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Flow cytometry of sputum: assessing inflammation and immune response elements in the bronchial airways**
Citation:
Lay, J., D. Peden, AND N. Alexis. Flow cytometry of sputum: assessing inflammation and immune response elements in the bronchial airways**. INHALATION TOXICOLOGY. Informa Healthcare USA, New York, NY, 23(7):392-406, (2011).
Impact/Purpose:
THIS MANUSCRIIPT WAS ORIGINALLY CLEARED VIA PAPER FILE EPHD-12-058 AND ENTERERD IN THE SCIENCE INVENTORY - title was changed for publication
Description:
Rationale: The evaluation of sputum leukocytes by flow cytometry 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 dithiothrietol, filtered and total leukocytes acquired. Multicolor flow cytometry 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, CDI4, HLA-DR) that differentiated granulocytes from monocytes and macrophages. These gating strategies allowed identification of small populations of eosinophils, CD11c+myeloiddendritic cells, B cells and NK cells. Conclusions. Multicolor flow cytometry can be successfully applied to sputum samples to identify and characterize leukocyte populations residing on the surfaces of the central airways.