Endobronchial Biopsy Cell Quantification

Endobronchial biopsies were performed using disposable biopsy forceps with jaws closed at the interbronchial carina before retracting. Tissue was fixed immediately in 4% paraformaldehyde and paraffin embedded. CD4⁺ T cells were identified by staining with mouse anti-CD4 (Dako) at 1:100 dilution using the EnVision peroxidase staining method (Dako) as previously described (62 (link)). Slides were coded to avoid observer bias and assessed using a Leitz Dialux 20 light microscope and Image 1.5 software. Total epithelial and subepithelial areas of 2 to 3 bronchial biopsies were counted at each time point. Cell counts were expressed as the number of cut cell profiles with visible nucleus per 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%.