Quantitative method of macrophage carbon content in induced sputum

Background  Macrophage carbon content, as a biomarker of novel particulate matter exposure, can be obtained by inducing sputum. At present, there is no effective method for induced sputum preservation in molecular epidemiological investigations, and there is no domestic report on a quantitative method for evaluating the macrophage carbon content in induced sputum. The application of this marker to the exposure assessment of carbon-containing particles requires standardized research and verification.

Objective  This study aims to explore and establish a new preservation method for freshly induced sputum and a quantitative method for the carbon content of airway macrophages in induced sputum.

Methods  In this study, workers without occupational exposure to particulate matters from waterworks were selected as a general population (n=169) for method validation and influencing factor study. The participants inhaled 4.5% hypertonic saline water and coughed up sputum, and 20-30 mL Saccomanno's fixative was added in the sputum specimens, shaken well, and stored in a cool place shielded from light. Transferred to the lab, the sputum specimens were digested by the addition of sputasol, washed with Dulbecco's phosphate-buffered saline, and centrifuged to obtain pure cell suspension. The concentrated cell suspension was deposited on a slide by blood smear method, followed by Diff-quick staining. Finally, 50 macrophages with an intact morphology were randomly selected from each sample and were photographed with an optical microscope at 100×with oil-immersed, and carbon content was quantified by Image J software:after the nuclei were removed, color pictures were converted to gray images, and gray values were calibrated specifically for each cell stain to ensure that all carbon particles in the cell could be calculated.

Results  The freshly induced sputum preserved with Saccomanno's fixative had fewer impurities under optical microscope, and the cells had an intact cell morphology, clearly round or kidney-shaped bluish-purple nuclei on the one side, light pink or light blue cytoplasm, and black carbon particles in the cytoplasm, most of which were clustered and distributed in small clumps, suggesting that the prepared induced sputum could be used to quantify the carbon content of airway macrophages. The success rate of induced sputum was 63.3% in the selected general population without occupational particulate matter exposure, and sex did not affect the success rate of sputum induction. Using the median of the proportion of cytoplasm area occupied by carbon particles as individual's carbon content of airway macrophage, the median (P₂₅, P₇₅) carbon content of airway macrophages in the general population was 0.83% (0.63%, 1.34%), that for males was 0.79% (0.59%-1.27%), and that for females was 0.95% (0.75%-1.34%).

Conclusion  This convenient, reliable, and efficient method can well preserve induced sputum samples, and can be used to the large-scale quantification of carbon content of airway macrophages based on induced sputum.