Validation of sputum cytokine immunoassays in COPD

<b>Background:</b> Drug regulators (EMA and FDA) have published recommendations for bioanalytical method validation, however assay performance in complex matrices such as sputum supernatant is rarely discussed. The aim of this study was to validate a panel (27 plex immunoassay) of inflammatory biomarkers measured in sputum supernatant according to regulatory standards. The validated biomarkers were then used in COPD patients to evaluate relationships with sputum cell counts and the presence of bacterial colonisation. <b>Methods:</b> Validation experiments for a 27-plex Luminex assay included matrix dilution, standard recovery, calibration curve establishment and assay reproducibility for PBS processed sputum samples. Samples from COPD patients (n=81) were then assessed. Real time qPCR was performed to identify bacterial colonisation, including H. influenzae (HI). <b>Results:</b> 20 of the 27 panel analytes were classified as validated assays, with 15 having detectable levels in COPD samples. IL-1β, IL-2, IL-4, IL-8, IL-17A, G-CSF, IFN-γ, IP-10, MIP-1α, MIP-1β and TNF-α significantly correlated with sputum neutrophil percentage in COPD. IL-1β, IL-4, IL-8, G-CSF and IFN-γ were significantly increased in patients with HI colonisation, with IL-17A, IP-10 and TNF-α tending towards significance (p=0.06, 0.07 and 0.05). <b>Conclusion:</b> This work validates a panel of biomarkers for use in COPD studies. This panel demonstrated different cytokine profiles in COPD patients with bacterial colonisation.