Philippe Rosias

Exhaled breath condensate in children: Pearls and pitfalls

Exhaled breath condensate (EBC) is a rapidly growing field of research in respiratory medicine. Airway inflammation is a central feature of chronic lung diseases, like asthma, cystic fibrosis, bronchopulmonary dysplasia and primary ciliary dyskinesia. EBC may be a useful technique for non‐invasive assessment of markers of airway inflammation. The non‐invasive character of EBC ‘inflammometry’ and the general lack of appropriate techniques makes it particularly interesting for paediatrics.

Breath condenser coatings affect measurement of biomarkers in exhaled breath condensate

Exhaled breath condensate collection is not yet standardised and biomarker measurements are often close to lower detection limits. In the current study, it was hypothesised that adhesive properties of different condenser coatings interfere with measurements of eicosanoids and proteins in breath condensate. In vitro, condensate was derived from a collection model using two test solutions (8-isoprostane and albumin) and five condenser coatings (silicone, glass, aluminium, polypropylene and Teflon). In vivo, condensate was collected using these five coatings and the EcoScreen® condenser to measure 8-isoprostane, and three coatings (silicone, glass, EcoScreen®) to measure albumin. In vitro, silicone and glass coatings had significantly higher albumin recovery compared with the other coatings. A similar trend was observed for 8-isoprostane recovery. In vivo, median (interquartile range) 8-isoprostane concentrations were significantly higher using silicone (9.2 (18.8) pg·mL-1) or glass (3.0 (4.5) pg·mL-1) coating, compared with aluminium (0.5 (2.4) pg·mL-1), polypropylene (0.5 (0.5) pg·mL-1), Teflon (0.5 (0.0) pg·mL-1), and EcoScreen® (0.5 (2.0) pg·mL-1). Albumin in vivo was mainly detectable using glass coating. In conclusion, a condenser with silicone or glass coating is more efficient for measurement of 8-isoprostane or albumin in exhaled breath condensate, than EcoScreen®, aluminium, polypropylene or Teflon. Guidelines for exhaled breath condensate standardisation should include the most valid condenser coating to measure a specific biomarker.

Biomarker reproducibility in exhaled breath condensate collected with different condensers

Optimal collection and analysis of exhaled breath condensate (EBC) are prerequisites for standardisation and reproducibility of assessments. The present study aimed to assess reproducibility of EBC volume, hydrogen peroxide (H2O2), 8-isoprostane and cytokine measurements using different condensers, including a newly developed glass condenser. At four points in time, 30 healthy subjects performed sequential EBC collections randomly using the following four condensers: glass, silicone, EcoScreen® (Erich Jaeger GmbH, Hoechberg, Germany) and an optimised glass condenser. In small EBC samples, H2O2 was measured by spectrophotometer, 8-isoprostane by enzyme immunoassay, and cytokines by multiplexed xMAP® technology (Luminex Corporation, Austin, TX, USA). The optimised glass condenser yielded significantly more EBC volume (median 2,025 µL, interquartile range 1,600–2,525). The reproducibility of EBC volume, yielded by the new glass condenser, was comparable with EcoScreen® (19–20 coefficients of variation (CV)%), but was significantly better compared with silicone and glass (29–37 CV%). The new condenser was associated with significantly more detections of H2O2, 8-isoprostane, interleukin-2, -4, -5 and -13, and tumour necrosis factor-α. Isoprostane concentrations were significantly higher using the new condenser, whereas H2O2 and cytokine concentrations were not. Reproducibility of biomarkers was equally variable for all condenser types. In conclusion, significantly more exhaled breath condensate volume and biomarker detections were found using the optimised glass condenser, including higher 8-isoprostane levels. However, biomarker reproducibility in exhaled breath condensate in healthy adults was not influenced by the type of condenser.

Cytokines in exhaled breath condensate of children with asthma and cystic fibrosis.

