Funding: This work was supported by funding from the Imperial National Institute for Health Research (NIHR) Biomedical Research Centre (BRC), the NIHR Health Protection Research Unit (HPRU) in Respiratory Infections at Imperial College London in partnership with Public Health England (PHE) and the NIHR Imperial Patient Safety Translational Research Centre. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR, the Department of Health or Public Health England.

The techniques used in the following protocol have been approved by the West London Research Ethics Committee (Reference number 15/lO/0444).
1. Nasal Absorption (NA)
<ol>
	<li>Preparation prior to NA sampling
	<ol>
		<li>When carrying out NA, first wash hands and put on gloves, preferably in front of the patient.</li>
		<li>Inspect the nasal cavity using a head torch, and have the clinician use their non-dominant thumb to retract the patient&#39;s nose to visualize the nasal cavity. 
		Note: A nasal speculum is not usually required.</li>
		<li>Visualize the nasal cavity and inferior turbinate prior to sampling. 
		Note: The nares (nostrils) are not round in cross-section and they go straight backwards. Generally, the inferior turbinate can be seen as a bulge or indentation on the lateral wall of the nostril, with the nasal septum forming the smooth, flat medial wall. We want to sample from this inferior turbinate, since the underlying epithelium is a simple ciliated epithelium of the respiratory tract44.</li>
	</ol>
	</li>
	<li>NA sampling
	<ol>
		<li>When sampling, pass the NA SAM gently up the lumen of the nostril, orientating it to be flat against the inferior turbinate.</li>
		<li>Ask the donor to use an index finger to press the SAM onto the nasal mucosa. NA can cause a slight tickling, with possible eye watering, as the MLF is absorbed. 
		Note: In adults, we generally perform NA for 60 s.</li>
		<li>After absorption of MLF, remove the NA device from the nostril and put back into the original tube.</li>
		<li>Process the samples immediately in the laboratory or freeze them, as detailed later in this protocol. 
		Note: NA is being studied in a variety of nasal mucosal challenge models, and also in different airways diseases. However, validation against other respiratory sampling methods should be performed for each study and patient population.</li>
	</ol>
	</li>
</ol>
2. Bronchial Absorption (BA)
<ol>
	<li>Preparation prior to BA sampling 
	Note: BA is performed by specialized personnel in a bronchoscopy suite.
	<ol>
		<li>Before placing the BA device down the catheter, check that the SAM is extruding and withdrawing back into the catheter.</li>
		<li>Prior to conducting BA on a patient, conduct a mock BA on a bronchoscopy simulator.</li>
	</ol>
	</li>
	<li>BA sampling
	<ol>
		<li>Pass the bronchoscope down the trachea and right main bronchus to the level of the bronchus intermedius, just proximal to the division into the right lower and right middle lobes. 
		NOTE: We typically sample from the bronchus intermedius, though other sites within the airway can be sampled. When observing the catheter and SAM, one cannot see the bronchoscope tip. We observe the following simple steps for BA, once the bronchoscope has reached the desired sampling site.</li>
		<li>CATHETER DOWN: Insert the BA catheter through the operating channel of the bronchoscope until the white tip is just visible in the airway, to a maximum of 1 cm distal to the end of the bronchoscope. Keep the bronchoscope and catheter tip in the center of the lumen of the airway. Be careful to minimize contact between the catheter tip and the bronchial mucosa to reduce the risk of abrasion to the mucosa.</li>
		<li>SAM OUT: Depress the handle of the BA device so that the SAM is extruded into the lumen of the right middle or right lower lobe airway. Under direct vision, bend the tip of the bronchoscope to ensure that the SAM is making contact with the MLF on the airway wall. Leave the SAM within the airway, flat to the mucosal wall for 30 s.</li>
		<li>SAM IN: Look through the bronchoscope to ensure that the moist SAM probe is straight and not bent back over the end of the catheter. If required, the catheter and bronchoscope tip can be brought back to straighten the SAM. Under direct vision, retract the straight SAM gently back into the catheter. 
		NOTE: If there is difficulty retracting the SAM back into the catheter, withdraw the whole device with SAM extruded back out of the airway.</li>
		<li>CATHETER UP: Withdraw the entire catheter from the operating channel of the bronchoscope.</li>
		<li>CUT OFF SAM: The SAM is cut off with sterile scissors and is then put into a cryotube on ice. These samples can be processed with varying methods, as detailed later in this protocol.</li>
	</ol>
	</li>
</ol>
3. Processing of NA and BA Samples
Note: There are numerous options for laboratory processing of samples resulting from NA and BA. These protocols seek to store samples for later use, and to elute the MLF sample from the SAM.
<ol>
	<li>Options for immediate handling of NA and BA samples
	<ol>
		<li>Option 1: Store the moist SAM on ice for a few hours prior to further processing.</li>
		<li>Option 2: Immediately deep freeze NA SAMs in the collection tube. Similarly, freeze BA SAMs in cryogenic vials after removal, with scissors, from the sampling device.</li>
		<li>Option 3: Place the detached SAM in elution buffer (300 &#181;L) prior to processing directly or deep freezing.</li>
	</ol>
	</li>
	<li>Options of elution buffer for NA and BA samples 
	Note: The choice of elution buffer depends on what is going to be analyzed in the MLF sample and we suggest four main alternatives:
	<ol>
		<li>Use a pre-prepared assay buffer suitable for immunoassay procedures. Such buffers should contain a small amount of detergent (0.05%) as well as protein, e.g., bovine serum albumin (BSA) at 1%.</li>
		<li>Alternatively, use a buffer containing a greater amount of detergent, so that cell lysis occurs. 
		Note: We use buffers containing Triton-X or NP40 at 1% concentration. Cell lysis buffers enable both intracellular and extracellular cytokines to be eluted from the SAM, and generally result in higher levels of cytokines and chemokines. These buffers should also contain protein, and are made up with BSA to 1%.</li>
		<li>For RNA measurements, such as quantitative PCR of viral RNA or measuring host RNA, add RNA extraction buffer directly to the moist SAM. 
		Note: Chaotropic RNA extraction buffers contain guanidinium that denatures proteins. An alternative is to add RNA extraction buffer to the eluted MLF fluid contained in immunoassay or cell lysis buffer.</li>
		<li>Use organic solvents, such as trifluoroacetic acid, for extraction of lipids and metabolites, for evaluation by mass-spectrometry. 
		Note: Details of all these reagents are included in the Materials section.</li>
	</ol>
	</li>
	<li>Elution technique
	<ol>
		<li>For any of the above elution techniques, insert the SAM into a 2 mL micro-centrifuge tube, along with the desired extraction buffer.</li>
		<li>Vortex mix the sample for 30 s to wash the SAM of loosely attached fluids and biomolecules.</li>
		<li>To ensure full sample recovery, perform centrifugal elution by adding the moist SAM to a spin filter mini-column that inserts into the same 2 mL micro-centrifuge tube used for washing. 
		Note: Two types of spin filter mini-column can be used. The first contains only a plastic mesh, which holds the SAM in place, allowing full elution of fluids. Alternatively, if working with infectious materials, use spin filters with a 0.22 &#181;m pore size. These filters will sterilize samples and are suitable for samples with suspected Mycobacterial infection. However, these filters should be pre-incubated with buffer, to minimize binding of mediators to the filter by non-specific interactions.</li>
		<li>Use sterilized forceps to transfer the moist SAM to the spin filter. Change forceps between samples to prevent contamination.</li>
		<li>Centrifuge samples for 20 min at 16,000 x g in a mini-centrifuge cooled to 4 &#176;C.</li>
	</ol>
	</li>
	<li>Summary example protocol
	<ol>
		<li>In the laboratory, label 2 mL micro-centrifuge tubes for sample collection and add the desired volume of elution buffer (typically 300 &#181;L for NA; 100 &#181;L for BA). Seal lid and place on ice.</li>
		<li>Following sampling (either immediately or in the lab) the SAM is removed from the handle using forceps and placed into the buffer containing collection tube (produced in previous step). Ensure the cap of the microcentrifuge tube is closed securely and transfer the samples, on ice, to the laboratory for further processing.</li>
		<li>Remove tubes containing SAM and elution buffer from their transfer container and vortex mix for 30 s.</li>
		<li>Using sterile forceps, remove SAM and place into a spin column (with or without 0.22 &#181;m cellulose acetate filter).</li>
		<li>Collect the elution buffer from the collection tube and retain.</li>
		<li>Place the spin column, with SAM, in the original collection tube (or a new one if sterile-filtering samples) and add collection buffer to spin column. In this manner, the wash fluid will pass through the spin column back into the collection tube, helping to elute the sample from the SAM.</li>
		<li>Centrifuge the samples with lids sealed (16,000 x g, 20 min, 4 &#176;C).</li>
		<li>Remove the samples from the centrifuge and place into a rack.</li>
		<li>Open the sealed lid of the tubes and remove the spin column containing the SAM. Dispose of SAM and spin column in an appropriate waste container.</li>
		<li>Carefully aliquot the eluate contained within the tube into labelled cryogenic tubes. Record the total volume of eluate and the volume in each aliquot.</li>
		<li>Transfer the sealed cryogenic tubes to a -80&#176;C freezer and store upright until use.&#8195;</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

