Novel flow cytometry approach to identify bronchial epithelial cells from healthy human airways.
Sampling various compartments within the lower airways to examine human bronchial epithelial cells (HBEC) is essential for understanding numerous lung diseases. Conventional methods to identify HBEC in bronchoalveolar lavage (BAL) and wash (BW) have throughput limitations in terms of efficiency and ensuring adequate cell numbers for quantification. Flow cytometry can provide high-throughput quantification of cell number and function in BAL and BW samples, while requiring low cell numbers. To date, a flow cytometric method to identify HBEC recovered from lower human airway samples is unavailable. In this study we present a flow cytometric method identifying HBEC as CD45 negative, EpCAM/pan-cytokeratin (pan-CK) double-positive population after excluding debris, doublets and dead cells from the analysis. For validation, the HBEC panel was applied to primary HBEC resulting in 98.6% of live cells. In healthy volunteers, HBEC recovered from BAL (2.3% of live cells), BW (32.5%) and bronchial brushing samples (88.9%) correlated significantly (p = 0.0001) with the manual microscopy counts with an overall Pearson correlation of 0.96 across the three sample types. We therefore have developed, validated, and applied a flow cytometric method that will be useful to interrogate the role of the respiratory epithelium in multiple lung diseases.
Chan-Yeung Center for Occupational and Environmental Respiratory Disease, Department of Medicine, Division of Respiratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada.
Danay
D
Maestre-Batlle
Chan-Yeung Center for Occupational and Environmental Respiratory Disease, Department of Medicine, Division of Respiratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada.
Olga M
OM
Pena
Chan-Yeung Center for Occupational and Environmental Respiratory Disease, Department of Medicine, Division of Respiratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada.
Jeremy A
JA
Hirota
Chan-Yeung Center for Occupational and Environmental Respiratory Disease, Department of Medicine, Division of Respiratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada.
Evelyn
E
Gunawan
Chan-Yeung Center for Occupational and Environmental Respiratory Disease, Department of Medicine, Division of Respiratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada.
Christopher F
CF
Rider
Chan-Yeung Center for Occupational and Environmental Respiratory Disease, Department of Medicine, Division of Respiratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada.
Darren
D
Sutherland
Department of Pediatrics, University of North Carolina at Chapel Hill, USA.
Neil E
NE
Alexis
Chan-Yeung Center for Occupational and Environmental Respiratory Disease, Department of Medicine, Division of Respiratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada.
Chris
C
Carlsten
D000328:Adult; D000368:Aged; D015415:Biomarkers / Q000378:metabolism; D001980:Bronchi / Q000166:cytology*; D002452:Cell Count; D002470:Cell Survival; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006262:Health; D006801:Humans; D008297:Male; D015203:Reproducibility of Results; D055815:Young Adult
D015415:Biomarkers; D005456:Fluorescent Dyes