Cargando…

Validation of Sputum Biomarker Immunoassays and Cytokine Expression Profiles in COPD

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 immun...

Descripción completa

Detalles Bibliográficos
Autores principales: Mulvanny, Alex, Pattwell, Caroline, Beech, Augusta, Southworth, Thomas, Singh, Dave
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9405928/
https://www.ncbi.nlm.nih.gov/pubmed/36009496
http://dx.doi.org/10.3390/biomedicines10081949
_version_ 1784773997925236736
author Mulvanny, Alex
Pattwell, Caroline
Beech, Augusta
Southworth, Thomas
Singh, Dave
author_facet Mulvanny, Alex
Pattwell, Caroline
Beech, Augusta
Southworth, Thomas
Singh, Dave
author_sort Mulvanny, Alex
collection PubMed
description 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.
format Online
Article
Text
id pubmed-9405928
institution National Center for Biotechnology Information
language English
publishDate 2022
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-94059282022-08-26 Validation of Sputum Biomarker Immunoassays and Cytokine Expression Profiles in COPD Mulvanny, Alex Pattwell, Caroline Beech, Augusta Southworth, Thomas Singh, Dave Biomedicines Article 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. MDPI 2022-08-11 /pmc/articles/PMC9405928/ /pubmed/36009496 http://dx.doi.org/10.3390/biomedicines10081949 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Mulvanny, Alex
Pattwell, Caroline
Beech, Augusta
Southworth, Thomas
Singh, Dave
Validation of Sputum Biomarker Immunoassays and Cytokine Expression Profiles in COPD
title Validation of Sputum Biomarker Immunoassays and Cytokine Expression Profiles in COPD
title_full Validation of Sputum Biomarker Immunoassays and Cytokine Expression Profiles in COPD
title_fullStr Validation of Sputum Biomarker Immunoassays and Cytokine Expression Profiles in COPD
title_full_unstemmed Validation of Sputum Biomarker Immunoassays and Cytokine Expression Profiles in COPD
title_short Validation of Sputum Biomarker Immunoassays and Cytokine Expression Profiles in COPD
title_sort validation of sputum biomarker immunoassays and cytokine expression profiles in copd
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9405928/
https://www.ncbi.nlm.nih.gov/pubmed/36009496
http://dx.doi.org/10.3390/biomedicines10081949
work_keys_str_mv AT mulvannyalex validationofsputumbiomarkerimmunoassaysandcytokineexpressionprofilesincopd
AT pattwellcaroline validationofsputumbiomarkerimmunoassaysandcytokineexpressionprofilesincopd
AT beechaugusta validationofsputumbiomarkerimmunoassaysandcytokineexpressionprofilesincopd
AT southworththomas validationofsputumbiomarkerimmunoassaysandcytokineexpressionprofilesincopd
AT singhdave validationofsputumbiomarkerimmunoassaysandcytokineexpressionprofilesincopd