Collection of Nasal Secretions and Tears and Their Use in Allergology

Sveva Castelli; Stefania Arasi; Ruby Pawankar; Paolo M. Matricardi

Disclosures

Curr Opin Allergy Clin Immunol. 2018;18(1):1-9. 

In This Article

Sampling Methods of Nasal Secretions

Current sampling techniques are quite heterogeneous and they are mainly based on the following principles: collection of spontaneous secretions, nasal washings and the use of different absorption devices (Table 1). They all reflect different compromises between the need to obtain a sufficient sample volume and the desire to avoid artefacts of the procedure.[8] The sampling method has an important impact on the level of any substance detected in nasal secretions.[9] The comparison of the outcomes of different studies is thus difficult.

Spontaneous Secretions

The amount of spontaneously secreted nasal fluid is often insufficient in healthy individuals under normal conditions.[8] On the contrary, patients with nasal mucosa diseases frequently present nasal hypersecretion and rhinorrhea. In this case, spontaneous secretion are a specimen simple to collect and offering valid results.[23]

Nose Blowing

Blowing the nose is a simple way to collect nasal secretions and can be performed with a plastic or an aluminum handkerchief in which each nostril should be blown separately. With the help of a Pasteur pipette the fluid can be recovered and transferred into an Eppendorf tube containing a fixed amount of buffer for dilution. Arguments for this method might be that it is quick and easy to perform, it allows a fixed dilution, and it does not irritate the mucosa. However, the quantities of nasal fluid obtained is very variable, and insufficient sample size for analysis is often observed, especially in healthy individuals.[8]

Aspiration

Aspiration techniques were designed to optimize the collection of spontaneous secreted nasal fluid. The microsuction device is a quick method with minimal irritation of the mucosa.[48] The direct aspiration system consists in an outside siliconized metal aspiration tube connected through a metal cap on one side to a tube linked to a vacuum pump and on the other to a disposable collection tube for the secretions.[48] Phosphate buffered saline (PBS) is added to the collected sample after aspiration. Though, the method is limited when too little secretion is present in the nasal cavity.[48] Another major concern is that the dilution factor may differ between the samples and it may be limited for IgE evaluation whose concentration is often under the detection level.[49] Technologic advances in laboratory analysis with the development of highly sensitive immunological assays have partly limited the problem.[50,51]

Recently, the aspiration properties of a device originally designed for the collection of middle ear effusion in otitis media, were used to obtain nasal secretions.[52] Nasal secretions were obtained easily and safely, and the characteristic of the aspirator, with its thin tip and the rapid preparation, makes it applicable to daily clinical practice and also to children.[52] With this sampling method, sIgE to Japanese Cedar Pollen was detected in nasal secretions with an immunoassay in most of the symptomatic subjects and in more than half asymptomatic but sensitized patients.[52]

Nasal Washing

The most commonly dilution technique applied is nasal washings. In literature, different variations of nasal lavages has been described, with differences in terms of application, instilled volume, and recollection device. This method overcomes the problem of sample volume, always providing a large quantity of fluid. Major concern of those techniques is the unknown dilution factor of the secretions by the washing fluid.[53] Partly it is because of the fact that unpredictable fractions of the washing solution may be swallowed, absorbed, or get lost. That results in great variability of the analyte concentrations, which often fall below the lower detection limit of the assays, resulting in high numbers of missing values.[26,54]

Nasal Irrigation

In the classical nasal lavage, an irrigation fluid (about 10 ml of buffer saline) is applied into the nose and the solution, mixed with a sample of epithelial lining fluid, is recovered by allowing it to run from the nasal cavity into a Petri dish.[34] Alternatively, a catheter can be inserted in the nose to recover the fluid.[14,55] This method seems to be not the most appropriate to detect IgE in nasal secretions, that is the immunoglobulin isotype present at lowest concentrations in humans. The high dilution factor of nasal irrigation may explain the very low nasal secretions concentrations of IgE and IgE binding activity for grass pollen described with this methodology in patients with seasonal allergic rhinitis.[34]

Nasal Spray

A spray pump or a syringe can be used to deliver small portions of isotonic or hypertonic sodium chloride. This method allows to keep the volume of washing fluid considerably smaller. The volume instilled with the dispenser in each nostril ranges in different studies from 1 to 5 ml NaCl 0.9–1.8%.[56,57] The patient has to sit with his head extended 45° from the horizontal during instillation and keep the position. The diluted mucus is recollected into a vessel after a prefixed time of incubation. This method produced secretions with levels of IgE high enough to be detected in standard immunological assays.[4,44,56]