Results from existing airway sampling techniques are regarded as highly variable; alternative sampling techniques are needed to standardize research within this field5. NA and BA permit sampling of MLF in a non-invasive manner, and have exciting potential to measure immune responses in healthy and diseased airways. These techniques offer numerous potential advantages over existing techniques, including greater tolerability, speed of sampling, the ability to frequently repeat sampling, lower inter-user variability, and decreased dilution of immune mediators5. The duration of absorption and the processing technique used should be optimized for each study and rigorously maintained between sampling events. Additionally, in the case of BA, the site of sampling within the airway should be carefully replicated between individuals.
NA, and particularly BA, are still relatively novel techniques for clinical research. However, the benefits of these techniques have resulted in their use in numerous studies, including careful validation against alternative techniques5. These devices are now available as CE-marked devices ready for widespread use in respiratory research. While NA and BA result in much smaller sample volumes than alternative sampling techniques, higher obtained concentrations can result in greater sensitivity for low abundance immune mediators.
Depending on desired downstream applications, NA and BA samples can be directly frozen for later processing, enhancing study feasibility in a clinical research environment. The protocol for sample handling can also be adapted to suit particular downstream applications. The suggested processing techniques can be used for collection of protein or lipid immune mediators or nucleic acids, but should be optimized for each study. In particular, MLF can be eluted with different buffers. Firstly, immunoassay buffer can be used to measure mucosal cytokines, chemokines, and antibodies6,45. Buffers with higher detergent levels can also be used to guarantee cell lysis occurs, allowing the inclusion of intra-cellular cytokines. Chaotropic RNA extraction buffers should be used for the determination of viral infection, viral load, host mRNA, and the microbiome. Alternatively, organic solvents can be used for lipidomics and mass spectrometry.
In conclusion, direct absorption of MLF from mucosal surfaces is an exciting technique with potential use in respiratory, gastro-intestinal, urogenital, and other mucosal diseases. However, these promising absorption techniques will require precise validation of sampling and processing technique for individual assays (biomarkers) in each disease setting. In addition, these novel precision mucosal sampling techniques will require validation against conventional samples, such as from blood, breath, and sputum. With these techniques, MLF can be used to measure microbes, cytokines, chemokines, prostanoids, and antibodies.

