Cytometric analysis of BAL T cells labeled with a standardized antibody cocktail correlates with immunohistochemical staining

Background: Determining T-cell phenotypes of lung cells obtained by bronchoalveolar lavage (BAL) is frequently clinically useful, particularly for evaluating causes of interstitial lung disease. The current standard of determining CD4/CD8 T-cell subsets by immunohistochemical (IHC) staining of cytocentrifuge slides is labor-intensive and subject to interpreter variation. Flow cytometry (FCM) is a precise and rapid method commonly used in research to characterize cells in the lung. However, few studies address the methodology of analysis of BAL lymphocytes by FCM. Methods: Patients underwent bronchoscopy for clinical purposes. A BAL cell differential and T-cell subtype was requested by the treating physician to supplement the evaluation of patients with suspected interstitial lung disease. We used a commercially available T-cell antibody reagent, approved for analysis of blood via FCM, for T-cell subtyping of clinical BAL specimens. Results: The percentages of CD4 and CD8 T-cell populations, as well as the CD4/CD8 ratios showed excellent correlation with IHC staining of cytocentrifuge slides regardless of the acquisition program used, as long as the gating strategy remained consistent (r ≥ 0.9693 for CD4, r ≥ 0.9589 for CD8, and r ≥ 0.9485 for the CD4/CD8 ratio). Conclusion: These findings validate the use of standardized, commercially available antibody cocktails for BAL lymphocyte subtyping, making this technique available to clinicians and researchers with access to a three-color or four-color flow cytometer.