Breath Collection from Children for Disease Biomarker Discovery
February 14th, 2019
This protocol describes a simple method for the acquisition of breath samples from children. Briefly, samples of mixed air are pre-concentrated in sorbent tubes prior to gas chromatography-mass spectrometry analysis. Breath biomarkers of infectious and non-infectious diseases can be identified using this breath collection method.
A standardized breath collection method in children could be used to answer key questions about the status of pediatric medical conditions noninvasively and to compare results between studies. The main advantage of this technique is that it allows the collection of breath samples from children in a simple, noninvasive and reproducible manner. The implications of this techniques extend to the diagnosis of a wide range of disease states including infections, metabolic disorders and cancer and for point of care testing for clinical use.
Visual instruction is very helpful because there are so many parts to our breath collection equipment and because setting up the equipment can be complicated. To assemble the breath sample equipment, attach a cardboard mouthpiece to the breath sampler and attach a short length of large diameter tubing to the other end of the sampler. Attach the breath connector to the bag valve via the tubing.
Turn the knurled thumb screw on the side of the inlet fitting counterclockwise to unlock the valve in the open position and push the stem of the bag valve down to open the inlet fitting for sampling. Turn the knurled thumb screw on the side of the inlet fitting clockwise to lock the valve open and confirm that the blue valve on the breath sampler is open and parallel with the connector. Then write the patient ID, date and time on the label of the polymer bag.
Before initiating the breath collection, use a breath sampler without a bag to demonstrate the breath exhalation to the patient. Explain that the patient should breath out like they would do when blowing up a balloon and to continue breathing out as far as they comfortably can. Provide the patient with the labeled breath sampler and ask the patient to exhale into the bag as in the demonstration.
Close the blue valve on the breath sampler device as soon as the patient has finished breathing out and after waiting at least 30 seconds between collections have the patient repeat the exhalation until at least one liter of breath has been collected. Note on the bag label how many breaths were collected from the patient and turn the knurled thumb screw on the side of the inlet fitting counterclockwise to push the stem of the valve up to close the inlet fitting. Then turn the knurled thumb screw on the side of the inlet fitting clockwise to lock the bag valve closed and detach the bag from the breath sampler.
Immediately after collecting the breath sample, attach a new sample bag to a pump outlet port. And push the stem of the bag valve down to open the inlet fitting for sampling. Turn the knurled thumb screw on the side of the inlet fitting clockwise to lock the valve open and set the pump to run at 100 milliliters per minute for 12 minutes.
The pump will collect 1, 200 milliliters of ambient air. At the end of the ambient air collection, turn the knurled thumb screw on the side of the inlet fitting counterclockwise and push the stem of the valve up to close the inlet fitting. Then turn the knurled thumb screw on the side of the inlet fitting clockwise to lock the bag valve closed and detach the bag from the pump.
To transfer the breath or ambient air sample to a thermal desorption tube within one hour of collection, remove the tube from the refrigerator and use the manufacturer's capping tube to remove the long term storage caps from the sorbent tube. Use tubing to attach the grooved end of the tube to the bag and insert the other end of the thermal desorption tube into a piece of tubing connected to a pump. Set the pump to run at 100 milliliters per minute for 10 minutes.
Turn the knurled thumb screw on the side of the inlet fitting counterclockwise to open the valve and push the stem of the valve down to open the inlet fitting before starting the pump. After 10 minutes, the pump will stop. Remove the thermal desorption sorbent tube, tighten the caps onto both ends and store the sorbent tube in the refrigerator until analysis.
In this representative study of breath and ambient air samples from 10 children undergoing evaluation at St.Louis Children's Hospital, significantly more volatile organic compounds or VOCs on average were found in the breath samples compared to the ambient environmental control samples. The increased number of VOCs in breath compared to ambient air can be visibly distinguished by comparing the representative total ion chromatograms in each sample. As a quality control measure of successful breath collection, the levels of two common breath VOCs were compared to room air controls.
Isoprene, one of the most abundant VOCs in breath was measured in a tenfold higher concentration in pediatric breath samples than in room air controls. Whereas beta-pinene, which is typically found in sub-parts-per-billion levels exhibited a threefold higher abundance in breath than air confirming the success of the breath collection. It is important to transfer the breath sample to the sorbent tubes within an hour of the breath collection and to note the tube orientation during the transfer.
Following this procedure, investigators may want to pursue studies in animal models to answer additional questions about the audience of breath volatile compounds.