Most respiratory diseases cause an inflammatory response, and there is an urgent need for sampling from the respiratory mucosal surface in allergic rhinitis, viral and fungal infections, tuberculosis, asthma, chronic obstructive pulmonary disease, pulmonary fibrosis and lung cancer 1. Nasopharyngeal aspirate (NPA), nasal lavage, and bronchoalveolar lavage (BAL) are common techniques for sampling the upper and lower airway. However, these techniques present considerable problems including poor tolerability, dilution of inflammatory mediators, and the inability to frequently repeat sampling2. One alternative to NPA sampling is the use of swabs, either nylon flocked, cotton, or rayon3,4, but these also have limitations, since they cause discomfort and damage to the nasal epithelium, and in some cases irreversible binding of inflammatory mediators5. These techniques cannot generally be repeated serially over an hour, and alternative techniques may be more effective for detection of low abundance cytokines and chemokines5,6. Additionally, the user variability associated with these techniques may generate inconsistencies in data, resulting in the requirement of larger patient cohorts.
Alternatively, absorption techniques using both natural and synthetic sponges have been used to collect MLF from mucosal surfaces. Ophthalmic sponges composed of natural cellulose (e.g., Weck-cel) have been used to sample saliva, cervical, and vaginal secretions7. In addition, synthetic sponges made from polyvinyl alcohol (PVA) and hydroxylated polyvinyl acetate (HPVA) have been utilised8. Seven different absorptive materials have been compared for sampling oral fluid prior to measuring antibodies9, while polyurethane mini-sponges have been used to collect human tears10.
Filter paper consisting of natural cellulose from the cotton plant has been widely used to absorb nasal secretions since the pioneering paper of Alam and colleagues in 199211,12,13,14,15,16,17. Filter paper discs have been produced from filter cards (e.g. Shandon), and have been utilized to measure histamines and cytokines after controlled nasal allergen challenges and with natural allergen exposure18,19,20,21. However, different batches of filter paper vary in their degree of protein binding and some fail to release cytokines. Methods using a synthetic absorptive matrix (SAM) have therefore been developed2,22,23. SAMs are now generally used to obtain nasal MLF by NA. These absorbent materials are comfortable to use and can obtain MLF even from inflamed noses at frequent intervals over extended periods of time.
Nasal absorption is a form of Precision Mucosal Sampling using a SAM for the sampling of MLF in the upper airway. NA devices are manufactured as CE-marked medical devices from medical grade materials using clean rooms and are free of dust and allergens. The NA sampler consists of a handle and SAM in a sterile cryotube. The SAM consists of polymers, typically fibers, but it is also available as foam, and these are selected to be soft and absorptive, with rapid wicking for sample collection. SAMs have minimal protein binding to allow the efficient elution of absorbed secretions. NA is a very gentle, non-invasive technique that can be performed on donors of all ages. In addition, serial sampling, even every few minutes, is possible. NA has been validated against existing upper airway sampling techniques5 and repetitive sampling has allowed generation of kinetic data following challenge of the airway with allergen23,24,25, bacterial endotoxin26 and viral-type TLR agonists (Jha, A. et al., manuscript in preparation). NA has also been used in infants to investigate the natural history of atopy27,28,29 and in viral bronchiolitis30.
Bronchoscopic microsampling (BMS) is a procedure for collection of MLF in the lower airway that has been developed by Olympus31,32,33. Unfortunately, this BMS system is only licensed in Japan. Olympus supply two BMS systems: one with a fibrous hydroxylated polyester (FHPE) probe34,35,36,37, and one with a cotton probe33,38,39,40,41,42,43. A major stumbling block has been that the BMS probe used in patients with asthma caused mucosal contact bleeding, with half of all samples contaminated with blood. The authors concluded that it was not feasible to sample MLF using this BMS system from peripheral airways in asthma patients43.
As an alternative, we have developed BA using a soft SAM that can be performed during bronchoscopic investigation of the lower airways, including following experimental infection of asthmatic subjects with rhinovirus6. The BA device consists of: an external hollow catheter, a hand-piece that on activation extrudes the SAM, and a central plastic guide wire that has the SAM on its end. As for NA, BA kits are manufactured from medical grade materials using clean rooms and are free of dust and allergens. Additionally, devices are CE marked and are provided gamma-irradiated. The SAM strip is soft, absorptive, and has rapid wicking for sample collection. It also has minimal protein binding to allow the efficient elution of absorbed secretions. The device can fit through the working channel of a bronchoscope and can be used to rapidly and accurately sample MLF at specific sites of interest within the airway. Unlike BAL or BMS, BA does not result in contact bleeding or additional patient discomfort post-procedure.
Careful consideration should be given to the processing of NA and BA samples. Samples can be directly frozen and processed in batches, or can be processed immediately. The type of processing can be tailored toward certain downstream applications, including immunoassays for cytokines, chemokines and immunoglobulins, or elutions of viral, bacterial, and host cell associated RNA. We present the clinical collection and laboratory processing techniques associated with NA and BA as a guide for clinical researchers.

