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. Attach a short length of large diameter tubing to the other extreme of the breath sampler, as shown in Supplemental Figure 1. Use new tubing for each patient.</li>
	<li>Attach the breath connector to the bag valve via the tubing. See Figure 1 for photo of the breath sampler and bag connected.</li>
	<li>Turn the knurled thumbscrew on the side of the inlet fitting counterclockwise to unlock the valve and push the stem of the bag valve down, to open the inlet fitting for sampling.</li>
	<li>Lock the valve open, by turning the knurled thumbscrew on the side of the inlet fitting clockwise.</li>
	<li>Confirm that the blue valve on the breath sampler is open (parallel with the connector).</li>
	<li>Write patient ID, date, and time on the label of the polymer bag.</li>
	<li>Condition TD sorbent tube prior to breath collection using recommended procedures (available from individual manufacturers). Cap and store thermal desorption tubes at 4&#8211;5 &#176;C prior to breath collection to minimize artifacts.</li>
</ol>
2. Breath collection
<ol>
	<li>Perform a demonstration of breath exhalation to the child by using a breath sampler (without a bag). Explain to the child that they should breathe out like they would do when &#8220;blowing up a balloon&#8221; and continue breathing out as far as they can comfortably. Put the cardboard mouthpiece between your lips and exhale as far as you can.</li>
	<li>Provide the child with a new breath sampler attached to a bag and ask them to exhale as in the demonstration, as illustrated in Figure 2.</li>
	<li>Close the blue valve on the breath sampler device as soon as the child has finished breathing out. Reopen the valve as needed prior to additional exhalations.</li>
	<li>Repeat step 2.2 and 2.3 until at least 1 L of breath has been collected. For a healthy child this may take 2 exhalations, and for a sick or younger child 2&#8211;4 exhalations. 1 L of breath is the minimum analytical requirement. Note on the bag label how many breaths were collected from the patient. See Supplemental Figure 2 for a photo of bag containing different volumes of breath.</li>
	<li>Before detaching the bag from the breath sampler, make sure to loosen the knurled thumbscrew on the side of the inlet fitting by turning it anticlockwise and push the stem of the valve up to close the inlet fitting. See Supplemental Figure 3 for photo of the bag valve in the open and closed position.</li>
	<li>Lock the bag valve closed by turning the knurled thumbscrew on the side of the inlet fitting clockwise.</li>
	<li>Detach the bag from the breath sampler.</li>
	<li>Dispose of the mouthpiece and put aside the breath sampler for cleaning prior to use with a different patient.</li>
</ol>
3. Breath transfer to thermal desorption tubes
<ol>
	<li>Remove the TD tube from the refrigerator. Remove the long-term storage caps of the sorbent tube, using the manufacturer-provided tube capping/uncapping tool.</li>
	<li>Attach the grooved end of the TD sorbent tube to the sampling bag using tubing. Note that tube orientation is important, as TD tubes are designed to have air flowing in one direction only, starting from the grooved end. Note that the transfer of breath from bag to TD should be done within 1 hour of breath collection.</li>
	<li>Insert the other end of the TD tube into the tubing, which is connected to a pump.</li>
	<li>Turn the pump on and set to run at 100 mL/min for 10 min.</li>
	<li>Open the valve on the bag by turning anticlockwise the knurled thumbscrew on the side of the inlet fitting and push the stem of the valve down to open the inlet fitting. This is illustrated in Supplemental Figure 4, which demonstrates breath transfer into a TD sorbent tube using a pump.</li>
	<li>Start the pump, which will stop after 10 min of collection.</li>
	<li>Remove the TD sorbent tube and tighten the caps onto both ends using the tube capping/uncapping tool. Long-term storage caps must be firmly tightened in order to ensure a leak-tight seal.</li>
	<li>Place a sticker on the end of one cap to indicate the tube has been used. On the sticker, indicate patient study identification (ID) number and date.</li>
	<li>Place tube in a small re-sealable plastic bag. Store sorbent tube at 4&#8211;5 &#176;C. Press the rest of the breath out of the bag and discard bag. Record patient study ID, TD tube serial number, collection day, time of breath collection, time of breath transfer, and food intake (time of food intake prior to breath collection and meals consumed).</li>
</ol>
4. Ambient air collection
<ol>
	<li>Collect the ambient air samples in patient&#8217;s environment immediately after breath collection.
	<ol>
		<li>Attach the bag to the pump outlet port by using tubing, as illustrated in Supplemental Figure 5.</li>
		<li>Push the stem of the bag valve down to open the inlet fitting for sampling.</li>
		<li>Lock the valve open by turning clockwise the knurled thumbscrew on the side of the inlet fitting.</li>
	</ol>
	</li>
	<li>Turn the pump on and set to run at 100 mL/min for 12 min. The pump will collect 1,200 mL of ambient air.</li>
	<li>After the requested volume has been collected, loosen the knurled thumbscrew on the side of the inlet fitting by turning it anticlockwise and push the stem of the valve up to close the inlet fitting.</li>
	<li>Lock the bag valve closed by turning clockwise the knurled thumbscrew on the side of the inlet fitting.</li>
	<li>Detach the bag from the pump.</li>
	<li>Follow the same steps as in section 3. The only difference is that ambient air VOCs will be transferred, not those from breath.</li>
</ol>
5. Sample and data analysis
NOTE: Conditions for analysis of breath and air samples have been described previously9.
<ol>
	<li>Analyze collected data and detect compounds in the chromatograms. Use typical software programs to find and identify all compounds detected by the instrument (Figure 3A). For example, use a deconvolution feature to identify compounds. Filter data using retention window size factor of 80, mass heights filters &#8805;100 counts, and compound absolute area filter &#8805;500 counts.</li>
	<li>Use chemical standards to identity compounds in breath and air samples. Extract the base ion peak area of compounds of interest, such as isoprene and &#946;-pinene (Figure 4), and compare the levels of volatiles in breath and air.</li>
</ol>

