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.

Today, we're going to demonstrate the technique of nasosorption, a form of precision mucosal sampling using a synthetic absorptive matrix to absorb mucosal lining fluid. The nasosorption sampler consists of a handle and SAM in a sterile cryotube. The SAM consists of polymers, either fibers or foam, and are specially selected to be soft and absorptive, with rapid wicking for sample collection.

SAMs also have minimal protein binding, to allow the efficient elution of absorbed secretions. Nasosorption is a very gentle, noninvasive technique that can be performed on patients of all ages, including babies. In addition, serial sampling, even every few minutes, is possible.

When carrying out nasosorption you should first wash your hands and put on gloves, preferably in front of the patient. First the nasal cavity is inspected. We recommend the use of a head torch and for the clinician to use their nondominant thumb to retract the patient's nose to visualize the nasal cavity.

A nasal speculum is not usually required. If you look into the nasal cavity, the nares, or nostrils, are not round in cross section and they go straight backwards. You generally see the inferior turbinate as a bulge, an 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 tract. When sampling, the nasosorption SAM is passed gently up the lumen, orientating it to be flat against the inferior turbinate. Then use the index finger to press the SAM onto the nasal mucosa.

This can cause a slight tickling with possible eye watering as the MLF is absorbed. We generally use a timed 60-second absorption. The nasosorption device is then removed from the nostril, put back into the cryotube and can be immediately stored on ice and then deep frozen.

Alternatively you can immediately proceed to elute the MLF. nasosorption is being studied in a variety of nasal mucosal challenge models and also in different airway diseases. However, this relatively new method still requires formal validation against other respiratory sampling methods.

The technique of bronchosorption uses a synthetic absorptive matrix to sample the mucosal lining fluid of the bronchus. The method is performed by specialized personnel via a fiberoptic bronchoscope. The bronchosorption device consists of an external hollow catheter with a central plastic guidewire.

This has a SAM at the end which can be extruded on activation of the handpiece. Like nasosorption the SAM strip is soft and absorptive with rapid wicking for sample collection. It also has minimal protein binding to allow the efficient elution of absorbed secretions.

Before placing the bronchosorption device down the bronchoscope, it is usual to check that the SAM is extruding and withdrawing back into the catheter with ease. We shall now demonstrate the method of bronchosorption using a bronchoscopy simulator. First a standard bronchoscopy is performed, passing the bronchoscope down the trachea, into the right main bronchus, and onto the level of the bronchus intermedius just proximal to the division of the right lower and right middle lobes.

Once inserted to this level, five steps are followed. One, catheter down. Insert the bronchosorption catheter through the operating channel of the bronchoscope until the white tip is visible in the airway to a maximum of one centimeter 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 mucosal damage. Two, SAM out.

Depress the handle of the bronchosorption device so that the SAM strip 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 mucosal lining fluid on the airway wall. Leave the SAM strip within the airway, flat to the mucosal wall for a timed 30 seconds.

Three, SAM in. Under direct vision, retract the SAM directly back into the catheter. As you retract make sure that the moist SAM probe is straight.

If needed, the catheter and bronchoscope tip can be brought back to straighten the SAM. If there is difficulty retracting the SAM, withdraw the whole device with SAM extruded back out of the airway. Four, catheter up.

Withdraw the entire catheter from the operating channel of the bronchoscope. Five, cut off SAM. The SAM is cut off with sterile scissors, put into a cryotube, and should be immediately placed on ice before being deep frozen or eluted directly.

The following video clip shows the extrusion of a SAM into the airway of a patient undergoing a research bronchoscopy. Bronchosorption is currently being studied in a variety of different lung diseases. We hope this method of precision mucosal sampling will prove useful in diagnostic workup and patient stratification and monitoring.

Laboratory Processing After nasosorption and bronchosorption. The Elution of Mucosal Lining Fluid from the Synthetic Absorptive Matrix. Today we're going to demonstrate the general principles of laboratory sample processing after the noninvasive mucosal sampling techniques of nasosorption and bronchosorption.