<table border="0" cellpadding="0" cellspacing="0" width="1562">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:253px;">Nasosorption (adult, 7mm width)</td>
			<td style="width:108px;">Hunt Developments</td>
			<td style="width:140px;">NSFL-FXI-11</td>
			<td style="width:1061px;">Different sizes are available for different patient groups/ages.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:253px;">Bronchosorption</td>
			<td style="width:108px;">Hunt Developments</td>
			<td style="width:140px;">BSFL-FXI-11</td>
			<td>Minimum bronchoscope channel size 2mm; Max working length 815mm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:253px;">Corning&#160;Costar&#160;Spin-X&#160;centrifuge tube filters (without membrane)</td>
			<td style="width:108px;">Sigma Aldrich</td>
			<td style="width:140px;">CLS9301-1000EA</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:253px;">Corning&#160;Costar&#160;Spin-X&#160;centrifuge tube filters (0.22um membrane)</td>
			<td style="width:108px;">Sigma Aldrich</td>
			<td style="width:140px;">CLS8160-24EA</td>
			<td>For sterilisation of samples with infection risk.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">Assay (elution) buffer</td>
			<td style="width:108px;">Millipore</td>
			<td style="width:140px;">AB-33k</td>
			<td>Not listed on the Millipore website but available through enquiry or general lab supply companies, such as Cedarlane. Contains 0.05% Tween-20 and 1% BSA.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:253px;">NP-40 Cell lysis buffer</td>
			<td style="width:108px;">Life Technologies</td>
			<td style="width:140px;">FNN0021</td>
			<td>Add bovine serum albumin to 1% (w/v). Can recover higher absolute mediator levels.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">Buffer RLT (RNA extraction)</td>
			<td style="width:108px;">Qiagen</td>
			<td align="right" style="width:140px;">79216</td>
			<td>Allows recovery of RNA from nasosorption and bronchosorption samples.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">Trifluoroacetic acid</td>
			<td style="width:108px;">Sigma Aldrich</td>
			<td style="width:140px;">302031-100ML-M</td>
			<td>For elution of samples to be used in HPLC applications</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">2.0ml micro-centrifuge tubes</td>
			<td style="width:108px;">Costar</td>
			<td align="right" style="width:140px;">3213</td>
			<td>2ml tubes are required for the Spin-X tube filters, traditional 1.5ml tubes will not fit these.</td>
		</tr>
	</tbody>
</table>

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.

