Discovery and Validation of a Volatile Signature of Eosinophilic Airway Inflammation in Asthma.

RATIONALE: Volatile organic compounds (VOCs) in asthmatic breath may be associated with sputum eosinophilia. We developed a volatile biomarker-signature to predict sputum eosinophilia in asthma. METHODS: VOCs emitted into the space above sputum samples (headspace) from severe asthmatics (n=36) were collected onto sorbent tubes and analysed using thermal desorption gas chromatography-mass spectrometry (TD-GC-MS). Elastic net regression identified stable VOCs associated with sputum eosinophilia ≥3% and generated a volatile biomarker signature. This VOC signature was validated in breath samples from: (I) acute asthmatics according to blood eosinophilia ≥0.3x109cells/L or sputum eosinophilia of ≥ 3% in the UK EMBER consortium (n=65) and U-BIOPRED-IMI consortium (n=42). Breath samples were collected onto sorbent tubes (EMBER) or Tedlar bags (U-BIOPRED) and analysed by gas-chromatography-mass spectrometry (GC×GC-MS -EMBER or GC-MS -U-BIOPRED). MAIN RESULTS: The in vitro headspace identified 19 VOCs associated with sputum eosinophilia and the derived VOC signature yielded good diagnostic accuracy for sputum eosinophilia ≥ 3% in headspace (AUROC (95% CI) 0.90(0.80-0.99), p<0.0001), correlated inversely with sputum eosinophil % (rs= -0.71, p<0.0001) and outperformed FeNO (AUROC (95% CI) 0.61(0.35-0.86). Analysis of exhaled breath in replication cohorts yielded a VOC signature AUROC (95% CI) for acute asthma exacerbations of 0.89(0.76-1.0) (EMBER cohort) with sputum eosinophilia and 0.90(0.75-1.0) in U-BIOPRED - again outperforming FeNO in U-BIOPRED 0.62 (0.33-0.90). CONCLUSIONS: We have discovered and provided early-stage clinical validation of a volatile biomarker signature associated with eosinophilic airway inflammation. Further work is needed to translate our discovery using point of care clinical sensors.

Quantitative detection of formaldehyde using solid phase microextraction gas chromatography-mass spectrometry coupled to cysteamine scavenging.

Formaldehyde (HCHO) is a toxic and carcinogenic pollutant and human metabolite that reacts with biomolecules under physiological conditions. Quantifying HCHO is essential for ongoing biological and biomedical research on HCHO; however, its reactivity, small size and volatility make this challenging. Here, we report a novel HCHO detection/quantification method that couples cysteamine-mediated HCHO scavenging with SPME GC-MS analysis. Our NMR studies confirm cysteamine as an efficient and selective HCHO scavenger that out-competes O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine, the most commonly used scavenger, and forms a stable thiazolidine amenable to GC-MS quantification. Validation of our GC-MS method using FDA and EMA guidelines revealed detection and quantification limits in the nanomolar and micromolar ranges respectively, while analysis of bacterial cell lysate confirmed its applicability in biological samples. Overall, our studies confirm that cysteamine scavenging coupled to SPME GC-MS analysis provides a sensitive and chemically robust method to quantify HCHO in biological samples.

Elevated levels of chloramines and chlorine detected near an indoor sports complex.