In particular, we shall demonstrate how we elute the mucosal lining fluid sample from the synthetic absorptive matrix. At the point of collection samples should be placed on ice for transportation to the laboratory. Samples can either be processed immediately or can be placed directly into deep freeze in their original sample tube for elution at a later date.

If immediate processing is being performed the sample can be transferred on ice to the laboratory in its original sample tube. Or the SAM can be detached and placed in elution buffer at the point of collection and then subsequently transferred on ice to the laboratory. Samples can remain on ice either in buffer or within their sample tube for several hours prior to processing.

A washing step prior to the centrifugation and spin elution of the SAM aims to optimize the sample yield. To achieve this the SAM is inserted into an Eppendorf tube along with the desired extraction bugger. The sample is then mixed on a vortex mixer for 30 seconds to wash the SAM of loosely attached fluids and biomolecules.

To ensure full sample recovery spin elution is then performed by adding the moist SAM to a spin insert inside the same Eppendorf tube used for washing. Clean forceps should be used for this procedure and should be changed between samples to prevent contamination. This method uses centrifugation to extract the fluid from the SAM.

We centrifuge samples for 20 minutes at 16, 000 g in a centrifuge cooled to four degrees celsius. The elution bugger used will be determined by the desired application. We commonly use protein-containing amino assay buffer or RNA lysis buffer.

Two types of spin filter 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, spin filters with a 0.2 micrometer pore size can be used.

These filters will decontaminate infected samples and are suitable for samples with suspected microbacterial infection. If such filters are required it's important to include a pre-incubation step where proteinaceous buffer is allowed to soak through the filter prior to the addition of sample. This step minimizes the binding of mediators to the filter by nonspecific protein absorption.

The following is an example method used by our laboratory for nasosorption and bronchosorption processing of samples not requiring a decontamination step including class two pathogens. Step one, the SAM is placed into an Eppendorf containing the appropriate buffer and vortex for 30 seconds. For nasosorption we typically use 300 microliters of buffer and for bronchosorption 100 microliters.

Step two, the SAM is removed using forceps and placed into a plastic mesh filter cup along with the buffer used to wash the SAM. Step three, the plastic mesh filter cup containing the SAM is returned to the original Eppendorf. Step four, the Eppendorf containing filter cup, buffer, and SAM is centrifuged for 20 minutes at 16, 000 g at four degrees celsius.

Following centrifugation the filter cup containing the SAM is removed and disposed of. The elute can then be aliquoted and deep frozen for analysis at a later time. Nasosorption sampling has been applied to a number of clinical respiratory research settings.

This includes measurements of inflammatory mediators during nasal allergen challenge such as prostaglandin D2 and interleukin 5. Importantly in these challenge model settings, sampling by nasosorption allows frequent kinetic profiling of mediator levels, where sampling can be repeated every few minutes. Nasosorption has also been used during a rhinovirus infection challenge in healthy subjects and allergic asthmatics where nasosorption was repeated daily over the study period.

Using nasosorption in the study allowed sensitive characterization of interferon responses to rhinovirus infection. Finally, we have used nasosorption sampling to study infants with bronchiolitis. In this study infection with respiratory syncytial virus was associated with higher levels of inflammatory mediators, such as interferon gamma.

Importantly, sampling with nasosorption allows inclusion of a healthy control group to studies in infants. Bronchosorption was also used in the rhinovirus challenge study in healthy subjects and allergic asthmatics. This study demonstrated significant increases in the inflammatory mediators interferon gamma, ITAC, IL-10, and IL-5 in the lower airway during rhinovirus infection.

Inclusion of this technique to studies of the lower airway creates the possibility for measuring levels of mediators, which might be undetectable by conventional methods such as bronchoalveolar lavage. In conclusion, mucosal lining fluid can be obtained noninvasively and has exciting potential to measure inflammatory responses in infection and inflammation. Methods of elution need to be adapted according to the nature of the samples and the parameters that are to be measured.

In particular, MLF can be used to measure mucosal cytokines and chemokines, viral infections, viral load, bacterial and fungal infections, and the microbiome, and mucosal antibodies. Finally, mucosal sampling and elution methods will require tailored development and careful validation for individual projects.