NA has been utilized in a number of studies to easily and non-invasively measure mucosal inflammation. Following administration of allergen to the nose, prostaglandin-D2 (PGD2) levels can be observed to rise and fall within minutes (Figure 1), in line with mast cell degranulation (Thwaites et al., manuscript in preparation). Additionally, mediators of type-II inflammation, such as IL-5 (Figure 2, reprinted with permission from 24), can be measured in the hours following nasal allergen challenge23,24. In experimental infection of allergic asthmatics and healthy controls, NA was used to measure a panel of mediators, including interferon-gamma (IFN-&#947;), over the course of 7 days (Figure 3, reprinted with permission from 6). Additionally, in natural respiratory syncytial virus (RSV) infection of infants, NA demonstrated RSV to be associated with elevated levels of inflammatory cytokines, such as IFN-&#947;, relative to non-RSV infants with bronchiolitis and healthy controls (Figure 4, reprinted with permission from 30). Interestingly, this discrimination between RSV and non-RSV bronchiolitis was not significant in time-matched NPA samples (Figure 4).
BA was also used during experimental infection of allergic asthmatics with rhinovirus. At day 4 of rhinovirus infection, levels of IFN-&#947;, CXCL11, IL-10, and IL-5 were elevated from baseline (Figure 5; A, B, C and D, respectively). Additionally, this technique demonstrated elevated IL-5 levels in the lower airway of allergic asthmatics during rhinovirus infection, relative to healthy controls (Figure 5D)(Figure 5 reprinted with permission from 6).
These representative results were generated from samples eluted using assay buffer containing 0.05% Tween-20 and 1% BSA (see Table of Materials).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56413/56413fig1.jpg" /> 
Figure 1: Rapid generation and clearance of prostaglandin-D2 following nasal allergen challenge. Levels of prostaglandin-D2 (PGD2) measured from nasal absorption eluates in serial samples following nasal allergen challenge with Timothy grass pollen (n=5). Each line represents data from one individual. <a href="https://cloudflare.jove.com/files/ftp_upload/56413/56413fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56413/56413fig2.jpg" /> 
Figure 2: Kinetic measurement of IL-5 following nasal allergen challenge. Production of IL-5 in serial nasal absorption samples following nasal allergen challenge. Three repeat allergen challenge studies were conducted, with the participants (n=19) receiving placebo (blue), low dose (10 mg) oral prednisone (orange), or high dose (25 mg) oral prednisone (red) one hour prior to allergen administration. Lines denote geometric mean and error bars are 95% confidence intervals of all participants. (Figure reprinted with permission from Leaker et al., Mucosal Immunology, 2017). <a href="https://cloudflare.jove.com/files/ftp_upload/56413/56413fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56413/56413fig3.jpg" /> 
Figure 3: Induction of Interferon-&#947; during rhinovirus infection. During infection challenge of healthy adults (n= 11, blue) and allergic asthmatics (n= 28, red) with rhinovirus-16, nasal absorption sampling was used to measure levels of interferon-&#947; (IFN- &#947;). Data are represented as A) raw spaghetti plots of individuals and B) median levels with error bars denoting interquartile ranges. (Figure reprinted with permission from Hansel et al.6). <a href="https://cloudflare.jove.com/files/ftp_upload/56413/56413fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56413/56413fig4.jpg" /> 
Figure 4: Nasosorption discriminates elevated Interferon-&#947; associated with RSV infection of infants. Nasal absorption and nasopharyngeal aspiration (NPA) were used to measure interferon-&#947; levels in infants with bronchiolitis associated with respiratory syncytial virus (RSV) infection (red, n=12), a non-RSV respiratory pathogen (green, n=12), and healthy controls (blue, n=9). Data were analyzed using a Kruskall-Wallis test with Dunns correction for multiple comparisons (***p&#60;0.001). Lines denotes median values and error bars are interquartile ranges. (Figure modified with permission from Thwaites et al.30). <a href="https://cloudflare.jove.com/files/ftp_upload/56413/56413fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56413/56413fig5.jpg" /> 
Figure 5: Inflammatory mediators in bronchial absorption samples during rhinovirus infection. 
Following rhinovirus-16 infection of healthy controls (n=10, blue) and allergic asthmatic volunteers (n=23, red), bronchial absorption was used to measure levels of A) IFN-&#947;, B) CXCL11, C) IL-10, and D) IL-5 at baseline and on day 4 of infection. Data analyzed by Wilcoxon signed rank test (matched samples) and Mann-Whitney test (unmatched samples). Figure reprinted with permission from Hansel et al.6 <a href="https://cloudflare.jove.com/files/ftp_upload/56413/56413fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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 ...

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29.2K Views.  St Mary's Hospital. 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.

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Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

Experimental examination of normal human mammary epithelial cell (HMEC) behavior, and how normal cells acquire abnormal properties, can be facilitated by in vitro culture systems that more accurately model in vivo biology. The use of human derived material for studying cellular differentiation, aging, senescence, and immortalization is particularly advantageous given the many significant molecular differences in these properties between human and commonly utilized rodent cells1-2. Mammary cells present a convenient model system because large quantities of normal and abnormal tissues are available due to the frequency of reduction mammoplasty and mastectomy surgeries.
The mammary gland consists of a complex admixture of many distinct cell types, e.g., epithelial, adipose, mesenchymal, endothelial. The epithelial cells are responsible for the differentiated mammary function of lactation, and are also the origin of the vast majority of human breast cancers. We have developed methods to process mammary gland surgical discard tissues into pure epithelial components as well as mesenchymal cells3. The processed material can be stored frozen indefinitely, or initiated into primary culture. Surgical discard material is transported to the laboratory and manually dissected to enrich for epithelial containing tissue. Subsequent digestion of the dissected tissue using collagenase and hyaluronidase strips stromal material from the epithelia at the basement membrane. The resulting small pieces of the epithelial tree (organoids) can be separated from the digested stroma by sequential filtration on membranes of fixed pore size. Depending upon pore size, fractions can be obtained consisting of larger ductal/alveolar pieces, smaller alveolar clusters, or stromal cells. We have observed superior growth when cultures are initiated as organoids rather than as dissociated single cells. Placement of organoids in culture using low-stress inducing media supports long-term growth of normal HMEC with markers of multiple lineage types (myoepithelial, luminal, progenitor)4-5. Sufficient numbers of cells can be obtained from one individual's tissue to allow extensive experimental examination using standardized cell batches, as well as interrogation using high throughput modalities.
Cultured HMEC have been employed in a wide variety of studies examining the normal processes governing growth, differentiation, aging, and senescence, and how these normal processes are altered during immortal and malignant transformation4-15,16. The effects of growth in the presence of extracellular matrix material, other cell types, and/or 3D culture can be compared with growth on plastic5,15. Cultured HMEC, starting with normal cells, provide an experimentally tractable system to examine factors that may propel or prevent human aging and carcinogenesis.

