Simple flow cytometric protocol of CD4+/CD8+ lymphocyte ratio assessment in bronchoalveolar lavage fluids from patients with interstitial lung diseases.

Abstract

Objective: To validate the fast and accurate flow cytometric (FCM) protocol using blood-standardized antibodies for alveolar lymphocyte subtyping with respect to standard immunocytochemistry (IC).

Study design: FCM and IC were applied to immunophenotype T cell subsets in bronchoalveolar lavage (BAL) fluids from patients with interstitial lung diseases. Diagnostic BAL specimens from 50 patients with suspected sarcoidosis, idiopathic pulmonary fibrosis, and hypersensitivity pneumonitis were evaluated by both IC and FCM. In FCM, CD4+ and CD8+ T cells were identified by light scatter gating with CD3 selection using basic tricolor cytometer.

Results: Relative amounts of CD4+, CD8+ T cells, and CD4+/CD8+ ratios demonstrated by the FCM showed excellent, significant correlations with IC results. FCM values did not differ significantly from IC results. However, the sensitivity and specificity of conventional IC staining were not sufficient to assess CD4+/ CD8+ ratio in most idiopathic pulmonary fibrosis cases. Additionally, performing IC immunophenotyping in BAL samples with low lymphocyte content introduced a remarkable error into CD4+/CD8+ ratio assessment.

Conclusion: FCM allowed reliable, precise, and fast T-cell subset measurement in all BAL samples, overcoming the IC disadvantages. Our validated FCM protocol provides diagnostically relevant CD4+/CD8+ ratio determination by simple light scatter gating strategy with CD3 selection.