Sputum Biomarkers in Chronic Obstructive Pulmonary Disease

Abstract

Introduction: Chronic obstructive pulmonary disease (COPD) is characterised by persistent inflammation and airflow limitation. The heterogeneity of the condition results in considerable variation in clinical manifestations and pathophysiological features present in different patients and it is recognized that different management strategies may be required for COPD subgroups. The complex network of cells, inflammatory mediators and signalling proteins involved in the chronic inflammation observed in COPD patients provides a wealth of potential biomarker measurements that may be used to monitor disease activity, identify patient endotypes, guide appropriate pharmacological treatments and measure intervention effects within early phase clinical trials. Immunoassays are commonly used to quantify sputum biomarkers, however assay performance and validation in this complex matrix is inconsistently reported. Aims: 1. To validate plate-based immunoassays to detect a range of inflammatory mediators in COPD sputum supernatant. 2. To apply validated immunoassays to sputum samples collected from healthy controls, COPD patients and healthy subjects after LPS challenge in order to evaluate biomarker potential. 3. To validate a flow cytometry assay for use on COPD sputum cells. Methods: Validation experiments included parallelism, standard recovery, calibration curve establishment, antibody optimisation, specificity assessment and assay reproducibility. Samples from stable and exacerbating COPD patients, smokers, healthy subjects and LPS challenged healthy subjects were assessed. Results: A comprehensive development and validation process resulted in the validation of 25 out of 32 analyte measurements in sputum samples using a combination of ELISA and Luminex assays and quantification of phospho-p38 within sputum neutrophils, macrophages and eosinophils via flow cytometry. I observed different validation results for immunoassays measuring the same analyte. Validated immunoassays applied to sputum supernatants demonstrated changes in inflammatory mediators induced by LPS challenge, differences between COPD patients and controls, changes during a COPD exacerbation, the effects of current smoking, correlations with sputum neutrophilia and associations between Haemophilus influenzae colonisation and higher levels of selected cytokines. Conclusion: My research highlights the importance of sputum immunoassay validation prior to application within a clinical study. The validation results for immunoassays that measure the same analyte can differ, which may influence the results obtained when comparing COPD patients to controls. My research demonstrates the importance of interpretation of assay validation data prior to analysis of data generated using the assay. Within this thesis, validation approaches that can be easily applied to plate-based immunoassays and flow cytometry assays for use within sputum samples are reported.

Date of Award	1 Aug 2023
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Thomas Southworth (Supervisor) & Sukh Singh (Supervisor)