Abstract
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.
Highlights
Proof of concept for the use of the HBEC antibody panel was determined by staining both stages of the Primary Human Bronchial Epithelial Cells (pHBEC) culture as a positive control, combining intracellular and cell surface (EpCAM) antibody markers (Fig. 1)
Cells were first gated by size and granularity, selecting a minimum/maximum side scatter area (SSC-A)/forward scatter area (FSC-A) to exclude cellular debris
BAL and BW contained a lower proportion of HBECs (2.3 ± 2.4% and 32.5 ± 24.0% respectively, when compared to bronchial brushings (88.9 ± 4.9%); Table 3). These values represent the percentages of HBEC after debris, doublets and dead cells were excluded which is the equivalent to the percentage of live, single, CD45-negative HBECs
Summary
Proof of concept for the use of the HBEC antibody panel was determined by staining both stages of the pHBEC culture as a positive control, combining intracellular (pan-CK) and cell surface (EpCAM) antibody markers (Fig. 1). BAL, BW and bronchial brushing samples from an ongoing clinical study were stained using the HBEC antibody panel and flow cytometry was performed (Fig. 4).
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