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><tbody><tr><td>Nasosorption (adult, 7mm width)</td><td>Hunt Developments</td><td>NSFL-FXI-11</td><td>Different sizes are available for different patient groups/ages.</td></tr><tr><td>Bronchosorption</td><td>Hunt Developments</td><td>BSFL-FXI-11</td><td>Minimum bronchoscope channel size 2mm; Max working length 815mm</td></tr><tr><td>Corning&amp;nbsp;Costar&amp;nbsp;Spin-X&amp;nbsp;centrifuge tube filters (without membrane)</td><td>Sigma Aldrich</td><td>CLS9301-1000EA</td><td></td></tr><tr><td>Corning&amp;nbsp;Costar&amp;nbsp;Spin-X&amp;nbsp;centrifuge tube filters (0.22um membrane)</td><td>Sigma Aldrich</td><td>CLS8160-24EA</td><td>For sterilisation of samples with infection risk.</td></tr><tr><td>Assay (elution) buffer</td><td>Millipore</td><td>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><td>NP-40 Cell lysis buffer</td><td>Life Technologies</td><td>FNN0021</td><td>Add bovine serum albumin to 1% (w/v). Can recover higher absolute mediator levels.</td></tr><tr><td>Buffer RLT (RNA extraction)</td><td>Qiagen</td><td>79216</td><td>Allows recovery of RNA from nasosorption and bronchosorption samples.</td></tr><tr><td>Trifluoroacetic acid</td><td>Sigma Aldrich</td><td>302031-100ML-M</td><td>For elution of samples to be used in HPLC applications</td></tr><tr><td>2.0ml micro-centrifuge tubes</td><td>Costar</td><td>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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30.0K 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.

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

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Immunology and Infection

A Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated procedures to obtain such measurements in the same animal are generally not feasible.  Here, we demonstrate protocols for obtaining from mice repeated measurements of AHR and bronchoalveolar lavage fluid samples.  Mice were challenged intranasally seven times over 14 days with a potent allergen or sham treated.  Prior to the initial challenge, and within 24 hours following each intranasal challenge, the same animals were anesthetized, orally intubated and mechanically ventilated.  AHR, assessed by comparing dose response curves of respiratory system resistance (RRS) induced by increasing intravenous doses of acetylcholine (Ach) chloride between sham and allergen-challenged animals, were determined.  Afterwards, and via the same intubation, the left lung was lavaged so that differential enumeration of airway cells could be performed.  These studies reveal that repeated measurements of AHR and BAL fluid collection are possible from the same animals and that maximal airway hyperresponsiveness and airway eosinophilia are achieved within 7-10 days of initiating allergen challenge.  This novel technique significantly reduces the number of mice required for longitudinal experimentation and is applicable to diverse rodent species, disease models and airway physiology instruments.  

Repeated measurements of rodent respiratory physiology and sampling of airway inflammatory cells are desirable, but generally not feasible. Here we describe a repeatable method for orally intubating mice that permits repeated measurements of airway hyperreactivity and sampling of airway inflammatory cells.

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated ...

Biology

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 ...

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 ...

Label-free Neutrophil Enrichment from Patient-derived Airway Secretion Using Closed-loop Inertial Microfluidics

Airway secretions contain a large number of immune-related cells, e.g., neutrophils, macrophages, and lymphocytes, which can be used as a major resource to evaluate a variety of pulmonary diseases, both for research and clinical purposes. However, due to the heterogeneous and viscous nature of patient mucus, there is currently no reliable dissociation method that does not damage the host immune cells in the patient airway secretion. In this research, we introduce a sample preparation method that uses inertial microfluidics for the patient&#39;s immune assessment. Regardless of the heterogeneous fluidic properties of the clinical samples, the proposed method recovers more than 95% of neutrophils from airway secretion samples that are diluted 1,000-fold with milliliters of clean saline. By recirculating the concentrated output stream to the initial sample reservoir, a high concentration, recovery, and purity of the immune cells are provided; recirculation is considered a trade-off to the single-run syringe-based operation of inertial microfluidics. The closed-loop operation of spiral microfluidics provides leukocytes without physical or chemical disturbance, as demonstrated by the phorbol 12-myristate 13-acetate (PMA)-induced elastase release of sorted neutrophils.

In this research, we demonstrate a label-free neutrophil separation method from clinical airway secretions using closed-loop operation of spiral inertial microfluidics. The proposed method would expand the clinical in vitro assays for various respiratory diseases.

Airway secretions contain a large number of immune-related cells, e.g., neutrophils, macrophages, and lymphocytes, which can be used as a major resource ...

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 ...