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Overview
This protocol outlines a noninvasive method for collecting breath samples from children, which can be analyzed for biomarkers of various diseases. The technique is designed to be simple and reproducible, facilitating comparisons across studies.
Key Study Components
Area of Science
- Neuroscience
- Biology
- Pediatrics
Background
- Breath analysis can provide insights into pediatric medical conditions.
- The method is noninvasive, making it suitable for children.
- Standardized techniques enhance the reliability of results.
- Visual instructions aid in the setup of complex equipment.
Purpose of Study
- To develop a method for noninvasive breath sample collection in children.
- To facilitate the diagnosis of various diseases.
- To enable comparisons of results across different studies.
Methods Used
- Collection of mixed air samples using sorbent tubes.
- Gas chromatography-mass spectrometry for analysis.
- Assembly of breath sample equipment with specific components.
- Use of visual aids for equipment setup.
Main Results
- Successful collection of breath samples from children.
- Identification of biomarkers related to infections and metabolic disorders.
- Demonstration of the method's reproducibility and simplicity.
- Potential applications in point-of-care testing.
Conclusions
- The breath collection method is effective for pediatric use.
- It opens avenues for noninvasive diagnostics in various diseases.
- Standardization can improve research comparability.
Frequently Asked Questions
Executive Industry Relevance
Noninvasive breath sampling enables early detection of pediatric disease biomarkers, supporting target validation in infectious, metabolic, and oncologic indications. The method’s reproducibility and minimal patient cooperation requirements enhance feasibility in resource-limited settings, facilitating cross-study comparability and translational continuity. Standardized collection reduces variability in volatile organic compound (VOC) profiling, improving predictive confidence in biomarker discovery pipelines.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of disease-associated VOC profiles to support hypothesis generation in pediatric populations.
- Operational Value: Provides a reproducible, noninvasive matrix for biomarker discovery without requiring invasive procedures.
- Predictive Value: Facilitates early-stage target validation by correlating breath VOC patterns with clinical phenotypes.
Screening & Assay Development
- Scientific Value: Generates stabilized breath samples suitable for GC-MS analysis, enabling untargeted VOC screening.
- Operational Value: Supports assay standardization through controlled sample volume, timing, and thermal desorption tube transfer within one hour.
- Scalability: Uses portable equipment, allowing deployment in field settings and multi-site studies.
Translational & Preclinical Research
- Translational Continuity: Bridges discovery and preclinical validation by providing human-relevant breath VOC data from pediatric cohorts.
- Biomarker Alignment: Enables identification of VOCs like isoprene and beta-pinene as potential translational biomarkers with fold-change validation against ambient controls.
- Risk De-risking: Reduces mechanistic ambiguity in biomarker studies through standardized, quantifiable outputs.
Pipeline & Workflow Integration
The method fits within the discovery workflow from hypothesis testing to lead identification, providing quantitative VOC measurements that inform target selection and assay readiness.
- Discovery Biology: Supports hypothesis testing by enabling comparison of breath VOC profiles between disease and control states.
- Screening: Produces standardized breath samples amenable to GC-MS, ensuring reproducible chemical readouts for compound screening.
- Analytics: Delivers quantitative VOC measurements (e.g., isoprene at 10x, beta-pinene at 3x ambient levels) that enable cross-sample comparison and threshold setting.
- Translational Research: Connects to preclinical work by establishing human-relevant VOC signatures that can be validated in model systems.
- Enterprise Reuse: Establishes a reusable sampling protocol applicable across infectious, metabolic, and neoplastic disease programs in pediatrics.
Operational & Enterprise Impact
- Scientific Value: Increases target confidence through reproducible, noninvasive biomarker detection in pediatric populations.
- Operational Value: Ensures standardization via fixed collection volume, timed transfer, and cold storage until GC-MS analysis.
- Strategic Value: Improves go/no-go decisions by reducing biological variability in early biomarker discovery.
- Portfolio Impact: Enables risk-adjusted prioritization of VOC targets based on fold-change and reproducibility across cohorts.
Implementation Considerations
- Requires training in breath sampler assembly, valve operation, and sample labeling to ensure reproducibility.
- Depends on access to thermal desorption tubes, pumps, and GC-MS infrastructure for downstream analysis.
- Necessitates cross-team standardization of collection timing, breath volume, and ambient air controls.
- Adaptation considerations include pediatric cooperation, equipment portability, and environmental VOC interference in field settings.
- Practical limitation: Sample integrity depends on transfer to sorbent tubes within one hour of collection to prevent analyte loss.
