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 > or = 0.9693 for CD4, r > or = 0.9589 for CD8, and r > or = 0.9485 for the CD4/CD8 ratio).


CONCLUSIONS
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.
Pulmonary Division, Indiana University School of Medicine, Indianapolis, 46202, USA.
Patricia A
PA
Smith
Lisa M
LM
Kohli
Karen L
KL
Wood
Chadi A
CA
Hage
Homer L
HL
Twigg
Kenneth S
KS
Knox
D000906:Antibodies / Q000276:immunology*; D001992:Bronchoalveolar Lavage Fluid / Q000166:cytology*; D017252:CD3 Complex / Q000032:analysis; D016516:CD4-CD8 Ratio / Q000379:methods; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007150:Immunohistochemistry; D016130:Immunophenotyping / Q000379:methods*; D017493:Leukocyte Common Antigens / Q000032:analysis; D017563:Lung Diseases, Interstitial / Q000276:immunology / Q000473:pathology; D018655:Lymphocyte Count; D015203:Reproducibility of Results; D017565:Sarcoidosis, Pulmonary / Q000276:immunology / Q000473:pathology; D012984:Software; D013194:Staining and Labeling / Q000379:methods; D016176:T-Lymphocyte Subsets / Q000166:cytology* / Q000276:immunology
D000906:Antibodies; D017252:CD3 Complex; D017493:Leukocyte Common Antigens