Impact of Storage and Transportation Conditions on Breath Samples Collected From Patients With Oesophageal Adenocarcinoma in an International Study

Background A distinct pattern of volatile organic compounds (VOCs) within exhaled breath have been linked to oesophageal cancer. Current methodologies for VOC detection necessitate a hub and spoke model for sample collection and analysis. In this study we reflect on the impact of pre-analysis sample handling and transportation conditions on the quality of the breath samples by mapping the degradation of breath acetone concentration over time since acetone has previously been reported as a marker of sample quality. Methods Breath samples were collected from 75 patients with OAC at a tertiary referral centre in the USA from 2020–2023, (i) at diagnosis, (ii) following neoadjuvant therapy, (iii) following surgical resection, and (iv) at routine follow-up (6–12 months). Breath was collected in PTFE bags and extracted onto Tenax and Carbograph 5 thermal desorption (TD) tubes within 10 minutes. The TD tubes were stored at −80°C and transported on dry ice to a central facility in UK for analysis using gas-chromatography mass-spectrometry (GC–MS). To assess quality of breath samples, peak acetone levels were plotted against the time between storage and analysis. Results Storage and transportation data was available for 71 patients and a total of 160 timepoints. The storage time ranged from 8 to 887 days; 171 ± 376.5 (Average ± 2SD). Until analysis, all samples were maintained at—80°C. Scatter plots of normalised acetone peak areas plotted against storage duration for two GC–MS methods are depicted in Fig. 1 and show no clear downward trend. We identified a few outliers that lay outside the central bulk of the data that most likely represent a sampling or analytical error. Conclusion In this longitudinal study, participants were sampled at clinically distinct timepoints, including pre-surgical fasting and cancer cachectic states, leading to variable ketogenic states. Despite this biological variability, acetone concentrations remained stable from collection to analysis. This demonstrates that storage in thermal desorption tubes at −80°C provides a reproducible method for VOC preservation, reinforcing its suitability for longitudinal breath analysis in clinical research and potential future diagnostic use.