Chloramines (NH2Cl, NHCl2, and NCl3) are toxic compounds that can be created during the use of bleach-based disinfectants that contain hypochlorous acid (HOCl) and the hypochlorite ion (OCl-) as their active ingredients. Chloramines can then readily transfer from the aqueous-phase to the gas-phase. Atmospheric chemical ionization mass spectrometry using iodide adduct chemistry (I-CIMS) made observations across two periods (2014 and 2016) at an urban background site on the University of Leicester campus (Leicester, UK). Both monochloramine (NH2Cl) and molecular chlorine (Cl2) were detected and positively identified from calibrated mass spectra during both sampling periods and to our knowledge, this is the first detection of NH2Cl outdoors. Mixing ratios of NH2Cl reached up to 2.2 and 4.0 parts per billion by volume (ppbv), with median mixing ratios of 30 and 120 parts per trillion by volume (pptv) during the 2014 and 2016 sampling periods, respectively. Levels of Cl2 were observed to reach up to 220 and 320 pptv. Analysis of the NH2Cl and Cl2 data pointed to the same local source, a nearby indoor sports complex with a swimming pool and a cleaning product storage shed. No appreciable levels of NHCl2 and NCl3 were observed outdoors, suggesting the indoor pool was not likely to be the primary source of the observed ambient chloramines, as prior measurements made in indoor pool atmospheres indicate that NCl3 would be expected to dominate. Instead, these observations point to indoor cleaning and/or cleaning product emissions as the probable source of NH2Cl and Cl2 where the measured levels provide indirect evidence for substantial amounts transported from indoors to outdoors. Our upper estimate for total NH2Cl emissions from the University of Leicester indoor sports complexes scaled for similar sports complexes across the UK is 3.4 × 105 ± 1.1 × 105 µg h-1 and 0.0017 ± 0.00034 Gg yr-1, respectively. The Cl-equivalent emissions in HCl are only an order of magnitude less to those from hazardous waste incineration and iron and steel sinter production in the UK National Atmospheric Emissions Inventory (NAEI).

Visualization of exhaled breath metabolites reveals distinct diagnostic signatures for acute cardiorespiratory breathlessness.

Acute cardiorespiratory breathlessness accounts for one in eight of all emergency hospitalizations. Early, noninvasive diagnostic testing is a clinical priority that allows rapid triage and treatment. Here, we sought to find and replicate diagnostic breath volatile organic compound (VOC) biomarkers of acute cardiorespiratory disease and understand breath metabolite network enrichment in acute disease, with a view to gaining mechanistic insight of breath biochemical derangements. We collected and analyzed exhaled breath samples from 277 participants presenting acute cardiorespiratory exacerbations and aged-matched healthy volunteers. Topological data analysis phenotypes differentiated acute disease from health and acute cardiorespiratory exacerbation subtypes (acute heart failure, acute asthma, acute chronic obstructive pulmonary disease, and community-acquired pneumonia). A multibiomarker score (101 breath biomarkers) demonstrated good diagnostic sensitivity and specificity (≥80%) in both discovery and replication sets and was associated with all-cause mortality at 2 years. In addition, VOC biomarker scores differentiated metabolic subgroups of cardiorespiratory exacerbation. Louvain clustering of VOCs coupled with metabolite enrichment and similarity assessment revealed highly specific enrichment patterns in all acute disease subgroups, for example, selective enrichment of correlated C5-7 hydrocarbons and C3-5 carbonyls in heart failure and selective depletion of correlated aldehydes in acute asthma. This study identified breath VOCs that differentiate acute cardiorespiratory exacerbations and associated subtypes and metabolic clusters of disease-associated VOCs.

Air pollution induces Staphylococcus aureus USA300 respiratory tract colonization mediated by specific bacterial genetic responses involving the global virulence gene regulators Agr and Sae.

Exposure to particulate matter (PM), a major component of air pollution, is associated with exacerbation of chronic respiratory disease, and infectious diseases such as community-acquired pneumonia. Although PM can cause adverse health effects through direct damage to host cells, our previous study showed that PM can also impact bacterial behaviour by promoting in vivo colonization. In this study we describe the genetic mechanisms involved in the bacterial response to exposure to black carbon (BC), a constituent of PM found in most sources of air pollution. We show that Staphylococcus aureus strain USA300 LAC grown in BC prior to inoculation showed increased murine respiratory tract colonization and pulmonary invasion in vivo, as well as adhesion and invasion of human epithelial cells in vitro. Global transcriptional analysis showed that BC has a widespread effect on S. aureus transcriptional responses, altering the regulation of the major virulence gene regulators Sae and Agr and causing increased expression of genes encoding toxins, proteases and immune evasion factors. Together these data describe a previously unrecognized causative mechanism of air pollution-associated infection, in that exposure to BC can increase bacterial colonization and virulence factor expression by acting directly on the bacterium rather than via the host.

Public health impact of mass sporting and cultural events in a rising COVID-19 prevalence in England.

