[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].
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).


CONCLUSIONS
Flow cytometry reveals similar results compared to immunocytochemistry in the determination of lymphocyte subsets characterised by CD3, CD4 and CD8 antigens.
Bergmann
M
M
Klinik für Allgemeine Innere Medizin, Christian-Albrechts-Universität Kiel.
Bewig
B
B
Tiroke
A
A
Barth
J
J
Fölsch
U R
UR
D000328:Adult; D000368:Aged; D000911:Antibodies, Monoclonal; D001992:Bronchoalveolar Lavage Fluid / Q000276:immunology*; D017252:CD3 Complex / Q000097:blood; D016516:CD4-CD8 Ratio; D015496:CD4-Positive T-Lymphocytes / Q000145:classification / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000145:classification / Q000276:immunology; D003937:Diagnosis, Differential; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007124:Immunoenzyme Techniques; D008297:Male; D008875:Middle Aged; D011658:Pulmonary Fibrosis / Q000175:diagnosis* / Q000276:immunology; D016176:T-Lymphocyte Subsets / Q000145:classification* / Q000276:immunology
D000911:Antibodies, Monoclonal; D017252:CD3 Complex