Name: breath collection from children for disease biomarker discovery
Uploaded: 2019-02-14T15:00:00
Description: Watch this Scientific Journal Video about Breath Collection from Children for Disease Biomarker Discovery at JoVE.com

We express our gratitude to the children and families of St Louis Children&#39;s Hospital who participated in this study. We acknowledge the unique efforts of Ms. Stacy Postma and Ms. Janet Sokolich during the breath collection. This work is supported by the St. Louis Children&#39;s Hospital Foundation.

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
<ol>
	<li>Using disposable gloves, attach a cardboard mouthpiece to the breath sampler, as shown in Supplemental Figure 1</stron...

<ol>
	<li>Ahmed, W. M., Lawal, O., Nilsen, T. M., Goodacre, R., Fowler, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exhaled+volatile+organic+compounds+of+infection:+a+systematic+review.">Exhaled volatile organic compounds of infection: a systematic review.</a> ACS Infectious Diseases. 3 (10), 695-710 (2017).</li><li>Berna, A. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+breath+specimens+for+biomarkers+of+Plasmodium+falciparum+infection.">Analysis of breath specimens for biomarkers of Plasmodium falciparum infection.</a> Journal of Infectious Diseases. 212 (7), 1120-1128 (2015).</li><li>Lawal, O., Ahmed, W. M., Nijsen, T. M. E., Goodacre, R., Fowler, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exhaled+breath+analysis:+a+review+of+'breath-taking'+methods+for+off-line+analysis.">Exhaled breath analysis: a review of 'breath-taking' methods for off-line analysis.</a> Metabolomics. 13 (10), (2017).</li><li>Kang, S., Thomas, C. L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+long+may+a+breath+sample+be+stored+for+at-80+degrees+C?+A+study+of+the+stability+of+volatile+organic+compounds+trapped+onto+a+mixed+Tenax:Carbograph+trap+adsorbent+bed+from+exhaled+breath.">How long may a breath sample be stored for at-80 degrees C? A study of the stability of volatile organic compounds trapped onto a mixed Tenax:Carbograph trap adsorbent bed from exhaled breath.</a> Journal of Breath Research. 10 (2), (2016).</li><li>Basanta, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+metabolomic+analysis+of+breath+using+differential+mobility+spectrometry+in+patients+with+chronic+obstructive+pulmonary+disease+and+healthy+smokers.">Non-invasive metabolomic analysis of breath using differential mobility spectrometry in patients with chronic obstructive pulmonary disease and healthy smokers.</a> Analyst. 135 (2), 315-320 (2010).</li><li>Mochalski, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+and+breath+levels+of+selected+volatile+organic+compounds+in+healthy+volunteers.">Blood and breath levels of selected volatile organic compounds in healthy volunteers.</a> Analyst. 138 (7), 2134-2145 (2013).</li><li>Mochalski, P., Wzorek, B., Sliwka, I., Amann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suitability+of+different+polymer+bags+for+storage+of+volatile+sulphur+compounds+relevant+to+breath+analysis.">Suitability of different polymer bags for storage of volatile sulphur compounds relevant to breath analysis.</a> Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences. 877 (3), 189-196 (2009).</li><li>Mochalski, P., King, J., Unterkofler, K., Amann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stability+of+selected+volatile+breath+constituents+in+Tedlar,+Kynar+and+Flexfilm+sampling+bags.">Stability of selected volatile breath constituents in Tedlar, Kynar and Flexfilm sampling bags.</a> Analyst. 138 (5), 1405-1418 (2013).</li><li>Schaber, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breathprinting+reveals+malaria-associated+biomarkers+and+mosquito+attractants.">Breathprinting reveals malaria-associated biomarkers and mosquito attractants.</a> Journal of Infectious Diseases. 217 (10), 1553-1560 (2018).</li><li>Herbig, J., Beauchamp, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+standardization+in+the+analysis+of+breath+gas+volatiles.">Towards standardization in the analysis of breath gas volatiles.</a> Journal of Breath Research. 8 (3), (2014).</li><li>Phillips, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variation+in+volatile+organic+compounds+in+the+breath+of+normal+humans.">Variation in volatile organic compounds in the breath of normal humans.</a> Journal of Chromatography B. 729 (1-2), 75-88 (1999).</li><li>Eckel, S. P., Baumbach, J., Hauschild, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+importance+of+statistics+in+breath+analysis-hope+or+curse?.">On the importance of statistics in breath analysis-hope or curse?.</a> Journal of Breath Research. 8 (1), (2014).</li></ol>

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</...