A subset of events within the UK Government Events Research Programme (ERP), developed to examine the risk of transmission of COVID-19 from attendance at events, was examined to explore the public health impact of holding mass sporting events. We used contact tracing data routinely collected through telephone interviews and online questionnaires, to describe the potential public health impact of the large sporting and cultural events on potential transmission and incidence of COVID-19. Data from the EURO 2020 matches hosted at Wembley identified very high numbers of individuals who tested positive for COVID-19 and were traced through NHS Test & Trace. This included both individuals who were potentially infectious (3036) and those who acquired their infection during the time of the Final (6376). This is in contrast with the All England Lawn Tennis Championships at Wimbledon, where there were similar number of spectators and venue capacity but there were lower total numbers of potentially infectious cases (299) and potentially acquired cases (582). While the infections associated with the EURO 2020 event may be attributed to a set of socio-cultural circumstances which are unlikely to be replicated for the forthcoming sporting season, other aspects may be important to consider including mitigations for spectators to consider such as face coverings when travelling to and from events, minimising crowding in poorly ventilated indoor spaces such as bars and pubs where people may congregate to watch events, and reducing the risk of aerosol exposure through requesting that individuals avoid shouting and chanting in large groups in enclosed spaces.

The variability of volatile organic compounds in the indoor air of clinical environments.

The development of clinical breath-analysis is confounded by the variability of background volatile organic compounds (VOCs). Reliable interpretation of clinical breath-analysis at individual, and cohort levels requires characterisation of clinical-VOC levels and exposures. Active-sampling with thermal-desorption/gas chromatography-mass spectrometry recorded and evaluated VOC concentrations in 245 samples of indoor air from three sites in a large National Health Service (NHS) provider trust in the UK over 27 months. Data deconvolution, alignment and clustering isolated 7344 features attributable to VOC and described the variability (composition and concentration) of respirable clinical VOC. 328 VOC were observed in more than 5% of the samples and 68 VOC appeared in more than 30% of samples. Common VOC were associated with exogenous and endogenous sources and 17 VOC were identified as seasonal differentiators. The presence of metabolites from the anaesthetic sevoflurane, and putative-disease biomarkers in room air, indicated that exhaled VOC were a source of background-pollution in clinical breath-testing activity. With the exception of solvents, and waxes associated with personal protective equipment (PPE), exhaled VOC concentrations above 3µg m-3are unlikely to arise from room air contamination, and in the absence of extensive survey-data, this level could be applied as a threshold for inclusion in studies, removing a potential environmental confounding-factor in developing breath-based diagnostics.

A systematic review of the diagnostic accuracy of volatile organic compounds in airway diseases and their relation to markers of type-2 inflammation.

BACKGROUND: Asthma and COPD continue to cause considerable diagnostic and treatment stratification challenges. Volatile organic compounds (VOCs) have been proposed as feasible diagnostic and monitoring biomarkers in airway diseases. AIMS: To 1) conduct a systematic review evaluating the diagnostic accuracy of VOCs in diagnosing airway diseases; 2) understand the relationship between reported VOCs and biomarkers of type-2 inflammation; 3) assess the standardisation of reporting according to STARD and TRIPOD criteria; 4) review current methods of breath sampling and analysis. METHODS: A PRISMA-oriented systematic search was conducted (January 1997 to December 2020). Search terms included: "asthma", "volatile organic compound(s)", "VOC" and "COPD". Two independent reviewers examined the extracted titles against review objectives. RESULTS: 44 full-text papers were included; 40/44 studies were cross-sectional and four studies were interventional in design; 17/44 studies used sensor-array technologies (e.g. eNose). Cross-study comparison was not possible across identified studies due to the heterogeneity in design. The commonest airway diseases differentiating VOCs belonged to carbonyl-containing classes (i.e. aldehydes, esters and ketones) and hydrocarbons (i.e. alkanes and alkenes). Although individual markers that are associated with clinical biomarkers of type-2 inflammation were recognised (i.e. ethane and 3,7-dimethylnonane for asthma and α-methylstyrene and decane for COPD), these were not consistently identified across studies. Only 3/44 reported following STARD or TRIPOD criteria for diagnostic accuracy and multivariate reporting, respectively. CONCLUSIONS: Breath VOCs show promise as diagnostic biomarkers of airway diseases and for type-2 inflammation profiling. However, future studies should focus on transparent reporting of diagnostic accuracy and multivariate models and continue to focus on chemical identification of volatile metabolites.

