title: 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).
  
  
  METHODS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Laboratory of Molecular Diagnostics and Immunology and Department of Pathomorphology, National Institute of Tuberculosis and Lung Diseases, Warsaw, Poland.
authors.forename: Adam
authors.initials: A
authors.lastname: Szpechcinski
authors.forename: Piotr
authors.initials: P
authors.lastname: Kopinski
authors.forename: Dorota
authors.initials: D
authors.lastname: Giedronowicz
authors.forename: Adriana
authors.initials: A
authors.lastname: Rozy
authors.forename: Paulina
authors.initials: P
authors.lastname: Jagus
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Szolkowska
authors.forename: Joanna
authors.initials: J
authors.lastname: Chorostowska-Wynimko
mesh_terms: D000328:Adult; D000368:Aged; D000542:Alveolitis, Extrinsic Allergic / Q000276:immunology / Q000473:pathology; D001992:Bronchoalveolar Lavage Fluid / Q000166:cytology* / Q000276:immunology*; D016516:CD4-CD8 Ratio / Q000379:methods*; D005260:Female; D005434:Flow Cytometry / Q000379:methods* / Q000592:standards; D006801:Humans; D016130:Immunophenotyping / Q000379:methods / Q000592:standards; D017563:Lung Diseases, Interstitial / Q000276:immunology* / Q000473:pathology*; D008297:Male; D008875:Middle Aged; D011658:Pulmonary Fibrosis / Q000276:immunology / Q000473:pathology; D015203:Reproducibility of Results; D017565:Sarcoidosis, Pulmonary / Q000276:immunology / Q000473:pathology; D055815:Young Adult