A Comprehensive Flow Cytometry Panel for Analysis of Idiopathic Subglottic Stenosis

Abstract

Objective: To present a comprehensive flow cytometry panel for idiopathic subglottic stenosis (iSGS).

Study design: Controlled ex vivo cohort study.

Setting: Tertiary care academic hospital in a metropolitan area.

Methods: Flow cytometry and single-cell RNA sequencing were performed on 9 paired normal and scar tissue samples from iSGS patients. Flow cytometry was used to assess the presence of myeloid (CD11b, CD14, CD15, Siglec8), lymphoid (CD3, CD4, CD8, gamma delta [γδ], FOXP3), endothelial (CD31), fibroblast (CD90, SMA), and epithelial (CD326, CK5) markers.

Results: On flow cytometry, iSGS scar is characterized by an increased presence of myeloid, lymphoid, endothelial, and fibroblast cell types, but a decreased presence of epithelial cells. In the myeloid lineage, iSGS scar samples demonstrated increased CD11b⁺ monocytes (P < .001), Siglec8⁺ eosinophils (P = .03), and CD14⁺ monocytes (P = .02). In the lymphoid lineage, iSGS scar demonstrated increased CD3⁺ T-cells (P < .001), CD4⁺ helper T-cells (P < .001), γδ⁺ T-cells (P < .001), and FOXP3⁺ regulatory T-cells (P = .002). iSGS scar exhibited specific increases in CD90⁺ (P = .04) and SMA⁺ (P < .001) fibroblasts but decreased CD326⁺ (E-cadherin) epithelial cells (P = .01) relative to normal samples.

Conclusion: We present a comprehensive flow cytometry panel for iSGS. This flow panel may serve as a common platform among airway scientists to elucidate the cellular mechanisms underpinning iSGS and other upper airway pathologies. Scar iSGS samples demonstrate a distinct cellular profile relative to normal iSGS specimens, exhibiting increased fibroblast, endothelial, and inflammatory cell types but decreased epithelium.

Keywords: flow cytometry; idiopathic subglottic stenosis; laryngotracheal stenosis; single‐cell RNA sequencing.