Breath Collection from Children for Disease Biomarker Discovery

Summary

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.

Abstract

Breath collection and analysis can be used to discover volatile biomarkers in a number of infectious and non-infectious diseases, such as malaria, tuberculosis, lung cancer, and liver disease. This protocol describes a reproducible method for sampling breath in children and then stabilizing breath samples for further analysis with gas chromatography-mass spectrometry (GC-MS). The goal of this method is to establish a standardized protocol for the acquisition of breath samples for further chemical analysis, from children aged 4-15 years. First, breath is sampled using a cardboard mouthpiece attached to a 2-way valve, which is connected to a 3 L bag. Breath analytes are then transferred to a thermal desorption tube and stored at 4-5 °C until analysis. This technique has been previously used to capture breath of children with malaria for successful breath biomarker identification. Subsequently, we have successfully applied this technique to additional pediatric cohorts. The advantage of this method is that it requires minimal cooperation on part of the patient (of particular value in pediatric populations), has a short collection period, does not require trained staff, and can be performed with portable equipment in resource-limited field settings.

Introduction

Biomarkers can yield valuable information about normal and pathological biological processes that may contribute to clinically identifiable disease. Recently, there has been increasing interest in the evaluation of breath volatiles as biomarkers for a variety of disease states, including infection, metabolic disorders, and cancer ¹. Exhaled breath contains quantifiable levels of volatile organic compounds (VOCs), semi-volatile organic compounds, and microbially derived material (e.g., nucleic acids from bacteria and viruses). The central goal of exhaled breath analysis is to gain insight into the status of a medical condition and/or environment....

Protocol

The study has been approved by the Institutional Review Board of Washington University School of Medicine (#201709030). Informed consent was obtained from a parent or legal guardian prior to inclusion in the study. Photographs in Figure 2 reproduced with written informed parental consent.

1. Breath sampler assembly

1. Using disposable gloves, attach a cardboard mouthpiece to the breath sampler, as shown in Supplemental Figure 1. Attach a short length of large diameter tubing to the other extreme of the breath sampler, as shown in Supplemental Figure 1. Use....

Results

In our study, breath samples were collected from 10 children (8-17 years old) undergoing evaluation at St. Louis Children's Hospital. Breath samples and ambient air samples (n = 10) were collected as described above. Samples were analyzed using gas chromatography quadrupole time-of-flight mass spectrometry (GC-QToF-MS) and thermal desorption, as previously described⁹. After removal of background contaminants, the implemented protocols yielded an averag.......

Discussion

Despite considerable progress in breath research over the last decade, standardized practices for the sampling and analysis of breath gas volatiles remain undefined¹⁰. A primary reason for this lack of standardization has been the diversity of breath collection methods, which have direct impact on the resulting chemical diversity present in any given exhaled breath sample. Breath exhalate contains an extensive range of volatile organic compounds at highly varied concentrations⁶

Materials

Name	Company	Catalog Number	Comments
Breath bag	SKC	237-03	These are 3 L bags
Cardboard mouthpiece	A-M systems	161902	0.86" OD, 2.00" L
Large diameter tubing	Cole Parmer	95802-11	Silicone Tubing, 1/4"ID x 5/16"OD,
Long-term storage caps	Markes International	C-CF010	Brass storage cap ¼" & PTFE ferrule, pk 10
Male adapter	Charlotte Pipe	2109	Part 1/3 of breath connector (1/2" Universal part No. 436-005)
Male adapter (made from Teflon)	In-house built		Part 3/3 of breath connector (1/4" ID x 1/2" MIP). This part was specially machined from rods made from virgin Teflon
Pump	SKC	220-1000TC-C	Pocket PumpTouch with Charger
Small diameter tubing	Supelco	20533	Teflon tubing  L × O.D. × I.D. 25 ft × 1/4 in. (6.35 mm) × 0.228 in. (5.8 mm)
Thermal desorption tubes	Markes International	C2-CAXX-5314	Tube, inert, TnxTA/Sulficarb, cond/cap, pk 10
Tube capping/uncapping tool	Markes International	C-CPLOK
Two-way ball valve connector	Homewerks Worldwide	VBV-P40-E3B	Part 2/3 of breath connector (1/2")