Assessment of breath volatile organic compounds in acute cardiorespiratory breathlessness: a protocol describing a prospective real-world observational study

Introduction Patients presenting with acute undifferentiated breathlessness are commonly encountered in admissions units across the UK. Existing blood biomarkers have clinical utility in distinguishing patients with single organ pathologies but have poor discriminatory power in multifactorial presentations. Evaluation of volatile organic compounds (VOCs) in exhaled breath offers the potential to develop biomarkers of disease states that underpin acute cardiorespiratory breathlessness, owing to their proximity to the cardiorespiratory system. To date, there has been no systematic evaluation of VOC in acute cardiorespiratory breathlessness. The proposed study will seek to use both offline and online VOC technologies to evaluate the predictive value of VOC in identifying common conditions that present with acute cardiorespiratory breathlessness. Methods and analysis A prospective real-world observational study carried out across three acute admissions units within Leicestershire. Participants with self-reported acute breathlessness, with a confirmed primary diagnosis of either acute heart failure, community-acquired pneumonia and acute exacerbation of asthma or chronic obstructive pulmonary disease will be recruited within 24 hours of admission. Additionally, school-age children admitted with severe asthma will be evaluated. All participants will undergo breath sampling on admission and on recovery following discharge. A range of online technologies including: proton transfer reaction mass spectrometry, gas chromatography ion mobility spectrometry, atmospheric pressure chemical ionisation-mass spectrometry and offline technologies including gas chromatography mass spectroscopy and comprehensive two-dimensional gas chromatography-mass spectrometry will be used for VOC discovery and replication. For offline technologies, a standardised CE-marked breath sampling device (ReCIVA) will be used. All recruited participants will be characterised using existing blood biomarkers including C reactive protein, brain-derived natriuretic peptide, troponin-I and blood eosinophil levels and further evaluated using a range of standardised questionnaires, lung function testing, sputum cell counts and other diagnostic tests pertinent to acute disease. Ethics and dissemination The National Research Ethics Service Committee East Midlands has approved the study protocol (REC number: 16/LO/1747). Integrated Research Approval System (IRAS) 198921. Findings will be presented at academic conferences and published in peer-reviewed scientific journals. Dissemination will be facilitated via a partnership with the East Midlands Academic Health Sciences Network and via interaction with all UK-funded Medical Research Council and Engineering and Physical Sciences Research Council molecular pathology nodes. Trial registration number NCT0367299

Development and validation of a panel of volatile biomarkers of airway eosinophilia in severe asthma.

Type 2 inflammation and airway eosinophilia have an incidence in 40-60% of severe asthmatics. Therefore, a number of biological therapies have been developed to target the type 2 inflammatory pathways involved in eosinophil activation, such as anti-IL5 and anti-IL5 receptor-α monoclonal antibodies. There is, therefore, the need to develop non-invasive biomarkers to assess airway eosinophilia, which seems to play an increasingly important role to improve severe asthmatics’ stratification for eosinophil targeted therapies This study aimed to develop a panel of volatile biomarkers of airway eosinophilia identifying, by GC-MS analysis and statistical modelling, volatile organic compounds (VOCs) in severe asthmatics’ sputum headspace samples, which were able to discriminate with a moderate accuracy eosinophil-enriched and non-eosinophil-enriched sputum headspaces. In order to optimise the headspace sampling method, the headspace background signal, generated within the sampling system in absence of sputum sample, was characterised, and its daily abundance variations were evaluated. Furthermore, the discovered panel of VOCs was validated in exhaled breath of severe asthmatics eligible for anti-IL5/Rα treatments by targeted GCxGC-FID/MS analysis. The statistical model developed showed a high accuracy to predict severe asthmatics’ one-year response to anti-IL5/Rα therapies. The in vitro selected VOCs were also validated in exhaled breath of exacerbating eosinophilic asthmatics and exacerbating non-eosinophilic asthmatics - whose classification was based on the blood eosinophil count threshold of 0.5x109/L - and healthy volunteers. The panel of VOCs revealed a high discriminatory accuracy among acute eosinophilic asthmatics, acute non-eosinophilic asthmatics and healthy volunteers, suggesting that the selected VOCs represent a promising, non-invasive clinical tool for asthma exacerbation prediction. A future challenge will be to identify the metabolic pathways, in activated eosinophil cultures, which may be involved in the origin of the selected VOCs, in order to confirm their inflammatory origin.</p

Volatile organic compounds in a headspace sampling system and asthmatics sputum samples.

Background:The headspace of a biological sample contains exogenous VOCs present within the sampling environment which represent the background signal.Study aims:This study aimed to characterise the background signal generated from a headspace sampling system in a clinical site, to evaluate intra- and inter-day variation of background VOC and to understand the impact of a sample itself upon commonly reported background VOC using sputum headspace samples from severe asthmatics.Methods:The headspace, in absence of a biological sample, was collected hourly from 11am to 3pm within a day (time of clinical samples acquisition), and from Monday to Friday in a week, and analysed by thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS). Chemometric analysis identified 1120 features, 37 of which were present in at least the 80% of all the samples. The analyses of intra- and inter-day background variations were performed on thirteen of the most abundant features, ubiquitously present in headspace samples. The concentration ratios relative to background were reported for the selected abundant VOC in 36 asthmatic sputum samples, acquired from 36 stable severe asthma patients recruited at Glenfield Hospital, Leicester, UK.Results:The results identified no significant intra- or inter-day variations in compounds levels and no systematic bias of z-scores, with the exclusion of benzothiazole, whose abundance increased linearly between 11am and 3pm with a maximal intra-day fold change of 2.13. Many of the identified background features are reported in literature as components of headspace of biological samples and are considered potential biomarkers for several diseases. The selected background features were identified in headspace of all severe asthma sputum samples, albeit with varying levels of enrichment relative to background.Conclusion:Our observations support the need to consider the background signal derived from the headspace sampling system when developing and validating headspace biomarker signatures using clinical samples

Volatile organic compounds in a headspace sampling system and asthmatics sputum samples

The headspace of a biological sample contains exogenous volatile organic compounds (VOCs) present within the sampling environment which represent the background signal. This study aimed to characterise the background signal generated from a headspace sampling system in a clinical site, to evaluate intra- and inter-day variation of background VOC and to understand the impact of a sample itself upon commonly reported background VOC using sputum headspace samples from severe asthmatics. The headspace, in absence of a biological sample, was collected hourly from 11am to 3pm within a day (time of clinical samples acquisition), and from Monday to Friday in a week, and analysed by thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS). Chemometric analysis identified 1120 features, 37 of which were present in at least the 80% of all the samples. The analyses of intra- and inter-day background variations were performed on 13 of the most abundant features, ubiquitously present in headspace samples. The concentration ratios relative to background were reported for the selected abundant VOC in 36 asthmatic sputum samples, acquired from 36 stable severe asthma patients recruited at Glenfield Hospital, Leicester, UK. The results identified no significant intra- or inter-day variations in compounds levels and no systematic bias of z-scores, with the exclusion of benzothiazole, whose abundance increased linearly between 11am and 3pm with a maximal intra-day fold change of 2.13. Many of the identified background features are reported in literature as components of headspace of biological samples and are considered potential biomarkers for several diseases. The selected background features were identified in headspace of all severe asthma sputum samples, albeit with varying levels of enrichment relative to background. Our observations support the need to consider the background signal derived from the headspace sampling system when developing and validating headspace biomarker signatures using clinical samples