Since the late 1990s, a surge in interest in the analysis of exhaled breath condensate (EBC) resulted in the American Thoracic Society and European Respiratory Society (ATS/ERS) organising a Task Force in 2001 to develop guidelines on EBC collection and measurement of biomarkers. This Task Force published their guidelines in 2005 based on literature and expert opinions at that time, and multiple shortcomings and knowledge deficits were also identified. The clinical application of EBC collection and its biomarkers are currently still limited by several of these knowledge gaps, hence further guidelines for standardisation are required to ensure external validity. Using related articles produced since the publication of the ATS/ERS Task Force report, this paper attempts to provide a comprehensive update to the original guideline and review the methodological shortcomings identified. This review can hopefully serve as a yardstick for future studies involving this emerging clinical tool.
Cigarette smoking, the principal aetiology of chronic obstructive pulmonary disease (COPD) in the developed countries, delivers and generates oxidative stress within the lungs. This imbalance of oxidant burden and antioxidant capacity has been implicated as an important contributing factor in the pathogenesis of COPD. Oxidative processes and free radical generation orchestrate the inflammation, mucous gland hyperplasia, and apoptosis of the airway lining epithelium which characterises COPD. Pivotal oxidative stress/pro-inflammatory molecules include reactive oxygen species such as the superoxides and hydroxyl radicals, pro-inflammatory cytokines including leukotrienes, interleukins, tumour necrosis factor alpha, and activated transcriptional factors such as nuclear factor kappa-B and activator protein 1. The lung has a large reserve of antioxidant agents such as glutathione and superoxide dismutase to counter oxidants. However, smoking also causes the depletion of antioxidants, which further contributes to oxidative tissue damage. The downregulation of antioxidant pathways has also been associated with acute exacerbations of COPD. The delivery of redox-protective antioxidants may have preventative and therapeutic potential of COPD. Although these observations have yet to translate into common clinical practice, preliminary clinical trials and studies of animal models have shown that interventions to counter this oxidative imbalance may have potential to better manage COPD. There is, thus, a need for the ability to monitor such interventions and exhaled breath condensate is rapidly emerging as a novel and noninvasive approach in the sampling of airway epithelial lining fluid which could be used for repeated analysis of oxidative stress and inflammation in the lungs.
Exhaled breath condensate (EBC) collection is an innovative method of non-invasively sampling the lung, and can detect a variety of volatile and non-volatile biomarkers, but the disadvantage is the small volume of sample collected. It was hypothesized that a collection system at a lower temperature would increase the volume collected, but may alter the relative concentration of the biomarkers of interest. EBC was collected in a cross-over study using a custom-made collection system, cooled using either wet (4 °C) or dry ice (-20 °C) in randomized order in normal non-smoking volunteers. The volume of the EBC collected per unit time was determined as were conductivity, the concentrations and total amount of protein, hydrogen peroxide, and nitrite/nitrate concentrations. Dry ice was associated with a 79% greater volume of EBC than the wet ice (1387 ± 612 µL; 773 ± 448 µL respectively, p < 0.0001). Conductivity was influenced by the temperature of collection (18.78 ± 6.71 µS cm(-1) for wet ice and 15.32 ± 6.28 µS cm(-1) for dry ice, p = 0.02) as was hydrogen peroxide (1.34 ± 0.88 µg mL(-1) for wet ice and 0.68 ± 0.32 µg mL(-1) for dry ice, p = 0.009) while the concentrations and total values for protein and nitrate/nitrite were not significantly different (p > 0.05). This pilot study suggests that lower collection temperatures facilitate the collection of a larger sample volume. This larger volume is not simply more dilute, with increased water content, nor is there a simple correction factor that can be applied to the EBC biomarkers to correct for the different methods.
Exhaled breath condensate (EBC) analysis is a non-invasive method to repeatedly evaluate airway inflammation. Dissolved carbon dioxide contributes to lowering EBC pH which is reversed by degassing with argon. Hypothetically, argon may also improve biomarker stability by removing reactive gases, since many markers are pH sensitive or easily oxidized. In this study, the influence of two degassing methods was assessed on (i) the volume of EBC, (ii) the EBC pH, and (iii) the concentration of H₂O₂ in EBC. EBC was collected from 13 healthy subjects and 12 chronic obstructive pulmonary disease subjects over 20 min, then aliquoted and either left on ice or de-aerated with argon by bubbling or surface delivery at 400 ml min(-1) for 0 to 600 s, to quantify the EBC volume loss and the efficiency of pH equilibration. Biomarker stability was measured by H₂O₂ concentration. Both degassing methods reached a pH equilibrium by 300 s. Bubbling reached pH equilibrium faster (60 s versus 300 s), while having significantly less EBC volume loss (bubbling 3.32 ± 1.31% versus surface 10.74 ± 1.46%, p < 0.0001). The H₂O₂ concentration was higher in non-degassed samples (0.47 ± 0.18 µM, p = 0.017) but similar in the bubbling and surface degassed samples (0.30 ± 0.08 µM versus 0.31 ± 0.10 µM, p = 0.54). The optimal degassing methods were to bubble the aliquots with argon at 400 ml min(-1) for 60 s or surface degassing for 300 s. Both methods resulted in significantly less EBC volume loss than the commonly adopted method of bubbling for 10 min. There was a significant difference in the H₂O₂ concentration between the degassed and the non-degassed samples.
Asthma, chronic obstructive pulmonary disease (COPD) and lung cancer cause extensive mortality and morbidity worldwide. However, the current state-of-the-art diagnosis and management schemes of these diseases are suboptimal as the incidence of asthma has risen by 250% over the last two decades and the 5-year mortality rate of lung cancer remains at 88%. Proteomic analysis is at the frontier of medical research and demonstrates tremendous potential in the early detection, diagnosis and staging, as well as providing novel therapeutic targets for improved management of smoking-related lung diseases. Advances in analytical tools, such as 2D gel electrophoresis, mass spectrometry, protein arrays and improved bioinformatics, allow sensitive and specific biomarker/protein profile discoveries and the infusion of new knowledge towards the molecular basis of lung diseases and their progression. Significant hurdles still stand between these laboratory findings and their applications in clinical practice. One of the challenges is the difficulty in the selection of samples that provide scope into the specific disease entity. Induced sputum, bronchoalveolar lavage, exhaled breath and exhaled breath condensate are methods of sampling airway and lung fluids that can serve as a window to assess the microenvironment of the lungs. With better study design standardization and the implementation of novel technologies to reach the optimal research standard, there is enough reason be optimistic about the future of proteomic research and its clinical implications.
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