Quality-Controlled Sputum Analysis by Flow Cytometry.

Area of Science:

• Immunology
• Pulmonology
• Biotechnology

Background:

• Sputum analysis is crucial for lung health assessment but traditional cytology is time-consuming and often yields inadequate samples due to debris and squamous epithelial cells (SECs).
• Flow cytometry provides a high-throughput alternative for cellular analysis, capable of excluding unwanted cellular components.

Purpose of the Study:

• To present an efficient flow cytometry protocol for analyzing sputum cellular content.
• To establish a method for high-throughput diagnostics of lung diseases using sputum samples.

Main Methods:

• Developed a protocol to dissociate sputum into single-cell suspensions, followed by antibody staining and fixation.
• Implemented a gating strategy to exclude debris, dead cells (including SECs), and cell doublets.
• Characterized hematopoietic and epithelial subsets using CD45 expression and identified lung-specific macrophages for quality control.

Main Results:

• The protocol successfully processed sputum samples for flow cytometric analysis, enabling exclusion of debris and SECs.
• Demonstrated the ability to differentiate between hematopoietic and epithelial cell lineages within sputum.
• Validated the method's applicability across different flow cytometers (Navios EX, LSR II, Lyric) and its adaptability for additional cellular markers.

Conclusions:

• Flow cytometry offers a robust, efficient, and adaptable method for comprehensive sputum analysis.
• This protocol enhances the utility of sputum for high-throughput diagnostics of lung diseases.
• The method provides a reliable quality control measure by identifying lung-specific cells.