<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>

The authors have nothing to disclose.

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><tbody><tr><td>Breath bag&amp;nbsp;</td><td>SKC</td><td>237-03</td><td>These are 3 L bags</td></tr><tr><td>Cardboard mouthpiece&amp;nbsp;</td><td>A-M systems</td><td>161902</td><td>0.86&quot; OD, 2.00&quot; L</td></tr><tr><td>Large diameter tubing</td><td>Cole Parmer</td><td>95802-11</td><td>Silicone Tubing, 1/4&quot;ID x 5/16&quot;OD,</td></tr><tr><td>Long-term storage caps&amp;nbsp;</td><td>Markes International</td><td>C-CF010</td><td>Brass storage cap &amp;frac14;&quot; &amp;amp; PTFE ferrule, pk 10</td></tr><tr><td>Male adapter</td><td>Charlotte Pipe</td><td>2109</td><td>Part 1/3 of breath connector (1/2&quot; Universal part No. 436-005)</td></tr><tr><td>Male adapter (made from Teflon)</td><td>In-house built</td><td></td><td>Part 3/3 of breath connector (1/4&quot; ID x 1/2&quot; MIP). This part was specially machined from rods made from virgin Teflon</td></tr><tr><td>Pump</td><td>SKC</td><td>220-1000TC-C</td><td>Pocket PumpTouch with Charger</td></tr><tr><td>Small diameter tubing&amp;nbsp;</td><td>Supelco</td><td>20533</td><td>Teflon tubing&amp;nbsp; L &amp;times; O.D. &amp;times; I.D. 25 ft &amp;times; 1/4 in. (6.35 mm) &amp;times; 0.228 in. (5.8 mm)&amp;nbsp;</td></tr><tr><td>Thermal desorption tubes&amp;nbsp;</td><td>Markes International</td><td>C2-CAXX-5314</td><td>Tube, inert, TnxTA/Sulficarb, cond/cap, pk 10</td></tr><tr><td>Tube capping/uncapping tool</td><td>Markes International</td><td>C-CPLOK</td><td></td></tr><tr><td>Two-way ball valve connector&amp;nbsp;</td><td>Homewerks Worldwide</td><td>VBV-P40-E3B</td><td>Part 2/3 of breath connector (1/2&quot;)</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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Research

JoVE Journal

Chemistry

6.9K Views.  Washington University School of Medicine. Een gestandaardiseerde ademinzamelingsmethode bij kinderen kan worden gebruikt om belangrijke vragen over de status van pediatrische medische aandoeningen niet-invasief te beantwoorden en om resultaten tussen studies te vergelijken. Het belangrijkste voordeel van deze techniek is dat het mogelijk maakt het verzamelen van adem monsters van kinderen in een eenvoudige, niet-invasieve en reproduceerbare manier. De implicaties van deze technieken breiden zich uit tot de diagnose van een breed scal...

Video: Breath Collection from Children for Disease Biomarker Discovery

Real-time Breath Analysis by Using Secondary Nanoelectrospray Ionization Coupled to High Resolution Mass Spectrometry

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental exposure in a non-invasive manner. In this work, we present a protocol to characterize the exhaled VOCs in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry (Sec-nanoESI-HRMS). The homemade Sec-nanoESI source was readily set up based on a commercial nanoESI source. Hundreds of peaks were observed in the background-subtracted mass spectra of exhaled breath, and the mass accuracy values are -4.0-13.5 ppm and -20.3-1.3 ppm in the positive and negative ion detection modes, respectively. The peaks were assigned with accurate elemental composition according to the accurate mass and isotopic pattern. Less than 30 s is used for one exhalation measurement, and it takes approximately 7 min for six replicated measurements.

