Quantitative method of macrophage carbon content in induced sputum
n=169）进行诱导痰中碳含量方法学验证和影响因素研究。受试者吸入超声雾化的4.5%高渗盐水并咳出痰液，向痰液中加入约20~30 mL的Saccomanno固定液倒转摇匀，置于阴凉避光处保存。待返回实验室用痰消化液消化痰液，杜氏磷酸缓冲液清洗，离心获得纯细胞悬液。血涂片法涂片，Diff-quick快速染液固定染色。使用光学显微镜在物镜100倍放大并加香柏油对每份样本随机选取的50个形态完好的巨噬细胞拍照，并通过Image J软件处理照片进行巨噬细胞碳含量定量：在挖去细胞核之后，转换灰度使照片呈黑白色，并根据每个细胞染色情况特异性调整灰度值，以保证能计算出细胞中所有的碳颗粒。 结果
用Saccomanno固定液保存的新鲜诱导痰液，经处理后，镜下观察视野杂质少，细胞形态学完整，核质清晰，细胞核呈蓝紫色，胞浆呈淡粉红色或浅蓝色，细胞核偏于一侧呈圆形或肾形，胞浆内黑色碳颗粒清晰可见，大多呈小团块聚集分布，可进行巨噬细胞碳含量定量。诱导痰成功率在无职业颗粒物暴露的一般人群中为63.3%，性别不会影响痰液诱导成功率。采用巨噬细胞碳面积与巨噬细胞胞浆面积比（the proportion of cytoplasm area occupied by carbon particles，PCOC）的中位数作为个体巨噬细胞碳含量的读数，一般人群巨噬细胞碳含量的
M（ P25， P75）为0.83%（0.63%，1.34%），男性为0.79%（0.59%~1.27%），女性为0.95%（0.75%~1.34%）。 结论
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.
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.
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.