title: Flow cytometry of sputum: assessing inflammation and immune response elements in the bronchial airways.
abstract: BACKGROUND
  The evaluation of sputum leukocytes by flow cytometry (FCM) is an opportunity to assess characteristics of cells residing in the central airways, yet it is hampered by certain inherent properties of sputum including mucus and large amounts of contaminating cells and debris.
  
  
  OBJECTIVE
  To develop a gating strategy based on specific antibody panels in combination with light scatter properties for flow cytometric evaluation of sputum cells.
  
  
  METHODS
  Healthy and mild asthmatic volunteers underwent sputum induction. Manually selected mucus "plug" material was treated with dithiothreitol, filtered and total leukocytes acquired. Multicolor FCM was performed using specific gating strategies based on light scatter properties, differential expression of CD45 and cell lineage markers to discriminate leukocytes from squamous epithelial cells and debris.
  
  
  RESULTS
  The combination of forward scatter and CD45 expression reliably segregated sputum leukocytes from contaminating squamous epithelial cells and debris. Overlap of major leukocyte populations (neutrophils, macrophages/monocytes) required the use of specific antibodies (e.g. CD16, CD64, CD14, HLA-DR) that differentiated granulocytes from monocytes and macrophages. These gating strategies allowed identification of small populations of eosinophils, CD11c+ myeloid dendritic cells, B-cells and natural killer cells.
  
  
  CONCLUSIONS
  Multicolor FCM can be successfully applied to sputum samples to identify and characterize leukocyte populations residing on the surfaces of the central airways.
  
  
  CONCLUSIONS
  This research describes detailed methods to overcome difficulties associated with FCM of sputum samples, which previously has been lacking in the literature. FCM of sputum samples can provide valuable information on inflammation and immunological response elements in the bronchial airways for both clinical diagnostic and research applications and can be a useful tool in inhalation toxicology for assessing health effects of inhaled environmental pollutants.
authors.affiliation: Center for Environmental Medicine, Asthma and Lung Biology, University of North Carolina School of Medicine, Chapel Hill, USA. jcl@med.unc.edu
authors.forename: John C
authors.initials: JC
authors.lastname: Lay
authors.forename: David B
authors.initials: DB
authors.lastname: Peden
authors.forename: Neil E
authors.initials: NE
authors.lastname: Alexis
mesh_terms: D001249:Asthma / Q000276:immunology* / Q000378:metabolism; D001402:B-Lymphocytes / Q000378:metabolism; D015415:Biomarkers; D001980:Bronchi / Q000473:pathology*; D003713:Dendritic Cells / Q000378:metabolism; D004804:Eosinophils / Q000276:immunology; D004847:Epithelial Cells / Q000166:cytology / Q000276:immunology / Q000473:pathology; D005434:Flow Cytometry; D006801:Humans; D007113:Immunity, Innate; D007249:Inflammation / Q000276:immunology / Q000473:pathology*; D019570:Inhalation Exposure; D007694:Killer Cells, Natural / Q000378:metabolism; D017493:Leukocyte Common Antigens / Q000378:metabolism; D007958:Leukocyte Count; D008264:Macrophages / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D009000:Monocytes / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D009093:Mucus / Q000378:metabolism; D009504:Neutrophils / Q000276:immunology / Q000473:pathology; D010126:Ozone / Q000633:toxicity; D013183:Sputum / Q000737:chemistry / Q000166:cytology* / Q000276:immunology*
chemical_list: D015415:Biomarkers; D010126:Ozone; D017493:Leukocyte Common Antigens; C516769:PTPRC protein, human