BACKGROUND Inflammatory mediators in exhaled breath condensate (EBC) indicate ongoing inflammation in the lungs and might differentiate between asthma and cystic fibrosis (CF). OBJECTIVES To evaluate the presence, concentration, and short-term variability of TH1- and TH2-mediated cytokines (interferon-gamma [IFN-gamma], tumor necrosis factor alpha [TNF-alpha], interleukin 10 [IL-10], IL-5, IL-4, and IL-2) in EBC of children with asthma or CF and in controls and to analyze the discriminating ability of inflammatory markers in EBC between children with asthma or CF and controls. METHODS Expired air was conducted through a double-jacketed glass tube cooled by circulating ice water. In 33 asthmatic children, 12 children with CF, and 35 control children, EBC was collected during tidal breathing. Cytokines were measured using flow cytometry. RESULTS Interleukin 2, IL-4, IFN-gamma, and IL-10 were detected in 16%, 16%, 11%, and 9%, respectively, of all samples in asthma and CF. Interleukin 5 and TNF-alpha were not detected in children with CF. Cytokine concentrations did not differ significantly in children with asthma vs CF. In controls, IFN-gamma, TNF-alpha, and IL-10 were detected in 9%, 14%, and 3%, respectively; IL-2, IL-4, and IL-5 were not detected in controls. CONCLUSIONS Cytokines such as IFN-gamma, TNF-alpha, IL-10, IL-5, IL-4, and IL-2 can be detected in EBC of children with asthma or CF. However, the concentrations found are close to the detection limits of the assay used. These findings emphasize the importance of developing more sensitive techniques for the analysis of EBC and of standardizing the EBC collection method.

Biomarkers in exhaled breath condensate indicate presence and severity of cystic fibrosis in children.

Chronic airway inflammation is present in cystic fibrosis (CF). Non‐invasive inflammometry may be useful in disease management. The aim of the present cross‐sectional study was to investigate: (i) the ability of fractional exhaled nitric oxide and inflammatory markers (IM) [exhaled breath condensate (EBC) acidity, nitrite, nitrate, hydrogen peroxide (H2O2), 8‐isoprostane, Th1/Th2 cytokines] to indicate (exacerbations of) CF; and (ii) the ability of these non‐invasive IM to indicate CF disease severity. In 98 children (48 CF/50 controls), exhaled nitric oxide was measured using the NIOX, and condensate was collected using a glass condenser. In CF interferon (IFN‐γ) and nitrite concentrations were significantly higher, whereas exhaled nitric oxide levels were significantly lower compared with controls (3.3 ± 0.3 pg/ml, 2.2 ± 0.2 μm, 10.0 ± 1.2 p.p.b. vs. 2.6 ± 0.2 pg/ml, 1.4 ± 0.1 μm, 15.4 ± 1.4 p.p.b. respectively). Using multivariate logistic regression models, the presence of CF was best indicated by 8‐isoprostane, nitrite and IFN‐γ [sensitivity 78%, specificity 83%; area under receiver operating characteristic curve (AUC) 0.906, p < 0.001]. An exacerbation of CF was best indicated by 8‐isoprostane and nitrite (sensitivity 40%, specificity 97%, AUC curve 0.838, p = 0.009). Most indicative biomarkers of CF severity were exhaled nitric oxide, and condensate acidity (sensitivity 96%, specificity 67%; AUC curve 0.751, p = 0.008). In this cross‐sectional study, the combination of different exhaled IM could indicate (exacerbations of) CF, and severity of the disease in children. Longitudinal data are necessary to further confirm the role of these markers for the management of CF in children.

A European Respiratory Society technical standard: exhaled biomarkers in lung disease

Breath tests cover the fraction of nitric oxide in expired gas (FENO), volatile organic compounds (VOCs), variables in exhaled breath condensate (EBC) and other measurements. For EBC and for FENO, official recommendations for standardised procedures are more than 10 years old and there is none for exhaled VOCs and particles. The aim of this document is to provide technical standards and recommendations for sample collection and analytic approaches and to highlight future research priorities in the field. For EBC and FENO, new developments and advances in technology have been evaluated in the current document. This report is not intended to provide clinical guidance on disease diagnosis and management. Clinicians and researchers with expertise in exhaled biomarkers were invited to participate. Published studies regarding methodology of breath tests were selected, discussed and evaluated in a consensus-based manner by the Task Force members. Recommendations for standardisation of sampling, analysing and reporting of data and suggestions for research to cover gaps in the evidence have been created and summarised. Application of breath biomarker measurement in a standardised manner will provide comparable results, thereby facilitating the potential use of these biomarkers in clinical practice. ERS technical standard: exhaled biomarkers in lung disease http://ow.ly/mAjr309DBOP