A protocol for characterizing chemical composition of exhaled breath in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry is demonstrated.

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental ...

Visualizing Field Data Collection Procedures of Exposure and Biomarker Assessments for the Household Air Pollution Intervention Network Trial in India

Here, we present a visual representation of standard procedures to collect population-level data on personal exposures to household air pollution (HAP) from two different study sites in a resource-constrained setting of Tamil Nadu, India. Particulate matter PM2.5 (particles smaller than 2.5 microns in aerodynamic diameter), carbon monoxide (CO), and black carbon (BC) were measured in pregnant mothers (M), other adult women (OAW), and children (C) at various times over a 4 year period. In addition, stove usage monitoring (SUMs) with data-logging thermometers and ambient measurements of air pollution were carried out. Furthermore, the feasibility of collecting biological samples (urine and dried blood spots [DBSs]) from study participants at the field sites was successfully demonstrated. Based on findings from this and earlier studies, the methods used here have enhanced the data quality and avoided issues with household air pollution and biological sample collection in resource-constrained situations. The procedures established may be a valuable educational tool and resource for researchers conducting similar air pollution and health studies in India and other low- and middle-income countries (LMICs).

We detail the consistent, high-quality procedures used throughout air and biological sampling processes at Indian field sites during a large randomized controlled trial. Insights gathered from the oversight of applications of innovative technologies, adapted for exposure assessment in rural regions, enable better field data collection practices with more reliable outcomes.

Here, we present a visual representation of standard procedures to collect population-level data on personal exposures to household air pollution (HAP) ...

Environment

Dried Blood Spot Collection of Health Biomarkers to Maximize Participation in Population Studies

Biomarkers are directly-measured biological indicators of disease, health, exposures, or other biological information. In population and social sciences, biomarkers need to be easy to obtain, transport, and analyze. Dried Blood Spots meet this need, and can be collected in the field with high response rates. These elements are particularly important in longitudinal study designs including interventions where attrition is critical to avoid, and high response rates improve the interpretation of results. Dried Blood Spot sample collection is simple, quick, relatively painless, less invasive then venipuncture, and requires minimal field storage requirements (i.e.&#160;samples do not need to be immediately frozen and can be stored for a long period of time in a stable freezer environment before assay). The samples can be analyzed for a variety of different analytes, including cholesterol, C-reactive protein, glycosylated hemoglobin, numerous cytokines, and other analytes, as well as provide genetic material. DBS collection is depicted as employed in several recent studies.

Biomarkers are directly-measured biological indicators of disease or health. In population and social sciences, biomarkers need to be easy to obtain, transport, and analyze. Dried Blood Spot (DBS) collection meets this need, can be collected in the field with high response rates&#160;and analyzed for a variety of biomarkers.

Biomarkers are directly-measured biological indicators of disease, health, exposures, or other biological information. In population and social sciences, ...

Medicine

Capturing Actively Produced Microbial Volatile Organic Compounds from Human-Associated Samples with Vacuum-Assisted Sorbent Extraction

Volatile organic compounds (VOCs) from biological samples have unknown origins. VOCs may originate from the host or different organisms from within the host&#39;s microbial community. To disentangle the origin of microbial VOCs, volatile headspace analysis of bacterial mono- and co-cultures of Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii, and stable isotope probing in biological samples of feces, saliva, sewage, and sputum were performed. Mono- and co-cultures were used to identify volatile production from individual bacterial species or in combination with stable isotope probing to identify the active metabolism of microbes from the biological samples.
Vacuum-assisted sorbent extraction (VASE) was employed to extract the VOCs. VASE is an easy-to-use, commercialized, solvent-free headspace extraction method for semi-volatile and volatile compounds. The lack of solvents and the near-vacuum conditions used during extraction make developing a method relatively easy and fast when compared to other extraction options such as tert-butylation and solid phase microextraction. The workflow described here was used to identify specific volatile signatures from mono- and co-cultures. Furthermore, analysis of the stable isotope probing of human associated biological samples identified VOCs that were either commonly or uniquely produced. This paper presents the general workflow and experimental considerations of VASE in conjunction with stable isotope probing of live microbial cultures.

This protocol describes the extraction of volatile organic compounds from a biological sample with the vacuum-assisted sorbent extraction method, gas chromatography coupled with mass spectrometry using the Entech Sample Preparation Rail, and data analysis. It also describes culture of biological samples and stable isotope probing.

