Reproducibility of fluid-phase measurements in PBS-treated sputum supernatant of healthy and stable COPD subjects

Fengyan Wang1 State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510120, People’s Republic of China, [email protected]

Zhenyu Liang1 State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510120, People’s Republic of China, [email protected]

Yuqiong Yang1 State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510120, People’s Republic of China, [email protected]

Luqian Zhou1 State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510120, People’s Republic of China, [email protected]

Lili Guan1 State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510120, People’s Republic of China, [email protected]

Weiliang Wu1 State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510120, People’s Republic of China, [email protected]

Mei Jiang1 State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510120, People’s Republic of China, [email protected]

Weijuan Shi1 State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510120, People’s Republic of China, [email protected]

Kuimiao Deng1 State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510120, People’s Republic of China, [email protected]

Jianhua Chen2 Respiratory Medicine Department, Guangzhou Panyu Central Hospital, Guangzhou 511400, People’s Republic of China

Rongchang Chen1 State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510120, People’s Republic of China, [email protected]Correspondence[email protected]

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Abstract

Purpose

The purpose of this study was to investigate the reproducibility of fluid-phase measurements in PBS-treated sputum supernatant, processed using the two-step method, of healthy and stable COPD individuals.

Methods

Nine healthy subjects and 23 stable COPD patients provided sputum twice within 6 days. A two-step sputum processing method was used to obtain PBS-treated supernatant and sputum cells. Soluble protein markers and IgG and IgM autoantibody profiles in PBS supernatant were analyzed using customized microarrays. Repeatability of measurements was assessed by paired-sample testing and an intraclass correlation coefficient, then graphically reported by Bland–Altman plot.

Results

There was no significant difference between the repeated detection of 8/10 types of soluble protein markers, all 13 types of IgG autoantibodies, and 12/13 types of corresponding IgM autoantibodies in PBS supernatant. The repeatability of measurements in PBS supernatant was substantial to very good for interleukin 6 (IL6), IL8, IL13, IL10, IL33, vascular endothelial growth factor, soluble receptor for advanced glycation end-products, and tumor necrosis factor-α; for IgG autoantibodies against aggrecan, centromere protein B (CENP-B), collagen II, collagen IV, cytochrome C, elastin, heat shock protein 47 (HSP47), HSP70, and La/Sjögren syndrome type B antigen; for IgM autoantibodies against CENP-B, collagen I, collagen II, collagen IV, cytokeratin 18, and HSP70; and for sputum neutrophils, macrophages and eosinophils count. Bland–Altman plots suggested good consistency within repeated measurements. Stable COPD patients differed from healthy subjects in the proportion of neutrophils and eosinophils; relative fluorescence intensity of anti-cytochrome C IgG, anti-aggrecan IgM, and anti-cytochrome C IgM. There was a significant positive correlation for stable COPD patients between sputum anti-collagen II IgG and post-bronchodilator FEV₁%.

Conclusion

We confirmed fluid-phase measurements in PBS-treated sputum supernatant by high-throughput techniques with good repeatability. We demonstrated the presence of IgG and IgM autoantibodies to multiple antigens in the airways of COPD patients.

Keywords:

Supplementary materials

Figure S1 Scatter plots of soluble protein markers with pair-wise comparisons of the two measurements. Scatter plots of (A) IL1β; (B) IL5; (C) IL6; (D) IL8; (E) IL10; (F) IL13; (G) IL33; (H) sRAGE; (I) TNFα; and (J) VEGF.

Abbreviations: IL, interleukin; sRAGE, soluble receptor for advanced glycation end-products; TNFα, tumor necrosis factor-α; VEGF, vascular endothelial growth factor.

Figure S2 Scatter plots of IgG autoantibodies with pair-wise comparisons of the two measurements. Scatter plots of (A) anti-aggrecan IgG; (B) anti-amyloid IgG; (C) anti-CENP-B IgG; (D) anti-collagen I IgG; (E) anti-collagen II IgG; (F) anti-collagen IV IgG; (G) anti-cytochrome C IgG; (H) anti-cytokeratin 18 IgG; (I) anti-elastin IgG; (J) anti-HSP47 IgG; (K) anti-HSP70 IgG; (L) anti-SS-B IgG; and (M) anti-vimentin IgG.

Abbreviations: CENP-B, centromere protein B; HSP47, heat shock protein 47; HSP70, heat shock protein 70; SS-B, La/Sjögren syndrome type B antigen.

Figure S3 Scatter plots of IgM autoantibodies with pair-wise comparisons of the two measurements. Scatter plots of (A) anti-aggrecan IgM; (B) anti-amyloid IgM; (C) anti-CENP-B IgM; (D) anti-collagen I IgM; (E) anti-collagen II IgM; (F) anti-collagen IV IgM; (G) anti-cytochrome C IgM; (H) anti-cytokeratin 18 IgM; (I) anti-elastin IgM; (J) anti-HSP47 IgM; (K) anti-HSP70 IgM; (L) anti-SS-B IgM; (M) anti-vimentin IgM.

Abbreviations: CENP-B, centromere protein B; HSP47, heat shock protein 47; HSP70, heat shock protein 70; SS-B, La/Sjögren syndrome type B antigen.

Figure S4 Scatter plots of sputum cell counts with pair-wise comparisons of the two measurements. Scatter plots of (A) sputum neutrophils; (B) sputum macrophages; (C) sputum eosinophils; and (D) scatter plots of sputum lymphocytes.

Table S1 All measurement results

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Table S2 Comparison of measurements between groups

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Acknowledgments

We thank Ryan Chastain-Gross, PhD, from Liwen Bianji, Edanz Group China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript. This study was supported by National Key R&D Program of China (2017YFC1310600, 2017YFC1310601), the Guangzhou Healthcare Collaborative Innovation Major Project (201604020012), Medical Scientific Research Foundation of Guangdong Province (C2017050), and Postdoctoral start-up funding of Guangzhou (191710).

Author contributions

All authors contributed to data analysis, drafting or revising the article, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.

Disclosure

The authors report no conflicts of interest in this work.