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The aim of this paper is to explore the philosophies, attitudes and beliefs that surround the teaching of breath. Voice and speech teachers want students and actors to be versatile; able to adjust to the demands of any role, and each student enters the classroom with a myriad of mental and physical breathing habits. Many voice and speech trainers, however, only address a limited number of breathing habits and primarily teach "deep breathing." Why has deep breathing dominated voice training, and how do we effectively teach breath for all students and all habits? I will examine six major voice practitioners: Arthur Lessac, Patsy Rodenburg, Kristin Linklater, Catherine Fitzmaurice, F.M. Alexander, and Jo Estill and their philosophies about breath. I will also investigate my own experiences with each practitioner, both in my own training, and in my teaching. I will conclude with my personal philosophy about breath; what an ideal breath is, and how to teach it.

Un accès direct et non invasif à la pathogénèse par l’évaluation de biomarqueurs volatils (FeNO) ou non volatils de l’air exhalé grâce à la méthode des condensats de l’air exhalé (EBC) est utile pour estimer précocement le degré d’atteinte pulmonaire en rapport avec une exposition professionnelle et surveiller son évolution en regard des mesures correctives mises en place. L’étude du NO exhalé (FeNO) est actuellement bien standardisée et l’augmentation exponentielle des publications concernant les EBC reflètent un intérêt croissant pour le diagnostic non invasif des atteintes pulmonaires. Les marqueurs étudiés dans l’air exhalé sont des produits de l'inflammation tels que le FeNO et les cytokines ainsi que des produits du stress oxydant incluant le peroxyde d'hydrogène, les produits de peroxydation lipidique (8-isoprostane, malondialdehyde) et les oxydes d'azote (NOx). S’agissant d’une technique d’exploration relativement récente, les modalités méthodologiques font encore l’objet de nombreux travaux, les premières recommandations internationales étant apparues en 2005. Les écueils méthodologiques sont nombreux et encore décrits dans la littérature en 2012. Ils concernent toutes les étapes du processus du recueil et d’analyses.Objectifs : L’objectif principal de notre recherche était dans un premier temps de développer la méthode des EBC pour l'étude des composés de l'air exhalé puis de détecter et quantifier des biomarqueurs tels que les protéines totales, les NOx et le 8-isoprostane dans les EBC d’une population d’adultes sains (n= 48) ou atteints de pathologies pulmonaires telles que l'asthme (n=24), la BPCO (n=20), la pneumopathie interstitielle diffuse (n=27) et la sclérodermie (n=27). L’objectif secondaire était la comparaison des niveaux de biomarqueurs dosés dans les EBC et du FeNO chez les sujets malades par rapport aux témoins.Résultats / Conclusion : Notre recherche, soutenue l’ANR et l’ANSES, a permis de standardiser la méthodologie de recueil et d’analyse des EBC avec une optimisation du système de recueil et de l’agent coatant pour la détermination de la concentration nécessaire et compatible avec les analyses. Nous maitrisons le dosage dans les EBC des biomarqueurs tels que les protéines, les NOx et le 8-isoprostane. D'autres biomarqueurs sont encore en cours d'étude tels que le malondialdehyde et les cytokines. Ce travail méthodologique, publié, a permit dans un second temps la détection (> à 95%) et la quantification de ces biomarqueurs dans les EBC des sujets sains et malades de notre population.Perspectives : La constitution des valeurs de référence pour les biomarqueurs dosés dans les EBC est nécessaire à l’interprétation des dosages chez les sujets malades. Il convient également de finaliser la standardisation de la mesure des cytokines et du malondialdehyde et d’explorer de nouveaux biomarqueurs produits lors du stress nitrosant tels que la 3-nitrotyrosine pour compléter les mécanismes physiopathologiques. Enfin le développement de cette méthode non invasive et peu contraignante apparait prometteuse en clinique médicale dans le suivi individuel de la maladie ou son traitement comme lors d’études épidémiologiques respiratoires dans des populations exposées professionnellement ou environnementalement. Dans ce cadre, l’étude de marqueurs d’exposition professionnelle tels que les métaux lourds dans les EBC est en cours de développement. / The study of both volatile (FeNO) and non-volatile respiratory biomarkers using the method of exhaled breath condensates can be useful in medical surveillance of exposed workers, the early identification of respiratory diseases or in the monitoring of their development. Studies of exhaled NO (FeNO) is now well standardized and the exponential increase in publications on exhaled breath condensate (EBC) reflects growing interest in a non-invasive diagnosis of pulmonary diseases. The biomarkers studied are products of inflammation, such as FeNO and cytokines, and products of oxidative stress, including hydrogen peroxide (H202), products of lipid peroxydation (8-isoprostane, malondialdehyde) and nitrogen oxides. The first recommendation was published in 2005 but although many recent publications have applied this new method, numerous methodological pitfalls remain and still described in 2012. They concern all the stages of the collection to the analysis.Objectives: The main objective of this research was initially to develop the method of EBC for the study of compounds of exhaled air and then detect and quantify biomarkers such as total protein, NOx and 8-isoprostane in exhaled air in a population of healthy adults (n = 48) or patients with lung inflammatory diseases such as asthma (n = 24), COPD (n = 20), diffuse interstitial pneumonia (n = 27) and scleroderma (n = 27). The secondary objective was to compare levels of biomarkers measured in the EBC and FeNO in patients compared to controls.Results / conclusion: Our research, supported by ANR and ANSES, allowed to standardize the methodology of collection and analysis of EBC with a choice of the collection system and coating which must be effective and compatible with the analyzes. In EBC, we control the dosage of biomarkers such as proteins, NOx and 8-isoprostane. Other biomarkers are still being studied such as malondialdehyde and cytokines. This published methodological study, allowed in a second step the detection (> 95%) and quantification of these biomarkers in EBC of healthy patients in our population.Perspectives: This standardization is a key epidemiological requirement for the task force on the establishment of reference values and the publication of methodological guidelines so as to realize the promise of this approach for clinical studies of lung diseases. We have also to finish the development of biomarkers such as cytokines or malondialdehyde and to investigate new biomarkers to complete the pathophysiological mechanisms. Finally our objective is the widespread use of this noninvasive method in daily epidemiological studies on subjects with professional and/or environmental exposure. In this context, the study of markers of the toxic burden in the lungs such as heavy metals in the EBC is being developed.

