Flow cytometric enrichment for respiratory epithelial cells in sputum

Authors

  • Petra S. Kraemer,

    1. Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington
    Search for more papers by this author
  • Carissa A. Sanchez,

    1. Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington
    2. Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington
    Search for more papers by this author
  • Gary E. Goodman,

    1. Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington
    2. Swedish Hospital Medical Center Tumor Institute, Seattle, Washington
    Search for more papers by this author
  • James Jett,

    1. Department of Pulmonary Medicine and Medical Oncology, Mayo Clinic, Rochester, Minnesota
    Search for more papers by this author
  • Peter S. Rabinovitch,

    1. Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington
    2. Department of Pathology, University of Washington, Seattle, Washington
    Search for more papers by this author
  • Brian J. Reid

    Corresponding author
    1. Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington
    2. Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington
    3. Department of Medicine, University of Washington, Seattle, Washington
    4. Department of Genome Sciences, University of Washington, Seattle, Washington
    • Member, Divisions of Human Biology and Public Health Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N., C1-157, PO Box 19024, Seattle, WA 98109-1024
    Search for more papers by this author

Abstract

Background

Induced sputum, in contrast to bronchoscopic biopsies and lavages, is an easily obtained source of biological specimens. However, obtaining abnormal exfoliated cells for detailed molecular studies is limited because respiratory epithelial cells comprise only about 1% of sputum cell populations.

Methods

We developed a multiparameter flow sorting strategy to purify epithelial cells from nonepithelial sputum cells, using anti-cytokeratin antibody AE1/AE3 to recognize human epithelial cells and DAPI to stain DNA. We excluded cells with a high degree of side-scatter, which were composed predominantly of squamous cells and contaminating macrophages. The remaining cytokeratin-positive respiratory epithelial cells were then sorted based on anti-cytokeratin (PE) vs DNA (DAPI) parameters.

Results

In this proof of principle study, the AE1AE3 cytokeratin/DNA flow sorting strategy enriched rare diploid respiratory epithelial cells from an average of 1.1% of cells in unsorted induced sputum samples to average purities of 42%. Thus, AE1AE3 flow-sorting results in a 38-fold enrichment of these cells.

Conclusions

We report a multiparameter flow cytometric assay to detect and enrich rare respiratory epithelial cells from induced sputum samples to average purities of 42%. With further development, this methodology may be useful as part of a molecular screening approach of populations at high risk for lung cancer. © 2004 Wiley-Liss, Inc.

Ancillary