Quantifying RSV and CD8+ T Cells in Endobronchial Biopsies

Endobronchial biopsies were fixed immediately in 4% paraformaldehyde and paraffin embedded. RSV was stained using a mouse anti-RSV antibody cocktail (NCL-RSV3, Leica Biosystems, UK) at 1:50 dilution. CD8⁺ T cells were identified by staining with mouse anti-CD8 (M0707, Dako, Denmark) at 1:100 dilution using the EnVision peroxidase-staining method (Dako) as previously described (62 (link)). Briefly, 5-μm sections were stained according to the manufacturer’s instructions, and an irrelevant mouse IgG1 kappa antibody (MOPC21) was used as negative control for staining specificity of mouse monoclonal antibodies. RSV-infected A549 cells were used as positive staining controls. Quantification was achieved as previously described with operators blinded to sample timing and infection status (62 (link)). Briefly, slides were coded to avoid observer bias, and areas of epithelium and subepithelium were assessed using a Leitz Dialux 20 light microscope, an Apple Macintosh computer, and Image 1.5 software. Total epithelial and subepithelial areas of two to three bronchial biopsies were counted for each bronchoscopy. Cell counts were expressed as the number of cut cell profiles with visible nucleus per 1 mm² of subepithelium and per 0.1 mm² of epithelium. The coefficient of variation for repeat counts of positive cells by a single observer ranged from 5 to 6%.