A method to process human mammary surgical discard material is described. Processed tissue, in the form of organoids, can be stored frozen indefinitely or placed in culture for long-term growth. This method enables experimental examination of normal human epithelial cell biology, and the effects of exogenous perturbations.

Experimental  examination of normal human mammary epithelial cell (HMEC) behavior, and how  normal cells acquire abnormal properties, can be facilitated ...

Methodology for Sputum Induction and Laboratory Processing

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which is interesting in various respiratory diseases like asthma, chronic obstructive pulmonary disease (COPD), chronic cough, or idiopathic pulmonary fibrosis. This technique is well tolerated, safe and non-invasive, but is currently limited to research services and specialized centers in clinical practice because it is technically demanding, time-consuming, and requires trained staff. The success rate of sputum induction and analysis is about 80%.
Here, we describe the induction and laboratory processing of sputum samples. Sputum is induced by inhalation of hypertonic or isotonic saline with salbutamol. For the processing, we use the whole sputum technique. Dithiothreitol (DTT) is used to allow mucolysis of sputum samples. The primary aim of sputum processing is to obtain a differential cell count to study the cell types present in the airway lumen. Additional analyses may also be performed on sputum supernatant and sputum cells, which may allow further investigation into inflammatory processes and immune mechanisms. Examples include studying mediators in sputum supernatant and performing a large spectrum of analysis on sputum cells such as flow cytometry, genomics, or proteomics.
Finally, representative results of sputum analysis in healthy controls, asthmatics, and COPD patients are presented.

Here we describe the technique of sputum induction. This protocol also explains the sputum processing to perform a differential cell count and to collect sputum supernatant and cells for further analysis.

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which ...

Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of genetically modified pig models, and the often-limited number of available animals of these models, establishment of (biobank) collections of adequately processed tissue samples suited for a broad spectrum of subsequent analyses methods, including analyses not specified at the time point of sampling, represent meaningful approaches to take full advantage of the translational value of the model. With respect to the peculiarities of porcine anatomy, comprehensive guidelines have recently been established for standardized generation of representative, high-quality samples from different porcine organs and tissues. These guidelines are essential prerequisites for the reproducibility of results and their comparability between different studies and investigators. The recording of basic data, such as organ weights and volumes, the determination of the sampling locations and of the numbers of tissue samples to be generated, as well as their orientation, size, processing and trimming directions, are relevant factors determining the generalizability and usability of the specimen for molecular, qualitative, and quantitative morphological analyses. Here, an illustrative, practical, step-by-step demonstration of the most important techniques for generation of representative, multi-purpose biobank specimen from porcine tissues is presented. The methods described here include determination of organ/tissue volumes and densities, the application of a volume-weighted systematic random sampling procedure for parenchymal organs by point-counting, determination of the extent of tissue shrinkage related to histological embedding of samples, and generation of randomly oriented samples for quantitative stereological analyses, such as isotropic uniform random (IUR) sections generated by the &#34;Orientator&#34; and &#34;Isector&#34; methods, and vertical uniform random (VUR) sections.

The practical application and performance of methods for generation of representative tissue samples of porcine animal models for a broad spectrum of downstream analyses in biobank projects are demonstrated, including volumetry, systematic random sampling, and differential processing of tissue samples for qualitative and quantitative morphologic and molecular analyses types.

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of ...

Remote Laboratory Management: Respiratory Virus Diagnostics

An uptick in recent pandemics (Ebola, Zika, MERS, influenza, etc.) underlines the need for a more 'nimble,' coordinated response that addresses a multitude of issues ranging from transportation, access, facilities, equipment, and communication to provider training. To address this need, we have developed an innovative, scalable, logistics-enhanced, mobile, laboratory facility for emergencies and epidemics in resource-constrained global settings. Utilizing a background in clinical operations as an academic medical center, we designed a rapidly-deployable, modular BSL-2 and BSL-3 facility with user-friendly software for tracking and management of drugs and supplies in remote regions during epidemics and outbreaks. Here, we present our intermodal, mobile, expandable shipping-container laboratory units. The design of the laboratory facilitates off-grid usage by minimizing power consumption and allowing alternate water sources. The unit's information communication technology (ICT) platform provides (i) user-friendly tablet-based documentation, (ii) enhanced tracking of patients and supplies, and (iii) integrated communication onsite with built-in telehealth capabilities. To ensure quality in remote environments, we have developed a checklist for a basic laboratory workflow and a protocol for respiratory viral diagnosis using reverse-transcription polymerase chain reaction (RT-PCR). As described, this innovative and comprehensive approach allows for the provision of laboratory capability in resource-limited global environments.

A rapidly-deployable, off-grid laboratory has been designed and built for remote, resource-constrained global settings. The features and critical aspects of the logistically-enhanced, expandable, multifunctional laboratory modules are explored. A checklist for a basic laboratory workflow and a protocol for a respiratory viral diagnostic test are developed and presented.

