Matrix effect in exhaled breath condensate interferon-gamma immunoassay.

Manuscripts presenting cytokine (and other protein) concentrations in exhaled breath condensate (EBC) are commonly published with insufficient control experiments performed to provide confidence that the assay was accurate. Our laboratory has identified false positives in EBC with several forms of immunoassay, including ELISA and multiplex bead arrays. In this current brief investigation, we studied EBC samples from ten subjects and examined for the effect of alteration of the matrix on the assay results reported by a commercially available ELISA assay for a commonly reported EBC cytokine (interferon-gamma (IFN-γ)). The commercial immunoassay reported measurable levels of the IFN-γ signal in all samples. However, when we added to the EBC samples the proteinaceous matrix of the zero-standard from the commercial kit, we could identify no IFN-γ signal. These simple findings are consistent with our earlier observations that the EBC matrix requires adjustment for there to be any validity to immunoassays.

Identification of acid reflux cough using serial assays of exhaled breath condensate pH.

BACKGROUND: Chronic cough is a common problem, frequently caused or exacerbated by acid reflux. Diagnosis of acid reflux cough is haphazard currently, often relying on long therapeutic trials of expensive medications. We tested the hypothesis that the most relevant mechanistic component of acid reflux in chronic cough is when it rises to the level of the airway where acid can potentially be aspirated. We further wished to determine if multi-sample exhaled breath condensate (EBC) pH profiles can identify chronic cough patients likely to respond to proton pump inhibitor therapy. METHODS: 59 subjects were recruited for this study. Initially we examined EBC pH (gas-standardized with Argon) in the setting of 15 experimental pharyngeal acid challenges to determine duration of EBC acidification. Subsequently, we enrolled 22 healthy subjects to determine a normal multi-sample exhaled breath condensate pH profile over 1-3 days. We additionally obtained multi-sample EBC pH profiles in 22 patients with chronic cough. These samples were timed to occur after coughing episodes. Exhaled breath condensate pH was measured after gas standardization. RESULTS: We found that exhaled breath condensate pH is substantially reduced for approximately 15 minutes after pharyngeal acid load. Healthy subjects rarely have any low EBC pH values (defined as < 7.4 based on a normative reference range from 404 healthy subjects). Patients with chronic cough who subsequently responded well to proton pump inhibition (n = 8) invariably had one or more cough episodes associated with EBC acidification. No patient who had normal EBC pH with each of their cough episodes reported a clinically relevant response to proton-pump inhibition. CONCLUSION: Patients whose cough responds to proton pump inhibition have transient exhaled breath condensate acidification with coughing episodes, supporting the role of airway acidification in reflux-triggered cough. Multi-sample EBC pH profiles, involving samples collected immediately subsequent to a coughing episode, may be useful appropriately to direct therapy to those patients with cough who have relevant acid reflux.

Normative data for pH of exhaled breath condensate.

INTRODUCTION: Measurement of pH is one of the simplest and most technically validated biomarkers studied in exhaled breath condensate (EBC). The pH of EBC has been found to be lower than controls in many respiratory disorders. Published data from normal control subjects have been reasonably consistent, but the data sets are not large. This study was undertaken to establish normative EBC pH reference values. PARTICIPANTS: Four hundred four healthy subjects of all ages were enrolled. INTERVENTIONS: Each participant provided a single EBC sample using a disposable collector at modest temperature so that EBC was collected as a liquid. MEASUREMENTS AND RESULTS: Samples of EBC were bubbled with argon gas to standardize for carbon dioxide, and pH was recorded with a calibrated and validated glass microelectrode on stabilization. The median EBC pH was 8.0 with interquartile (25 to 75%) range of 7.8 to 8.1. There were no differences based on age, sex, or race. The distribution is skewed, with 6.4% of EBC samples having a pH range < 7.4. CONCLUSIONS: An extensive normal data set now exists that reveals EBC pH is maintained in a modestly alkaline and tight range in subjects who consider themselves healthy.