Assessing the feasibility and acceptability of online measurements of exhaled volatile organic compounds (VOCs) in children with preschool wheeze: a pilot study.

Background: Investigating airway inflammation and pathology in wheezy preschool children is both technically and ethically challenging. Identifying and validating non-invasive tests would be a huge clinical advance. Real-time analysis of exhaled volatile organic compounds (VOCs) in adults is established, however, the feasibility of this non-invasive method in young children remains undetermined. Aim: To determine the feasibility and acceptability of obtaining breath samples from preschool children by means of real-time mass spectrometry analysis of exhaled VOCs. Methods: Breath samples from preschool children were collected and analysed in real time by proton transfer reaction-time of flight-mass spectrometry (PTR-TOF-MS) capturing unique breath profiles. Acetone (mass channel m/z 59) was used as a reference profile to investigate the breath cycle in more detail. Dynamic time warping (DTW) was used to compare VOC profiles from adult breath to those we obtained in preschool children. Results: 16 children were recruited in the study, of which eight had acute doctor-diagnosed wheeze (mean (range) age 3.2 (1.9-4.5) years) and eight had no history of wheezing (age 3.3 (2.2-4.1) years). Fully analysable samples were obtained in 11 (68%). DTW was used to ascertain the distance between the time series of mass channel m/z 59 (acetone) and the other 193 channels. Commonality of 12 channels (15, 31, 33, 41, 43, 51, 53, 55, 57, 60, 63 and 77) was established between adult and preschool child samples despite differences in the breathing patterns. Conclusion: Real-time measurement of exhaled VOCs by means of PTR-MS is feasible and acceptable in preschool children. Commonality in VOC profiles was found between adult and preschool children.

Diagnosis of COVID-19 by exhaled breath analysis using gas chromatography-mass spectrometry.

BACKGROUND: The ongoing coronavirus disease 2019 (COVID-19) pandemic has claimed over two and a half million lives worldwide so far. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is perceived to be seasonally recurrent, and a rapid noninvasive biomarker to accurately diagnose patients early on in their disease course will be necessary to meet the operational demands for COVID-19 control in the coming years. OBJECTIVE: The aim of this study was to evaluate the role of exhaled breath volatile biomarkers in identifying patients with suspected or confirmed COVID-19 infection, based on their underlying PCR status and clinical probability. METHODS: A prospective, real-world, observational study was carried out, recruiting adult patients with suspected or confirmed COVID-19 infection. Breath samples were collected using a standard breath collection bag, modified with appropriate filters to comply with local infection control recommendations, and samples were analysed using gas chromatography-mass spectrometry (TD-GC-MS). RESULTS: 81 patients were recruited between April 29 and July 10, 2020, of whom 52 out of 81 (64%) tested positive for COVID-19 by reverse transcription-polymerase chain reaction (RT-PCR). A regression analysis identified a set of seven exhaled breath features (benzaldehyde, 1-propanol, 3,6-methylundecane, camphene, beta-cubebene, iodobenzene and an unidentified compound) that separated PCR-positive patients with an area under the curve (AUC): 0.836, sensitivity: 68%, specificity: 85%. CONCLUSIONS: GC-MS-detected exhaled breath biomarkers were able to identify PCR-positive COVID-19 patients. External replication of these compounds is warranted to validate these results.

Enhanced wintertime oxidation of VOCs via sustained radical sources in the urban atmosphere.

Daytime atmospheric oxidation chemistry is conventionally considered to be driven primarily by the OH radical, formed via photolytic sources. In this paper we examine how, during winter when photolytic processes are slow, chlorine chemistry can have a significant impact on oxidative processes in the urban boundary layer. Photolysis of nitryl chloride (ClNO2) provides a significant source of chlorine atoms, which enhances the oxidation of volatile organic compounds (VOCs) and the production of atmospheric pollutants. We present a set of observations of ClNO2 and HONO made at urban locations in central England in December 2014 and February 2016. While direct emissions and in-situ chemical formation of HONO continue throughout the day, ClNO2 is only formed at night and is usually completely photolyzed by midday. Our data show that, during winter, ClNO2 often persists through the daylight hours at mixing ratios above 10-20 ppt (on average). In addition, relatively high mixing ratios of daytime HONO (>65 ppt) provide a strong source of OH radicals throughout the day. The combined effects of ClNO2 and HONO result in sustained sources of Cl and OH radicals from sunrise to sunset, which form additional ozone, PAN, oxygenated VOCs, and secondary organic aerosol. We show that radical sources such as ClNO2 and HONO can lead to a surprisingly photoactive urban atmosphere during winter and should therefore be included in atmospheric chemical models.