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 environmental exposures non-invasively. There are various methods for collecting and analyzing exhaled breath, depending on the constituents of interest. Currently there is no standardized exhaled breath collection method, which complicates comparative analysis of results across studies. Standardizing breath collection procedures is essential, as the sampling procedure itself has a considerable effect on the downstream results of breath analyses.
In many studies, late respiratory breath sampling is employed2,3. This sampling involves discarding the initial portion of exhaled breath (&#34;dead space&#34;), in order to preferentially capture the air at the end of the breath cycle. The advantage of this strategy is that it minimizes the levels of exogenous VOC (e.g., environmental VOCs), while enriching for endogenous, patient-specific VOCs. This method excludes the first few seconds of exhalation from an individual before collecting the breath sample. Other investigators have employed a pressure sensor to activate sampling during a predefined phase of expiration4,5. Because pressure sensors require complex engineering, this alternative method requires a dedicated and relatively costly sampling device.
Pediatric breath sampling can be particularly challenging. A key concern is that young children may be unable to cooperate with protocols for voluntary exhalation of &#34;dead space&#34; air. For this reason, it is easier to obtain mixed respiratory breath from children. However, a major caveat with mixed respiratory breath samples is the risk of environmental and material contamination. Therefore, the feasibility of pediatric collection is a driving concern in the field.
In addition, to collection methods, storage of breath samples can also influence sample quality. The high humidity in breath exhalate and the ultra-low concentrations (parts-per-trillion) of volatile organic breath compounds make breath samples particularly susceptible to problems related to storage6,7. Despite the great potential of real-time techniques like proton transfer reaction-mass spectrometry (PTR-MS), GC-MS remains the gold standard for the analysis of breath samples. Since GC-MS analysis of breath samples is an offline technique, it is coupled with pre-concentration methods such as thermal desorption (TD) tubes, solid phase micro-extraction, and needle trap devices. Prior to pre-concentration, breath samples need to be temporarily stored in polymer bags8. Polymer bags are popular because of their moderate price, relatively good durability, and reusability. While bags may be reused, time and effort are required to ensure efficient cleaning7,8. Each specific bag type also requires empirically determined and standardized procedures for quality control, reusability, and recovery.
TD tubes are widely used for breath pre-concentration because they capture a large number of volatiles and can be customized. The absorbent materials used for packing TD tubes may be adapted to particular applications and particular target volatiles of interest. TD tubes substantially improve the convenience of breath biomarker studies, especially at remote field sites, because TD tubes safely store breath volatiles for at least two weeks and are easy to transport3.
In an effort to standardize pediatric breath collection for biomarker discovery, here we describe a simple method to collect breath from young children. To illustrate the representative results of the implemented protocols, de-identified data are presented from an on-going cohort of children (age 8-17) undergoing evaluation for nonalcoholic fatty acid liver disease (NAFLD). Full results and analysis of this study will be reported in a later publication. In this work, we report a sub-set of data to demonstrate application of our protocol. In brief, children are instructed to exhale normally via mouthpiece into a polymer bag, as if &#34;blowing a balloon.&#34; The process is repeated 2-4 times until 1 L of breath is collected. The sample is then transferred into a TD tube and stored at 5 &#176;C prior to GC-MS analysis.

<table border="0" cellpadding="0" cellspacing="0" style="width:1303px;" width="1301">
	<tbody>
		<tr height="23">
			<td height="23" style="height:23px;width:243px;">Breath bag&#160;</td>
			<td style="width:171px;">SKC</td>
			<td style="width:119px;">237-03</td>
			<td style="width:771px;">These are 3 L bags</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cardboard mouthpiece&#160;</td>
			<td style="width:171px;">A-M systems</td>
			<td style="width:119px;">161902</td>
			<td>0.86&#34; OD, 2.00&#34; L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Large diameter tubing</td>
			<td style="width:171px;">Cole Parmer</td>
			<td style="width:119px;">95802-11</td>
			<td>Silicone Tubing, 1/4&#34;ID x 5/16&#34;OD,</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Long-term storage caps&#160;</td>
			<td style="width:171px;">Markes International</td>
			<td>C-CF010</td>
			<td>Brass storage cap &#188;&#34; &#38; PTFE ferrule, pk 10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Male adapter</td>
			<td style="width:171px;">Charlotte Pipe</td>
			<td style="width:119px;">2109</td>
			<td>Part 1/3 of breath connector (1/2&#34; Universal part No. 436-005)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Male adapter (made from Teflon)</td>
			<td style="width:171px;">In-house built</td>
			<td></td>
			<td>Part 3/3 of breath connector (1/4&#34; ID x 1/2&#34; MIP). This part was specially machined from rods made from virgin Teflon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pump</td>
			<td style="width:171px;">SKC</td>
			<td>220-1000TC-C</td>
			<td>Pocket PumpTouch with Charger</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Small diameter tubing&#160;</td>
			<td style="width:171px;">Supelco</td>
			<td style="width:119px;">20533</td>
			<td style="width:771px;">Teflon tubing&#160; L &#215; O.D. &#215; I.D. 25 ft &#215; 1/4 in. (6.35 mm) &#215; 0.228 in. (5.8 mm)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermal desorption tubes&#160;</td>
			<td style="width:171px;">Markes International</td>
			<td style="width:119px;">C2-CAXX-5314</td>
			<td>Tube, inert, TnxTA/Sulficarb, cond/cap, pk 10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tube capping/uncapping tool</td>
			<td style="width:171px;">Markes International</td>
			<td>C-CPLOK</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Two-way ball valve connector&#160;</td>
			<td style="width:171px;">Homewerks Worldwide</td>
			<td style="width:119px;">VBV-P40-E3B</td>
			<td>Part 2/3 of breath connector (1/2&#34;)</td>
		</tr>
	</tbody>
</table>

breath collection from children for disease biomarker discovery

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 &#176;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.

In our study, breath samples were collected from 10 children (8-17 years old) undergoing evaluation at St. Louis Children&#39;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...

Watch this Scientific Journal Video about Breath Collection from Children for Disease Biomarker Discovery at JoVE.com

Breath Collection from Children for Disease Biomarker Discovery

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.

Breath collection and analysis can be used to discover volatile biomarkers in a number of infectious and non-infectious diseases, such as malaria, ...

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