Nasal Brushing Sampling and Processing Using Digital High Speed Ciliary Videomicroscopy &ndash; Adaptation for the COVID-19 Pandemic

Primary Ciliary Dyskinesia (PCD) is a genetic motile ciliopathy, leading to significant otosinopulmonary disease. PCD diagnosis is often missed or delayed due to challenges with different diagnostic modalities. Ciliary videomicroscopy, using Digital High-Speed Videomicroscopy (DHSV), one of the diagnostic tools for PCD, is considered the optimal method to perform ciliary functional analysis (CFA), comprising of ciliary beat frequency (CBF) and beat pattern (CBP) analysis. However, DHSV lacks standardized, published operating procedure for processing and analyzing samples. It also uses living respiratory epithelium, a significant infection control issue during the COVID-19 pandemic. To continue providing a diagnostic service during this health crisis, the ciliary videomicroscopy protocol has been adapted to include adequate infection control measures.
Here, we describe a revised protocol for sampling and laboratory processing of ciliated respiratory samples, highlighting adaptations made to comply with COVID-19 infection control measures. Representative results of CFA from nasal brushing samples obtained from 16 healthy subjects, processed and analyzed according to this protocol, are described. We also illustrate the importance of obtaining and processing optimal quality epithelial ciliated strips, as samples not meeting quality selection criteria do now allow for CFA, potentially decreasing the diagnostic reliability and the efficiency of this technique.

Nasal Brushing Sampling and Processing Using Digital High Speed Ciliary Videomicroscopy &#8211; Adaptation for the COVID-19 Pandemic

To guarantee a successful and high-quality ciliary functional analysis for PCD diagnosis, a precise and careful method for respiratory epithelium sampling and processing is essential. To continue providing PCD diagnostic service during the COVID-19 pandemic, the ciliary videomicroscopy protocol has been updated to include appropriate infection control measures.

Primary Ciliary Dyskinesia (PCD) is a genetic motile ciliopathy, leading to significant otosinopulmonary disease. PCD diagnosis is often missed or delayed ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Human Respiratory Bacterial Infection Diagnosis Using Isothermal Amplification

Rapid Detection of Bacterial Pathogens Causing Lower Respiratory Tract Infections via Microfluidic-Chip-Based Loop-Mediated Isothermal Amplification

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will provide practical guidelines for managing and treating RTIs. This study describes a method for rapidly detecting bacterial pathogens that cause respiratory tract infections via multi-channel loop-mediated isothermal amplification (LAMP). LAMP is a sensitive and specific diagnostic tool that rapidly detects bacterial nucleic acids with high accuracy and reliability. The proposed method offers a significant advantage over traditional bacterial culturing methods, which are time-consuming and often require greater sensitivity for detecting low levels of bacterial nucleic acids. This article presents representative results of K. pneumoniae infection and its multiple co-infections using LAMP to detect samples (sputum, bronchial lavage fluid, and alveolar lavage fluid) from the lower respiratory tract. In summary, the multi-channel LAMP method provides a rapid and efficient means of identifying single and multiple bacterial pathogens in clinical samples, which can help prevent the spread of bacterial pathogens and aid in the appropriate treatment of RTIs.

Author Spotlight: Advancing Rapid Detection of Respiratory Pathogens Using Microfluidic Chip 

Various bacterial pathogens can cause respiratory tract infections and lead to serious health issues if not detected accurately and treated promptly. Rapid and accurate detection of these pathogens via&#160;loop-mediated isothermal amplification provides effective management and control of respiratory tract infections in clinical settings.

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will ...

Label-Free Neutrophil Separation from Tracheal Secretion Using Spiral Microfluidics Channel

Source: Ryu, H., et al., <a href="https://app.jove.com/v/57673">Label-free Neutrophil Enrichment from Patient-derived Airway Secretion Using Closed-loop Inertial Microfluidics.</a>&#160;J. Vis. Exp.&#160;(2018).
This video demonstrates separating neutrophils from tracheal secretions using a microfluidic device with spiral channels. The device achieves efficient separation based on cell size by utilizing varying thicknesses and trapezoidal-shaped channels. The interplay between forces results in large neutrophils accumulating near the inner wall while erythrocytes and mucin aggregates remain near the outer wall, ensuring effective neutrophil separation.

Source: Ryu, H., et al., Label-free Neutrophil Enrichment from Patient-derived Airway Secretion Using Closed-loop Inertial Microfluidics.&#160;J. Vis. ...

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

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Methodology for Sputum Induction and Laboratory Processing

Nasal Brushing Sampling and Processing Using Digital High Speed Ciliary Videomicroscopy – Adaptation for the COVID-19 Pandemic

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

Rapid Detection of Bacterial Pathogens Causing Lower Respiratory Tract Infections via Microfluidic-Chip-Based Loop-Mediated Isothermal Amplification

Label-Free Neutrophil Separation from Tracheal Secretion Using Spiral Microfluidics Channel