Differential cell count in BAL by flow cytometry using CD15 FITC/CD16PE/CD45 PERCP/HLA-DR APC monoclonal antibodies

Introduction: Usually inflammatory cell counts in bronchoalveolar lavage (BAL) are done manually on optical microscopy (OM) despite the high intra-and interobserver variability. The objective was to compare inflammatory cell counts performed by OM with these counts by flow cytometry (FC) with a new combination of monoclonal antibodies. Methods: 34 BAL samples were analysed in a 2-laser cytometer (FACSCalibur). The results were compared with those obtained by optical microscopy. The proposed combination of monoclonal antibodies identify leukocytes as CD45 + cells and lymphocytes as CD15 - , CD16 - and CD16 dim+ (NK lymphocytes), HLA-DR - and HLA-DR + (B cells and activated lymphocytes) cells; neutrophils as CD15 bright+ , CD16 bright+ , HLA-DR - cells; eosinophils as CD15 bright+ , CD16 - , HLA-DR - cells and alveolar macrophages as CD15 dim+ , CD16 bright+ , HLA-DR bright+ cells. Macrophage9s autofluorescence (AF) was overcome using the monoclonal antibody anti-HLA-DR conjugated with the dye APC as the main identification marker. Results: Agreement analysis for both methods shown high correlations (r=0.70 to 0.93; p Conclusions: The monoclonal antibodies combination proposed is effective and reliable to identify leukocyte populations in BAL. The process is simpler and faster than manual optical microscopy but some differences in macrophages and lymphocytes counts should be considered.