Volatile organic compounds (VOCs) from biological samples have unknown origins. VOCs may originate from the host or different organisms from within the ...

Biochemistry

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Immunology and Infection

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

Oral Biofilm Sampling for Microbiome Analysis in Healthy Children

Oral biofilm and its molecular analysis provide a basis for investigating various dental research and clinical questions. Knowledge of biofilm composition leads to a better understanding of cariogenic and periopathogenic mechanisms. Microbial changes taking place in the oral cavity during childhood are of interest for several reasons. The evolution of the child oral microbiota and shifts in its composition need to be analyzed further to understand and possibly prevent the onset of disease. At the same time, advanced knowledge of the natural composition of oral biofilm is needed. Early stages of caries-free permanent dentition with healthy gums provide a widely unaffected subgingival habitat that can serve as an in situ baseline for studying features of oral health and disease. Analysis of children's oral biofilm during different stages in life is thus an important theme in the field. Modern molecular analysis methods can provide comprehensive information about the bacterial diversity of such biofilms. To enable microbiota data comparison, it is important to standardize each step in the procedure for molecular data generation. This procedure spans from clinical sampling, Next Generation Sequencing (NGS), bioinformatic data processing, to taxonomic interpretation. One of the most critical factors here is biofilm sampling. Sampling in children is even more challenging in particular due to limited space in subgingival areas. We thus focus on the use of paper points for subgingival sampling. This article provides a detailed protocol for oral biofilm sampling of the subgingival sulcus, the mucosa, and saliva in children.

Changes in the oral microbiome throughout childhood are of growing interest. Comparison of different microbiome studies reveals a lack of standardized sampling protocols. Limited space makes sampling the sound subgingival sulcus of children challenging. Paper point sampling is presented here in detail as the method of choice for this area.

Oral biofilm and its molecular analysis provide a basis for investigating various dental research and clinical questions. Knowledge of biofilm composition ...

Absorption of Nasal and Bronchial Fluids: Precision Sampling of the Human Respiratory Mucosa and Laboratory Processing of Samples

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the human respiratory tract. NA is a non-invasive technique which absorbs fluid from the inferior turbinate, and causes minimal discomfort. NA has yielded reproducible results with the ability to frequently repeat sampling of the upper airway. 
By comparison, alternative methods of sampling the respiratory mucosa, such as nasopharyngeal aspiration (NPA) and conventional swabbing, are more invasive and may result in greater data variability. Other methods have limitations, for instance, biopsies and bronchial procedures are invasive, sputum contains many dead and dying cells and requires liquefaction, exhaled breath condensate (EBC) contains water and saliva, and lavage samples are dilute and variable.
BA can be performed through the working channel of a bronchoscope in clinic. Sampling is well tolerated and can be conducted at multiple sites in the airway. BA results in MLF samples being less dilute than bronchoalveolar lavage (BAL) samples.
This article demonstrates the techniques of NA and BA, as well as the laboratory processing of the resulting samples, which can be tailored to the desired downstream biomarker being measured. These absorption techniques are useful alternatives to the conventional sampling techniques used in clinical respiratory research.

This manuscript describes the use of nasal and bronchial absorption techniques, specifically using synthetic absorptive matrices (SAM) to sample the mucosal lining fluid (MLF) of the upper and lower airway. These methods provide better standardization and tolerability than existing respiratory sampling techniques.

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the ...

Simplified Whole Body Plethysmography to Characterize Lung Function During Respiratory Melioidosis

Surrogate animal models of disease are subject to the 3Rs of Responsible Research. There is a frequent revisiting of refinements to animal models to ensure that both animal welfare and scientific insights advance with the availability of new technologies. This article demonstrates the use of Simplified Whole Body Plethysmography (sWBP) to non-invasively study respiratory failure in a model of lethal respiratory melioidosis. sWBP has the sensitivity to detect breathing in mice through the entirety of the course of the disease, allowing for the moribund-associated symptoms (bradypnea and hypopnea) to be measured and potentially used to develop humane endpoint criteria.
Some of the benefits of sWBP in the context of respiratory disease are that host breath monitoring comes the closest of any physiologic measurement at assessing dysfunction of the primary infected tissue, namely, the lung. In addition to biological significance, the use of sWBP is rapid and non-invasive, minimizing stress in research animals. This work demonstrates the use of in-house sWBP apparatus to monitor disease throughout the course of respiratory failure in the murine model of respiratory melioidosis.

This protocol presents the construction and use of a Simplified Whole Body Plethysmography apparatus to monitor bacterial respiratory disease progression non-invasively.

Surrogate animal models of disease are subject to the 3Rs of Responsible Research. There is a frequent revisiting of refinements to animal models to ...

Breath Collection from Children for Disease Biomarker Discovery

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