Nasal Pool

Nasal pool is a variation of the 'classical' nasal washing.[58,59] The 'nasal pool' device is a soft plastic container with a nasal adapter. The adapter exists in different sizes and is also applicable for children. By gently compressing the device, 10 ml of saline are instilled in each nostril of the subject sitting in a flexed neck position. Challenge – agents, in form of standardized be added to the lavage fluid if a concomitant mucosal provocation is wished. The washing fluid is maintained in contact with the mucosal surface of the nasal cavity for up to 15–20 min in adults. When the pressure is released, the fluid returns into the container. The technique is atraumatic, requires little cooperation from the patient and is easy to handle.[60] When compared to the nasal spray method,[57] this modified method gave more reproducible ECP measurement in patients with rhinitis[61] and a higher total number of inflammatory cells in both normal individuals and patients with rhinitis.[61] This may be because of the higher exposure time and the larger volume instilled by the nasal pool method, suggesting that these two factors are relevant for the results. A limitation of the technique is again the unknown, high dilution factor, that may reduce the concentrations of sIgE antibodies in the collected nasal fluids below the detection limits of the standard tests.[40]

The Dilution Factor Issue

The unknown dilution factor is the main problem of the lavage techniques and should be always measured. Plasma albumin passes freely into extravascular spaces and is passively ultra-filtered into nasal secretions.[18] Therefore, albumin was described as a valuable 'endogenous' marker of dilution of nasal secretions.[62] The relative concentration of any analyte can be referred to albumin concentration and expressed as an index.[62] However, the use of the ratio analyte-to-albumin only allows relative but not absolute comparisons.[62] The levels of total protein are also used to correct for the unknown dilution and allow comparison of levels of substances between individuals.[48,49,53,63] To calculate the actual concentration of a substance 'exogenous' markers may be more effective. Among other markers, inulin[55] and lithium chloride[64] have been tested. These substances, not physiologically present in nasal secretions, are added in fixed concentrations to the washing solution, and then detected in the recovered lavage fluid to calculate the dilution factor. The most promising marker seems to be lithium chloride, outperforming inulin and other substances because of its harmless, easy, and low-cost application.[64] However, the application of markers to determine the dilution factor increases the complexity of the procedure and no standard marker has yet been set.[8]

Absorption

The use of a sampler with absorptive proprieties placed within the nasal cavity is another concept of nasal secretion collection. Different materials have been tested for this purpose: cotton,[65] filter paper,[33] rubber foam,[26,66] and cellular sponges of different sizes and forms.[54,67] Absorption method is based on capillary suction, recovering mainly the aqueous sol phase of the bilayer fluid on the nasal mucosa.[8] Absorbent materials are inserted in both nasal cavities on the floor between the septum and inferior turbinate and left in place for 5–10 min (Figure 1).[54] After removal, rapid sealing and centrifugation is needed to extract the absorbed fluid and avoid drying.[8] Sufficient amounts of undiluted nasal secretions have been demonstrated with cotton wool samplers[8] and rubber-foam samplers[26] with no need of PBS addition. Processing the fluid collected with cellular sponges, such as sinus packs and filter disks, require a small amount of physiologic solution to mobilize the nasal secretions out of the absorbent material.[54] This procedure can be performed with fixed dilution or with fixed volume.[68] The latter requires the calculation of dilution factor to correct the concentrations measured.[54]

Figure 1.

Insertion of sinus pack in the nasal cavity.

When compared to other sampling methods, absorption demonstrated a superior detectability and reproducibility for analyzing local inflammatory mediators.[8,26,66,69] The procedure requires minimum cooperation from the patient.[26] Another argument for the method is the possibility of a clear differentiation between nasal sides.[54] However, the sponge may cause little irritation of the mucosa and possibly produce artefacts.[54] Sponges of bigger size, like sinus packs and rubber-foam samplers, guarantee sufficient amount of absorbed secretions but may not be applicable in case of severe nasal polyposis and anterior septal deviation.[66] In these cases, filter paper strips are more suitable, thought the volume collected with this material is often insufficient because of contact with a limited area of the nasal mucosa.[66] Patients should be allowed to freely breath through the nose during sampling time. However, the nasal air flow is hindered by most of the absorbent samplers.[66] Inserting a plastic tube in the center of the RFS could improve both air flowing and sampler handling.[66]

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