Introduction: Asthma and chronic obstructive pulmonary disease (COPD) are heterogeneous diseases with a wide range of clinical manifestations not adequately described within the current diagnostic criteria. Exhaled breath analysis may provide a novel method for diagnosing and phenotyping these diseases. Our aim was to ascertain patterns of breath volatile organic compounds (VOCs) and nuclear magnetic resonance (NMR) spectral regions identifying diseased patients and subgroups determined by treatment requirement, asthma control, exacerbation frequency and inflammatory phenotypes. The validity and reproducibility of the methodology and the outcome were also investigated. Methods: Three separate clinical studies (two involving exhaled gas and one involving breath condensate) were conducted, as well as validation studies. In exhaled gas analysis, the adaptive breath sampler developed by Basanta et al was modified; efficiency of air supply and air filter and the reproducibility and stability of VOCs in storage were determined by comparing breath chromatograms. Concentrated late-expiratory breath samples were collected from asthmatics, COPD subjects and healthy controls. In the asthmatic group, sputum induction with hypertonic saline, fraction exhaled nitric oxide (FeNO) measurement and asthma control questionnaire (ACQ) were performed. In COPD subjects, sputum induction and exacerbation frequency were collected. In the exhaled breath condensate (EBC) study, similar data were collected in asthmatics and healthy controls. Breath samples were analysed using gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) while EBC was analysed using NMR spectroscopy. Discriminatory compounds or NMR spectral regions were identified by univariate logistic regression, followed by multivariate analysis: 1. principal component analysis (PCA); 2. multivariate logistic regression; 3. receiver operating characteristic (ROC) analysis. The reproducibility was assessed using intraclass correlation coefficient (ICC).Results: In the COPD exhaled breath study, 11 VOCs significantly discriminated the COPD and healthy controls with AUROC of 0.74. The AUROC for phenotype discrimination was 0.83, 0.90, 0.94, 0.96 and 0.97 for inhaled corticosteroid (ICS) use, sputum eosinophilia (1% and 2% cut-off), neutrophilia (median cut-off) and exacerbation frequency respectively. In the asthma study, 15 VOCs significantly discriminated the two groups with AUROC of 0.93. The AUROC for phenotype discrimination was 0.96, 0.98, 0.90 and 0.97 for ICS use, eosinophils (2% cut-off), neutrophils (40% cut-off) and asthma control respectively. In EBC analysis, AUROC for asthmatics vs controls comparison was 0.96. Phenotyping results in this study were less good: only ICS use and sputum neutrophilia (65% cut-off) were clearly classified with AUROC of 0.89 and 0.88 while eosinophilia (3% cut-off) and asthma control had poor discrimination; 0.69 and 0.62 respectively. Breath VOC reproducibility varied greatly depending on the class of compounds studied, while for the EBC analysis, reproducibility was moderate to very good (ICCs in the range of 0.42-0.99).Conclusions: We have demonstrated the ability of breath analysis in discriminating asthmatics and COPD subjects from controls. Exhaled breath analysis was also able to phenotype these patients based on steroid treatment, sputum inflammatory cells, exacerbation frequency and asthma control. This metabolomic approach could provide a novel, non-invasive method of diagnosing and phenotyping obstructive lung diseases in the future.

