and lymphocytes in BAL were not significantly different between patients with COPD and control subjects. The percentage and absolute numbers of neutrophils were higher in BAL from patients with moderate to severe COPD. Conclusions: This novel flow cytometric assay for identification of various cell types from heterogenous samples of BAL and bronchial brushing will allow further investigation of cell characteristics, such as cytokine production and receptor expression, and an accurate evaluation of apoptosis for different cell types and provide a rationale for urgently required effective treatments for COPD. (C) 2004 Wiley-Liss, Inc.