An uptick in recent pandemics (Ebola, Zika, MERS, influenza, etc.) underlines the need for a more 'nimble,' coordinated response that addresses a ...

Culture and Imaging of Human Nasal Epithelial Organoids

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis Transmembrane conductance Regulator (CFTR) activity and restoration from small molecule compounds. Our group recently focused on establishing a well-differentiated organoid model directly derived from primary human nasal epithelial cells (HNE). Histology of sectioned organoids, whole-mount immunofluorescent staining, and imaging (using confocal microscopy, immunofluorescent microscopy, and bright field) are essential to characterize organoids and confirm epithelial differentiation in preparation for functional assays. Furthermore, HNE organoids produce lumens of varying sizes that correlate with CFTR activity, distinguishing between CF and non-CF organoids. In this manuscript, the methodology for culturing HNE organoids are described in detail, focusing on the assessment of differentiation using the imaging modalities, including the measurement of baseline lumen area (a method of CFTR activity measurement in organoids that any laboratory with a microscope can employ) as well as the developed automated approach to a functional assay (which requires more specialized equipment).

A detailed protocol is presented here to describe an in vitro organoid model from human nasal epithelial cells. The protocol has options for measurements requiring standard laboratory equipment, with additional possibilities for specialized equipment and software.

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis ...

Collection, Expansion, and Differentiation of Primary Human Nasal Epithelial Cell Models for Quantification of Cilia Beat Frequency

Measurements of cilia function (beat frequency, pattern) have been established as diagnostic tools for respiratory diseases such as primary ciliary dyskinesia. However, the wider application of these techniques is limited by the extreme susceptibility of ciliary function to changes in environmental factors e.g., temperature, humidity, and pH. In the airway of patients with Cystic Fibrosis (CF), mucus accumulation impedes cilia beating. Cilia function has been investigated in primary airway cell models as an indicator of CF Transmembrane conductance Regulator (CFTR) channel activity. However, considerable patient-to-patient variability in cilia beating frequency has been found in response to CFTR-modulating drugs, even for patients with the same CFTR mutations. Furthermore, the impact of dysfunctional CFTR-regulated chloride secretion on ciliary function is poorly understood. There is currently no comprehensive protocol demonstrating sample preparation of in vitro airway models, image acquisition, and analysis of Cilia Beat Frequency (CBF). Standardized culture conditions and image acquisition performed in an environmentally controlled condition would enable consistent, reproducible quantification of CBF between individuals and in response to CFTR-modulating drugs. This protocol describes the quantification of CBF in three different airway epithelial cell model systems: 1) native epithelial sheets, 2) air-liquid interface models imaged on permeable support inserts, and 3) extracellular matrix-embedded three-dimensional organoids. The latter two replicate in vivo lung physiology, with beating cilia and production of mucus. The ciliary function is captured using a high-speed video camera in an environment-controlled chamber. Custom-built scripts are used for the analysis of CBF. Translation of CBF measurements to the clinic is envisioned to be an important clinical tool for predicting response to CFTR-modulating drugs on a per-patient basis.

This protocol describes nasal epithelial cell collection, expansion, and differentiation to organotypic airway epithelial cell models and quantification of cilia beat frequency via live-cell imaging and custom-built scripts.

Measurements of cilia function (beat frequency, pattern) have been established as diagnostic tools for respiratory diseases such as primary ciliary ...

A Traditional Chinese Medicine Characteristic Therapy for Bronchial Asthma: Moxibustion

Bronchial asthma is a chronic inflammatory disease of the airway which can lead to symptoms such as recurrent wheezing, shortness of breath, chest tightness, or cough, as a result of increased airway reactivity. With high diurnal variation, these symptoms often occur or worsen at night or in the morning. By burning and roasting the Chinese medica above human acupoints, moxibustion is a type of treatment that stimulates the activity of the human meridians through drugs and heat stimulation to prevent and treat diseases. According to the principle of syndrome differentiation and treatment of traditional Chinese medicine, acupoints are selected on the corresponding parts, which has a definite effect. It is regarded as a characteristic traditional Chinese medicine therapy for bronchial asthma. This protocol elaborates the methods and steps of patient management, material preparation, selection of acupoints, operation, and postoperative nursing to ensure safe and effective moxibustion treatment to significantly improve the clinical symptoms and quality of life of patients with bronchial asthma.

This protocol presents a curative procedure of moxibustion in treating patients with bronchial asthma.

Bronchial asthma is a chronic inflammatory disease of the airway which can lead to symptoms such as recurrent wheezing, shortness of breath, chest ...