Breathomics for the clinician: the use of volatile organic compounds in respiratory diseases.

Exhaled breath analysis has the potential to provide valuable insight on the status of various metabolic pathways taking place in the lungs locally and other vital organs, via systemic circulation. For years, volatile organic compounds (VOCs) have been proposed as feasible alternative diagnostic and prognostic biomarkers for different respiratory pathologies.We reviewed the currently published literature on the discovery of exhaled breath VOCs and their utilisation in various respiratory diseasesKey barriers in the development of clinical breath tests include the lack of unified consensus for breath collection and analysis and the complexity of understanding the relationship between the exhaled VOCs and the underlying metabolic pathways. We present a comprehensive overview, in light of published literature and our experience from coordinating a national breathomics centre, of the progress made to date and some of the key challenges in the field and ways to overcome them. We particularly focus on the relevance of breathomics to clinicians and the valuable insights it adds to diagnostics and disease monitoring.Breathomics holds great promise and our findings merit further large-scale multicentre diagnostic studies using standardised protocols to help position this novel technology at the centre of respiratory disease diagnostics.

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 ofz-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.

Use of the ReCIVA device in breath sampling of patients with acute breathlessness: a feasibility study.

INTRODUCTION: Investigating acute multifactorial undifferentiated breathlessness and understanding the driving inflammatory processes can be technically challenging in both adults and children. Being able to validate noninvasive methods such as breath analysis would be a huge clinical advance. The ReCIVA® device allows breath samples to be collected directly onto sorbent tubes at the bedside for analysis of exhaled volatile organic compounds (eVOCs). We aimed to assess the feasibility of using this device in acutely breathless patients. METHODS: Adults hospitalised with acute breathlessness and children aged 5-16 years with acute asthma or chronic stable asthma, as well as healthy adult and child volunteers, were recruited. Breath samples were collected onto sorbent tubes using the ReCIVA® device and sent for analysis by means of two-dimensional gas chromatography-mass spectrometry (GCxGC-MS). The NASA Task Load Index (NASA-TLX) was used to assess the perceived task workload of undertaking sampling from the patient's perspective. RESULTS: Data were available for 65 adults and 61 children recruited. In total, 98.4% of adults and 75.4% of children were able to provide the full target breath sample using the ReCIVA® device. NASA-TLX measurements were available in the adult population with mean values of 3.37 for effort, 2.34 for frustration, 3.8 for mental demand, 2.8 for performance, 3.9 for physical demand and 2.8 for temporal demand. DISCUSSION: This feasibility study demonstrates it is possible and acceptable to collect breath samples from both adults and children at the bedside for breathomics analysis using the ReCIVA® device.

LabPipe: an extensible bioinformatics toolkit to manage experimental data and metadata.

BACKGROUND: Data handling in clinical bioinformatics is often inadequate. No freely available tools provide straightforward approaches for consistent, flexible metadata collection and linkage of related experimental data generated locally by vendor software. RESULTS: To address this problem, we created LabPipe, a flexible toolkit which is driven through a local client that runs alongside vendor software and connects to a light-weight server. The toolkit allows re-usable configurations to be defined for experiment metadata and local data collection, and handles metadata entry and linkage of data. LabPipe was piloted in a multi-site clinical breathomics study. CONCLUSIONS: LabPipe provided a consistent, controlled approach for handling metadata and experimental data collection, collation and linkage in the exemplar study and was flexible enough to deal effectively with different data handling challenges.

Two Aldehyde Clearance Systems Are Essential to Prevent Lethal Formaldehyde Accumulation in Mice and Humans.