Membrane extraction with a sorbent interface (MESI) is a sample preparation technique with a rugged and simple design allowing for solvent-free, on-line performance. When coupled to gas chromatography (GC), MESI is an extremely promising tool for the analysis of volatile organic compounds (VOCs), as it is selective and sensitive for detecting trace levels of analytes. A new calibration method to be used with the MESI technique is presented herein. The aim of this project was to characterize and quantify the biomarker ethylene in human breath and plant emissions. The MESI-GC system was optimized, and an external calibration curve for ethylene standard was obtained. Qualitative measures were obtained from emissions of the higher plant Arabidopsis thaliana. The dominant calibration method was validated by examining changes in mass transfer trends when flow and temperature conditions were altered. Finally, the dominant calibration method was used to quantify ethylene in real human breath samples from non-smoking and smoking volunteers. Results were consistent with those reported in literature. These findings suggest that the dominant calibration technique is a useful tool for monitoring ethylene in human breath and Arabidopsis.

Membrane extraction with a sorbent interface (MESI) is a sample preparation technique with a rugged and simple design allowing for solvent-free, on-line performance. When coupled to gas chromatography (GC), MESI is an extremely promising tool for the analysis of volatile organic compounds (VOCs), as it is selective and sensitive for detecting trace levels of analytes. A new calibration method to be used with the MESI technique is presented herein. The aim of this project was to characterize and quantify the biomarker ethylene in human breath and plant emissions. The MESI-GC system was optimized, and an external calibration curve for ethylene standard was obtained. Qualitative measures were obtained from emissions of the higher plant Arabidopsis thaliana. The dominant calibration method was validated by examining changes in mass transfer trends when flow and temperature conditions were altered. Finally, the dominant calibration method was used to quantify ethylene in real human breath samples from non-smoking and smoking volunteers. Results were consistent with those reported in literature. These findings suggest that the dominant calibration technique is a useful tool for monitoring ethylene in human breath and Arabidopsis.

Current flow estimation methods use tracheal sounds in all except one step of the process: ‘breath phase detection’, is done by assuming alternating breath phases or using a second acoustic channel. The alternating assumption is unreliable in long recordings; non-breathing events (apnea, swallow or cough) change the alternating pattern. Although phases can be detected using lung sounds intensity, the additional channel and associated labor is clinically impractical. We present a method using breath sound parameters to differentiate between the two respiratory phases. The novel method is independent of flow level, requiring only one prior- and one post- breath segment to identify the phase. This was tested on data from 93 healthy individuals, without any history of pulmonary diseases, at 4 different flow levels. The most prominent features were duration, volume and shape of the sound envelope. This method showed accuracy of 95.6%, 95.5% sensitivity and 95.6% specificity.

