Source: Annual Congress 2011 - Bronchoalveolar lavage and biomarkers in diffuse parenchymal lung disease
Disease area: Airway diseases

Abstract

NTRODUCTION: 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. Macrophage‘s 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<0.001) but FCM overestimates lymphocyte population +13(15.8)%, and conversely underestimates alveolar macrophage population –15.6(19.6)%.
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.

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M. Arias, B. Palomo, J. Belda, L. Tricas, M. P. Paniagua, C. Martinez, M. Gonzalez, A. Quero, P. Casan (Oviedo, Spain). Differential cell count in BAL by flow cytometry using CD15 FITC/CD16PE/CD45 PERCP/HLA-DR APC monoclonal antibodies. Eur Respir J 2011; 38: Suppl. 55, 4770

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