Feasibility of a new method to collect exhaled breath condensate in pre‐school children

Rosias PPR, Robroeks CM, van de Kant KD, Rijkers GT, Zimmermann LJ, van Schayck CP, Heynens JW, Jöbsis Q, Dompeling E. Feasibility of a new method to collect exhaled breath condensate in pre‐school children. 
Pediatr Allergy Immunol 2010: 21: e235–e244.
© 2009 John Wiley & Sons A/S

The effect of 3% and 6% hypertonic saline in viral bronchiolitis: a randomised controlled trial

Bronchiolitis is a common disorder in young children that often results in hospitalisation. Except for a possible effect of nebulised hypertonic saline (sodium chloride), no evidence-based therapy is available. This study investigated the efficacy of nebulised 3% and 6% hypertonic saline compared with 0.9% hypertonic saline in children hospitalised with viral bronchiolitis. In this multicentre, double-blind, randomised, controlled trial, children hospitalised with acute viral bronchiolitis were randomised to receive either nebulised 3%, 6% hypertonic saline or 0.9% normal saline during their entire hospital stay. Salbutamol was added to counteract possible bronchial constriction. The primary endpoint was the length of hospital stay. Secondary outcomes were need for supplemental oxygen and tube feeding. From the 292 children included in the study (median age 3.4 months), 247 completed the study. The median length of hospital stay did not differ between the groups: 69 h (interquartile range 57), 70 h (IQR 69) and 53 h (IQR 52), for 3% (n=84) and 6% (n=83) hypertonic saline and 0.9% (n=80) normal saline, respectively, (p=0.29). The need for supplemental oxygen or tube feeding did not differ significantly. Adverse effects were similar in the three groups. Nebulisation with hypertonic saline (3% or 6% sodium chloride) although safe, did not reduce the length of stay in hospital, duration of supplemental oxygen or tube feeding in children hospitalised with moderate-to-severe viral bronchiolitis. Hypertonic saline nebulisation did not reduce hospital stay for children with viral bronchiolitis http://ow.ly/xRVVx

Methodological aspects of exhaled breath condensate collection and analysis.

The collection and analysis of exhaled breath condensate (EBC) may be useful for the management of patients with chronic respiratory disease at all ages. It is a promising technique due to its apparent simplicity and non-invasiveness. EBC does not disturb an ongoing respiratory inflammation. However, the methodology remains controversial, as it is not yet standardized. The current diversity of the methods used to collect and preserve EBC, the analytical pitfalls and the high degree of within-subject variability are the main issues that hamper further development into a clinical useful technique. In order to facilitate the process of standardization, a simplified schematic approach is proposed. An update of available data identified open issues on EBC methodology. These issues were then classified into three separate conditions related to their influence before, during or after the condensation process: (1) pre-condenser conditions related to subject and/or environment; (2) condenser conditions related to condenser equipment; and (3) post-condenser conditions related to preservation and/or analysis. This simplified methodological approach highlights the potential influence of the many techniques used before, during and after condensation of exhaled breath. It may also serve as a methodological checklist for a more systematical approach of EBC research and development.

Free radicals in exhaled breath condensate in cystic fibrosis and healthy subjects

Many markers of airway inflammation and oxidative stress can be measured non-invasively in exhaled breath condensate (EBC). However, no attempt has been made to directly detect free radicals using electron paramagnetic resonance (EPR) spectroscopy. Condensate was collected in 14 children with cystic fibrosis (CF) and seven healthy subjects. Free radicals were trapped by 5,5-dimethyl-1-pyrroline-N-oxide. EPR spectra were recorded using a Bruker EMX® spectrometer. Secondly, to study the source of oxygen centered radical formation, catalase or hydrogen peroxide was added to the condensate. Radicals were detected in 18 out of 21 condensate samples. Analysis of spectra indicated that both oxygen and carbon centered radicals were trapped. Within-subject reproducibility was good in all but one subject. Quantitatively, there was a trend towards higher maximal peak heights of both oxygen and carbon centered radicals in the children with CF. Catalase completely suppressed the signals in condensate. Addition of hydrogen peroxide resulted in increased radical signal intensity. Detection of free radicals in EBC of children with CF and healthy subjects is feasible using EPR spectroscopy.