Tedlar™ bags are a promising medium for remote breath collection and later analysis using SIFT-MS for disease diagnosis. It is important to understand the changes in integrity of samples stored in Tedlar™ bags. However, there is little work into this problem completed to date, and thus little known about these issues. Therefore, a study into the integrity of samples stored in Tedlar™ bags and analysed using SIFT-MS was undertaken. The sample integrity of ammonia, acetone, ethanol, isoprene and pentane, all initially at 3ppm in breath and nitrogen substrates, and stored in Tedlar™ bags was investigated. Experiments tested the effect of storage size (0.5, 1, 3L), storage time (6-48 hours), storage temperature (23℃ - 25℃, 37℃), humidity (0.4 - 4.5% absolute) and inter-bag variation using triplicate bags. The SIFT-MS instrument used was LDI2 located at Christchurch Hospital. The repeatability and precision of LDI2 was established using prepared cylinder samples (0.05% absolute humidity) of acetone, pentane and ethanol tested at seven times over a 250 min time period. A generalised Cauchy distribution was used to give a combined distribution from multiple bags for the sample humidity and compound concentration. A combined measure of the repeatability and precision, T s , ranged between 217 - 349 ppb for ethanol, acetone and pentane. The factors affecting the repeatability and precision were both machine and compound dependant. The effect of the factors differed over time, with different precursors and compounds. No obvious effects of bag storage size on the sample integrity of pentane, isoprene, ethanol and acetone were observed. The absolute humidity change within bag samples was linked to the volume to surface area ratio because it was more affected by permeation and condensation. All compounds in the nitrogen substrate (except for 37℃ stored acetone (NO+)) displayed decreases in sample integrity with time. All compounds in the breath substrate displayed regular losses of sample integrity, except for the 37℃ and 23℃ - 25℃ stored ethanol (NO+) and 37℃ stored ethanol (H3O+), pentane (O2+) and ammonia (H3O+, O2+). The average change of sample integrity for pentane, isoprene, ethanol and acetone ranged from 0.2 to 3.6 times the maximum T s , while ammonia ranged from 0.9 - 10 times. All observed behaviour was reproducible. Absolute humidity and storage temperature affected the sample integrity of acetone, ethanol and ammonia. Generally, the intra-bag variance was comparable between all storage temperatures and substrates while the inter-bag variation was affected by the absolute humidity. Only the initial and final concentrations between precursors for the 23℃ - 25℃ stored breath and nitrogen substrates agreed. The breath substrate samples gave erroneous values for ammonia. Permeation of compounds into the bags from the atmosphere was not significant. The overall issues surrounding storing breath in Tedlar™ bags for analysis using SIFTMS is not the loss of sample integrity, but the kinetics, precision and repeatability of the SIFT-MS instrument. The current kinetics are not adequate to accurately monitor acetone, isoprene, pentane, ammonia and ethanol in breath and stored in Tedlar™ bags at breath absolute humidity levels greater than 3%. Generally, the loss of sample integrity was only marginally outside the repeatability and precision of the machine.

Current flow estimation methods use tracheal sounds in all except one step of the process: ‘breath phase detection’, is done by assuming alternating breath phases or using a second acoustic channel. The alternating assumption is unreliable in long recordings; non-breathing events (apnea, swallow or cough) change the alternating pattern. Although phases can be detected using lung sounds intensity, the additional channel and associated labor is clinically impractical. We present a method using breath sound parameters to differentiate between the two respiratory phases. The novel method is independent of flow level, requiring only one prior- and one post- breath segment to identify the phase. This was tested on data from 93 healthy individuals, without any history of pulmonary diseases, at 4 different flow levels. The most prominent features were duration, volume and shape of the sound envelope. This method showed accuracy of 95.6%, 95.5% sensitivity and 95.6% specificity.

Background and objective: Asbestos can cause various pulmonary diseases including asbestosis, pleural plaques and pleural thickening. Animal and in vitro studies suggest that the toxicity of asbestos is due to the iron content of the fibres which not only generate oxidants directly, but also activate the inflammatory cells in the lung that contribute to oxidative stress. This study therefore sought to establish data in man to corroborate the animal and in vitro evidence. Exhaled breath condensate (EBC) collection is a novel, non-invasive technique to collect samples from the lung for investigating inflammatory biomarkers of lung diseases. This technique is harmless, rapid and easily repeatable which leads itself to the investigation of lung diseases such as asbestos-related diseases and pulmonary fibrosis (PF) that are otherwise difficult to study. The hypothesis tested was that oxidative and nitrosylative stress markers will be elevated in the EBC of patients with asbestos-related diseases and PF compared to normal control subjects. Methods: The study design was a cross-sectional and observational in vivo study whereby EBC was collected and fractional exhaled nitric oxide (FeNO) and carbon monoxide (eCO) were measured. EBC markers including pH, hydrogen peroxide (H2O2), total nitrogen oxides (NOx), 3-nitrotyrosine (3-NT), 8-isoprostane (8-iso), total protein and transforming growth factor-β1 (TGFβ1) were measured by microelectrode analysis, colorimetric and enzyme immunoassays. 3-NT and 8-iso were further examined by immunohistochemical techniques in samples of lung tissue. Results: Subjects with asbestosis had significantly raised levels of EBC H2O2, 8-iso, total protein and FeNO compared with healthy individuals. The same markers except H2O2, but with 3-NT and eCO were again significantly increased in those with other causes of PF, compared with control subjects. Heavy nitrotyrosine staining was found on the lung sections from patients with asbestosis and PF. Conclusions: This study confirmed that increased production of reactive oxygen and nitrogen species is associated with asbestos exposure and pulmonary fibrosis in vivo confirming animal and in vitro studies. Analysis of EBC may prove a useful non-invasive tool in exploring the basic pathophysiology of lung diseases in clinical research and may in the future be used to monitor progress in asbestosis and pulmonary fibrosis.