Quirijn Jöbsis

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.

Exhaled nitric oxide and biomarkers in exhaled breath condensate indicate the presence, severity and control of childhood asthma

Background Exhaled nitric oxide and inflammatory biomarkers in exhaled breath condensate may be useful to diagnose and monitor childhood asthma. Their ability to indicate an asthma diagnosis, and to assess asthma severity and control, is largely unknown.

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.

Volatile organic compounds in exhaled breath as a diagnostic tool for asthma in children

Background The correct diagnosis of asthma in young children is often hard to achieve, resulting in undertreatment of asthmatic children and overtreatment in transient wheezers.

Clinical use of exhaled volatile organic compounds in pulmonary diseases: a systematic review.

There is an increasing interest in the potential of exhaled biomarkers, such as volatile organic compounds (VOCs), to improve accurate diagnoses and management decisions in pulmonary diseases. The objective of this manuscript is to systematically review the current knowledge on exhaled VOCs with respect to their potential clinical use in asthma, lung cancer, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), and respiratory tract infections. A systematic literature search was performed in PubMed, EMBASE, Cochrane database, and reference lists of retrieved studies. Controlled, clinical, English-language studies exploring the diagnostic and monitoring value of VOCs in asthma, COPD, CF, lung cancer and respiratory tract infections were included. Data on study design, setting, participant characteristics, VOCs techniques, and outcome measures were extracted. Seventy-three studies were included, counting in total 3,952 patients and 2,973 healthy controls. The collection and analysis of exhaled VOCs is non-invasive and could be easily applied in the broad range of patients, including subjects with severe disease and children. Various research groups demonstrated that VOCs profiles could accurately distinguish patients with a pulmonary disease from healthy controls. Pulmonary diseases seem to be characterized by a disease specific breath-print, as distinct profiles were found in patients with dissimilar diseases. The heterogeneity of studies challenged the inter-laboratory comparability. In conclusion, profiles of VOCs are potentially able to accurately diagnose various pulmonary diseases. Despite these promising findings, multiple challenges such as further standardization and validation of the diverse techniques need to be mastered before VOCs can be applied into clinical practice.

Hydrogen peroxide in exhaled air of healthy children: reference values

An increased content of hydrogen peroxide (H2O2), a marker of inflammation, has been described in the condensate of exhaled air from adults and children with inflammatory lung disorders, including asthma. However, the normal range of [H2O2] in the exhaled air condensate from healthy children has not been established. Therefore, the aim of this study was to determine the reference range of exhaled [H2O2] in healthy school-aged children. Ninety-three healthy nonsmoking children (48 female and 45 male, mean age 10 yrs, range 8-13 yrs), with a negative history for allergy, eczema or respiratory disease and with a normal lung function, participated. Exhaled air condensate was examined fluorimetrically for the presence of H2O2. In addition, the reproducibility of [H2O2] within subjects and between days and the stability of [H2O2] during storage at -20 degrees C were assessed. The median [H2O2] in the exhaled air condensate of all children was 0.13 microM, with a 2.5-97.5% reference range of <0.01-0.48 microM. No significant difference existed between males and females. There was no correlation between exhaled [H2O2] and age or lung function. Repeated [H2O2] measurements on 2 consecutive days showed satisfactory within-subject reproducibility and [H2O2] in stored samples remained stable for at least 1 month at -20 degrees C. In conclusion, this study provides reference data for exhaled hydrogen peroxide in a large group of healthy children. The observed levels were lower than those reported previously for healthy adults and were independent of age, sex and lung function.

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.

Increased cytokines, chemokines and soluble adhesion molecules in exhaled breath condensate of asthmatic children.

Background Airway inflammation in asthma is characterized by the production of cytokines, chemokines and soluble adhesion molecules. The assessment of these inflammatory biomarkers in exhaled breath condensate (EBC) is hampered by low detection rates. However, the use of a glass condenser system combined with a sensitive analytical technique may increase the possibility to assess these biomarkers in EBC in a reliable way.

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.