[Analysis of lymphocyte populations with flow cytometry in routine bronchoalveolar lavage diagnosis: comparison of a 3-color method with the alkaline phosphatase anti-alkaline phosphatase immunohistochemistry]

Abstract

Background: Identification of lymphocyte phenotypes in bronchoalveolar lavage (BAL) plays a crucial role in the diagnosis of interstitial lung disease. Cells staining positive for specific monoclonal antibodies may be detected by immunocytochemistry or flow cytometry (FCM). The present study compares a three-colour FCM-approach to a standard APAAP protocol for immunocytochemistry.

Methods: BAL-specimens of 22 patients with various lung diseases were investigated. Inclusion criteria was a lymphocytosis of > 10% of all BAL-cells. After the preparation of cytocentrifuge slides, staining was performed with monoclonal antibodies to CD3, CD4 and CD8 following the APAAP-protocol. FCM-analysis was performed with the following panel of conjugates: CD3-FITC, CD4- or CD8-PE, CD45-perCP. Lymphocytes were gated by their SSC/CD45 characteristics. T-helper and T-suppressor percentages were evaluated by quadrant analysis of CD3/CD4 or CD3/CD8 histograms.

Results: With the exception of CD3, where the range of values was quite narrow (10% variance), the correlation between the two methods was excellent (CD4: r = 0.98; CD8: r = 0.99; CD4/CD8: r = 0.96; p < 0.0001).

Conclusion: Flow cytometry reveals similar results compared to immunocytochemistry in the determination of lymphocyte subsets characterised by CD3, CD4 and CD8 antigens.