Dataset Information

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

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-?; 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.

SUBMITTER: Wang F

PROVIDER: S-EPMC6469484 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Publications

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

Wang Fengyan F Liang Zhenyu Z Yang Yuqiong Y Zhou Luqian L Guan Lili L Wu Weiliang W Jiang Mei M Shi Weijuan W Deng Kuimiao K Chen Jianhua J Chen Rongchang R

International journal of chronic obstructive pulmonary disease 20190411

<h4>Purpose</h4>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.<h4>Methods</h4>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 ...[more]

PMID: 31043775

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Project description:PurposeWe recently introduced a sputum cell quality score to rate how cell morphology, cellular debris and squamous cell contamination influence inflammatory cell identification during microscopic evaluation. However, sputum cell quality is generally not considered for the interpretation of sputum fluid phase biomarkers. Therefore, we compared the soluble protein concentrations between sputum samples with different cell quality. The impact of cell quality was compared to other factors potentially affecting soluble biomarker concentrations.MethodsA comprehensive sputum dataset from 154 clinically stable COPD patients was used to analyse the differences and the variability of sputum supernatant concentrations for 23 proteins between low, medium, and high sputum cell quality samples. A model was developed and tested to compare the impact of different factors on sputum supernatant protein levels.ResultsMean percentages of sputum macrophages, neutrophils, eosinophils, monocytes and lymphocytes showed no significant differences between low, medium and high cell quality levels. The mean percentage of squamous cells were lower, while total cell count/mL sputum and cell viability were significantly higher in sputum samples with higher cell quality. The concentrations of Interleukin-6, Interleukin-8 and Tumor Necrosis Factor Receptor 2 were significantly increased in sputum samples of higher cell quality. The variability of most protein concentrations declined with increasing cell quality levels. Sixteen proteins showed significantly negative correlations with the percentage of squamous cells. For 14 proteins we observed a positive correlation with cell number/mL sputum. Multiple regression analysis shows that generally less than 30% of the protein variability can be explained by the included factors.ConclusionSputum cell quality has a significant impact on some soluble biomarker concentrations in sputum supernatant. Sputum samples with low sputum cell quality show a higher variability of fluid phase proteins in comparison to medium and high sputum cell quality levels.

Dataset Information

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

Purpose

Methods

Results

Conclusion

Publications

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

Similar Datasets

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