Reactive aldehydes arise as by-products of metabolism and are normally cleared by multiple families of enzymes. We find that mice lacking two aldehyde detoxifying enzymes, mitochondrial ALDH2 and cytoplasmic ADH5, have greatly shortened lifespans and develop leukemia. Hematopoiesis is disrupted profoundly, with a reduction of hematopoietic stem cells and common lymphoid progenitors causing a severely depleted acquired immune system. We show that formaldehyde is a common substrate of ALDH2 and ADH5 and establish methods to quantify elevated blood formaldehyde and formaldehyde-DNA adducts in tissues. Bone-marrow-derived progenitors actively engage DNA repair but also imprint a formaldehyde-driven mutation signature similar to aging-associated human cancer mutation signatures. Furthermore, we identify analogous genetic defects in children causing a previously uncharacterized inherited bone marrow failure and pre-leukemic syndrome. Endogenous formaldehyde clearance alone is therefore critical for hematopoiesis and in limiting mutagenesis in somatic tissues.

Automating and Extending Comprehensive Two-Dimensional Gas Chromatography Data Processing by Interfacing Open-Source and Commercial Software.

Comprehensive two-dimensional gas chromatography (GC×GC) is a powerful analytical tool for both nontargeted and targeted analyses. However, there is a need for more integrated workflows for processing and managing the resultant high-complexity datasets. End-to-end workflows for processing GC×GC data are challenging and often require multiple tools or software to process a single dataset. We describe a new approach, which uses an existing underutilized interface within commercial software to integrate free and open-source/external scripts and tools, tailoring the workflow to the needs of the individual researcher within a single software environment. To demonstrate the concept, the interface was successfully used to complete a first-pass alignment on a large-scale GC×GC metabolomics dataset. The analysis was performed by interfacing bespoke and published external algorithms within a commercial software environment to automatically correct the variation in retention times captured by a routine reference standard. Variation in 1tR and 2tR was reduced on average from 8 and 16% CV prealignment to less than 1 and 2% post alignment, respectively. The interface enables automation and creation of new functions and increases the interconnectivity between chemometric tools, providing a window for integrating data-processing software with larger informatics-based data management platforms.

What does success look like for air quality policy? A perspective.

This paper explores the drivers and role of science in air quality policy over the last 100 years or so. Case studies on the smogs of Los Angeles and London, acid rain, health impacts of particulate matter, diesel and lead in fuel are used to explore the drivers and models for the interaction of science, evidence and air quality policy. It suggests there are two phases to air quality mitigation, the first driven by the air quality emergency as the pollution is visible and the effects can be relatively obvious and the second driven by science that is directed towards continuous improvement. A critical element of the 'science phase' is the evidence base, the models of evidence-based and -informed policy-making are explored with the conclusion that it is optimal when guided by the ideal of co-creation of knowledge and policy options between scientists and policy-makers. The future and wider drivers for air quality are detailed with a number of key areas for 'success' indicated as important for air quality policy development such as continuous improvement. Overall, we find there is tension between two factors: the ambition to reduce emissions, improve air quality and reduce the impacts on public health and the environment on one hand, and questions of cost, technical feasibility and societal acceptability on the other. This article is part of a discussion meeting issue 'Air quality, past present and future'.

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: NCT03672994.

Breath analysis by two-dimensional gas chromatography with dual flame ionisation and mass spectrometric detection - Method optimisation and integration within a large-scale clinical study.

Precision medicine has spurred new innovations in molecular pathology leading to recent advances in the analysis of exhaled breath as a non-invasive diagnostic tool. Volatile organic compounds (VOCs) detected in exhaled breath have the potential to reveal a wealth of chemical and metabolomic information. This study describes the development of a method for the analysis of breath, based on automated thermal desorption (TD) combined with flow modulated comprehensive two-dimensional gas chromatography (GC×GC) with dual flame ionisation and quadrupole mass spectrometric detection (FID and qMS). The constrained optimisation and analytical protocol was designed to meet the practical demands of a large-scale multi-site clinical study, while maintaining analytical rigour to produce high fidelity data. The results demonstrate a comprehensive method optimisation for the collection and analysis of breath VOCs by GC×GC, integral to the standardisation and integration of breath analysis within large clinical studies.