Flow cytometric enrichment for respiratory epithelial cells in sputum

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.