Capturing Oral Submucous Fibrosis Tissue Samples for Spatial-Omics Analysis

Isolation of Cells with Morphological and Spatial Information from Oral Submucous Fibrosis Samples by Laser Capture Microdissection

Oral submucous fibrosis (OSF) is a common type of potentially malignant disorder in the oral cavity. The atrophy of epithelium and fibrosis of the lamina propria and the submucosa are often found on histopathological slides. Epithelial dysplasia, epithelial atrophy, and senescent fibroblasts have been proposed to be associated with the malignant transformation of OSF. However, because of the heterogeneity of potentially malignant oral disorders and oral squamous cell carcinoma, it is difficult to identify the specific molecular mechanisms of malignant transformation in OSF. Here, we present a method to obtain a small number of epithelial or mesenchymal cells carrying morphological data and spatial information by laser capture microdissection on formalin-fixed paraffin-embedded tissue slides. Using a microscope, we can precisely capture microscale (~500 cells) dysplastic or atrophic epithelial tissue and fibrotic subepithelial tissue. The extracted cells can be evaluated by genome or transcriptome sequencing to acquire genomic and transcriptomic data with morphological and spatial information. This approach removes the heterogeneity of bulk OSF tissue sequencing and the interference caused by cells in non-lesioned areas, allowing for precise spatial-omics analysis of OSF tissue.

Author Spotlight: Unlocking the Mysteries of Oral Potential Malignancies 

Laser capture microdissection of oral submucous fibrosis tissues allows for precise extraction of cells from histological regions of interest for the analysis of multi-omics data with morphological and spatial information.

Oral submucous fibrosis (OSF) is a common type of potentially malignant disorder in the oral cavity. The atrophy of epithelium and fibrosis of the lamina ...

Multicolor Immunofluorescence of the Nasal Tissue in an Allergic Rhinitis Rat Model

Immunofluorescent Labeling in Nasal Mucosa Tissue Sections of Allergic Rhinitis Rats via Multicolor Immunoassay

Allergic rhinitis (AR) is a chronic, non-infectious inflammatory disease of the nasal mucosa, primarily mediated by specific immunoglobulin E (IgE), affecting approximately 10%-20% of the world's population. While immunofluorescence (IF) staining has long been a standard technique for detecting disease-specific protein expression, conventional IF techniques are limited in their ability to detect the expression levels of three or more proteins in the same sample. Consequently, multicolor IF techniques have been developed in recent years, which allow the simultaneous labeling of multiple targets in cells or tissues.
This protocol provides a comprehensive overview of the process for establishing a rat model of AR, obtaining nasal mucosal samples, and the technical procedures for multicolor immunofluorescence. All rats in the AR group exhibited typical symptoms such as sneezing, a runny nose, and an itchy nose, with behavioral observations scoring &#8805;5 points. Hematoxylin and eosin (H&amp;E) staining revealed increased inflammatory cell counts and disrupted nasal mucosal integrity in the AR group. Multicolor immunofluorescence (mIF) demonstrated increased expression of ROR&#947;t and TICAM-1, while Foxp3 expression decreased in the nasal mucosa tissue of AR rats.

Author Spotlight: Advancing Allergic Rhinitis Research with Multicolor Immunofluorescence

This protocol describes a multicolor immunofluorescence technique for evaluating the rat model of allergic rhinitis.

Allergic rhinitis (AR) is a chronic, non-infectious inflammatory disease of the nasal mucosa, primarily mediated by specific immunoglobulin E (IgE), ...

Anesthetic Thoracoscopy for Diagnosis of Pleural Effusions

Local Anesthetic Thoracoscopy for Undiagnosed Pleural Effusion

Local anesthetic thoracoscopy (LAT) is a minimally invasive diagnostic procedure gaining recognition among chest physicians for managing undiagnosed pleural effusions. This single-port procedure is conducted with the patient under mild sedation and involves a contralateral decubitus position. It is performed in a sterile setting, typically a bronchoscopy suite or surgical theater, by a single operator with support from a procedure-focused nurse and a patient-focused nurse.
The procedure begins with a thoracic ultrasound to determine the optimal entry point, usually in the IV-V intercostal space along the midaxillary line. Lidocaine/mepivacaine, with or without adrenaline, is used to anesthetize the skin, thoracic wall layers, and parietal pleura. A designated trocar and cannula are inserted through a 10 mm incision, reaching the pleural cavity with gentle rotation. The thoracoscope is introduced through the cannula for systematic inspection of the pleural cavity from the apex to the diaphragm. Biopsies (typically six to ten) of suspicious parietal pleura lesions are obtained for histopathological evaluation and, when necessary, microbiological analysis. Biopsies of the visceral pleura are generally avoided due to the risk of bleeding or air leaks. Talc poudrage may be performed before inserting a chest tube or indwelling pleural catheter through the cannula. The skin incision is sutured, and intrapleural air is removed using a three-compartment or digital chest drainage system. The chest tube is removed once there is no airflow, and the lung has satisfactorily re-expanded. Patients are usually discharged after 2-4 h of observation and followed up on an outpatient basis. Successful LAT relies on careful patient selection, preparation, and management, as well as operator education, to ensure safety and a high diagnostic yield.

Local anesthetic thoracoscopy (LAT) is essential for diagnosing recurrent, undiagnosed pleural effusion when a guideline-based workup fails to provide a specific cause. LAT can be performed as a day-case procedure by chest physicians. Here, we present a step-by-step approach for a successful and safe procedure.

Local anesthetic thoracoscopy (LAT) is a minimally invasive diagnostic procedure gaining recognition among chest physicians for managing undiagnosed ...