Validation of Sputum Biomarker Immunoassays and Cytokine Expression Profiles in COPD

Abstract

Immunoassays are commonly used to assess airway inflammation in sputum samples from chronic obstructive pulmonary disease (COPD) patients. However, assay performance and validation in this complex matrix is inconsistently reported. The aim of this study was to assess the suitability of various immunoassays for use with sputum samples, followed by use of validated immunoassays to evaluate biomarker levels in COPD patients. Assays were assessed for recombinant reference standard suitability, optimal sample dilution, standard recovery in the biological matrix and reproducibility. Validated assays were used to assess sputum supernatants in Cohort A (n = 30 COPD, n = 10 smokers, n = 10 healthy) and Cohort B (n = 81 COPD, n = 15 smokers, n = 26 healthy). Paired baseline and exacerbation samples from 14 COPD patients were assessed in cohort A, and associations with sputum cell counts and bacterial colonisation investigated in cohort B. 25/32 assays passed validation; the primary reason for validation failure was recombinant reference standard suitability and sample dilution effects. Interleukin (IL-)6 and IL-8 were significantly increased in COPD patients compared to healthy subjects and smokers for both cohorts. Tumour necrosis factor (TNF)α and IL-1β were higher in COPD compared to smokers using one immunoassay but not another, partly explained by different absolute recovery rates. IL-1β, IL-2, IL-4, IL-8, IL-17A, Granulocyte colony stimulating factor (G-CSF), Interferon (IFN-)γ, Interferon gamma induced protein (IP-)10, Macrophage inflammatory protein (MIP)-1α, MIP-1β and TNF-α levels correlated with sputum neutrophil percentage in COPD patients. IL-1β, IL-4, IL-8, G-CSF and IFN-γ levels were associated with Haemophilus influenzae colonisation in COPD patients. Current smokers had lower levels of IL-1β, IL-4, IL-8, G-CSF, IFN-γ, IP-10, Monocyte chemoattractant protein (MCP)-1, MIP-1α, MIP-1β and TNF-α. Validated immunoassays applied to sputum supernatants demonstrated differences between COPD patients and controls, the effects of current smoking and associations between Haemophilus influenzae colonisation and higher levels of selected cytokines. Immunoassay validation enabled inflammatory mediators associated with different COPD characteristics to be determined.

Identification of COPD Inflammatory Endotypes Using Repeated Sputum Eosinophil Counts

“…Patients were not included if they were using maintenance antibiotics or oral corticosteroids, or had a previous asthma diagnosis. Sputum data from 48 patients in this analysis have been previously reported, although not for the purpose of assessing eosinophil repeatability [ 9 , 21 ]. All patients provided written informed consent using protocols approved by the local Ethics Committees (05/Q1402/41, 10/H1016/25 and 16/NW/0836).…”

“…The detection of H. influenza , M. catarrhalis , Streptococcus pneumoniae ( S. pneumoniae ), and Pseudomonas Aeruginosa ( P. aeruginosa ), were analysed according to eosinophil sub-group as previously described [ 9 , 21 ]. Further methodological detail can be found in the online data supplement.…”

Comparing chronic obstructive pulmonary disease and depressive disorder in terms of inflammation-related biomarkers

“…IFN-γ levels are highly increased in COPD patients during exacerbation but have also been observed to be lower in stable COPD patients than in control individuals [ 30 ]. IFN-γ levels were associated with Haemophilus influenzae colonization in COPD patients [ 31 ]. IFN-γ also plays an important role in depression.…”

Mucus Structure, Viscoelastic Properties, and Composition in Chronic Respiratory Diseases

“…There is a very poor literature concerning their performance and validation in this complex matrix. Recently, Mulvanny et al [301] published a study about the validation of sputum supernatant biomarkers as well as their practical utility in COPD clinical studies. In the first part of the study, candidate biomarkers were validated through a series of experiments.…”