Objective Assessment of Nasal Function

Key Points

Ciliary activity causes the transport of mucus in the airways, an essential defense mechanism of the respiratory tract.
Mucociliary transport can be measured in vivo either by using the saccharine and/or color test or by radioisotope transport testing, and in vitro by measuring ciliary activity.
Nasal nitric oxide was found to be 10-fold lower in patients with primary ciliary dyskinesia (PCD), but it cannot be used to exclude or to prove PCD.
Nasal obstruction is one of the most common reasons for consultations in primary and specialized care.
Nasal obstruction can be evaluated subjectively by means of visual analog scales or quality of life questionnaires.
Nasal obstruction should also be evaluated by means of objective methods for the measurement of nasal airway such as rhinomanometry (RM), acoustic rhinometry (AR), and peak nasal inspiratory flow (PNIF).
Assessing different aspects of the nasal airway, RM, AR, and PNIF can be considered complementary and can be used individually or in combination to assess patients with rhinitis, disturbance of the nasal airway during sleep, or when surgical treatments are indicated.

Objective Assessment of Nasal Function

Objective parameters that assess nasal function allow measurement of changes from therapeutic intervention, disease, trauma, or any event that alters the status of the nose or sinuses. Although the metrics of symptoms, examination, and quality-of-life (QoL) tools have been largely used to assess changes with medical or surgical therapy for nasal conditions, all have a subjective component that can increase the potential noise in the data.

Apart from the nasal breathing function and its measurement, this chapter also covers the assessment of mucociliary function, including nitric oxide levels and genetic analyses.

Mucociliary Transport and Function

Healthy airway surfaces are lined by ciliated epithelial cells and are covered with an airway surface liquid composed of two layers: the periciliary layer and the mucus layer. The low-viscosity periciliary layer approximates the height of cilia and provides an optimal environment for ciliary beating. The protective mucus layer on top of it is the secretory product of the goblet cells and the submucosal glands. It is a nonhomogeneous, adhesive, viscoelastic gel composed of water, carbohydrates, proteins, and lipids. This mucus layer traps inhaled foreign particles such as dust, allergens, toxic substances, bacteria, and viruses from the air. Mucus is transported from the respiratory tracts into the pharynx by mucociliary clearance, where it is either swallowed or expelled via coughing. Mucociliary clearance in the airways is driven by the coordinated beating of ciliated cells in the airway epithelium. The permanent clearance of the mucus toward the pharynx is the most important defense mechanism in the upper and lower respiratory tracts.

Each ciliated cell has approximately 200 motile cilia, which are thin protrusions that extend up to 6 µm from the cell surface and beat in a coordinated fashion. The motile cilium structure is made of nine peripheral doublet microtubules and two central single microtubules (central pair complex) and includes inner and outer dynein arms, radial spokes, and nexin links. In normal airways, cilia beat with a rapid frequency that ranges from 8 to 20 Hz, mobilizing the mucus that sits atop the cilia. Inborn disorders of mucociliary transport (MCT) are due to ciliary dysfunction, as in primary ciliary dyskinesia (PCD), or increased viscosity of the respiratory secretions, as in cystic fibrosis. Also, MCT is frequently impaired because of inflammation, infection, and exposure to ciliotoxic agents, which are then called “secondary” ciliary abnormalities.

MCT can be studied by recording it in vivo as well as by measuring ciliary activity in vitro. Methods based on nasal ciliary motility for the diagnosis of PCD are often hampered by the presence of acquired abnormalities, such as secondary ciliary dyskinesia.

Measurement of Mucociliary Transport

Testing of Transport in Vivo

The MCT rate can be measured in vivo either by using the saccharine test or the radioisotope technique. If active MCT can be demonstrated with one of these methods, it is accepted that the diagnosis of PCD is excluded. An abnormal result cannot be considered proof of the disease; rather, it indicates only that further investigation is needed.

Saccharine Test

With the saccharine test, a particle of saccharine is placed on the inferior turbinate, and the time that elapses before the patient tastes the saccharine is measured. Patients are instructed to swallow at least once per minute, and the appearance of saccharine in the pharynx can be verified by the blue color in the pharynx. With this technique, the mean normal MCT time is about 10 minutes. A MCT time of up to 30 minutes is still considered normal. If transport takes more than 30 minutes, the test is considered abnormal. For this test, cooperation of the individual is needed, because the subject must report the sweet taste. Also sniffing, sneezing, and blowing the nose is prohibited, because this may affect the position of the particles; this limits the use of the test in children. The saccharine has to be placed on respiratory (ciliated) epithelium; otherwise no transport will be found.

Most frequently a color (methylene blue, indigo blue, and charcoal) is added to the saccharine as a visual control. Repeated examination will allow verification of transport of the particle and comparison of the appearance of the color in the pharynx with the perception of the sweet taste.

When combined with nasal endoscopy, the colored particles can be followed to evaluate the transport pattern. That technique can also be used to follow and study the pathways within the (maxillary) sinuses.

The test is cheap and does not require special equipment, which is useful. However, cases of PCD with dyskinetically beating cilia may be missed.

Nuclear Testing

When a minute amount of radiolabeled Tc 99m albumin colloid particles is placed on the inferior turbinate or on the nasal septum, the migration can be followed with a γ-camera. Normally, the majority of the radioactivity disappears from the nasal cavity within 30 minutes. The percentage of radioactivity that remains in the nasal cavity can be calculated, and in sagittal views, the migration of the spot can be measured. It has been shown that the dose of radioactivity is low enough that immotility does not create problems. In contrast to the saccharine test, this test is not influenced by sniffing. A normal test result is considered an exclusion criterion of PCD. If the particles moved insufficiently, further investigation is needed, because dysmotility could be due to upper airway infections or PCD. Moreover, in up to 25% of individuals with secondary ciliary dyskinesia, and also in controls, no migration of the tracer is found.

Marthin et al. studied an alternative method, the pulmonary radioaerosol mucociliary clearance technique, which has a higher specificity for PCD, because secondary dysmotility is much less prevalent in the lower airways. The radioactive labeled Tc 99m albumin transportation test is more reliable than the saccharine test, but it requires expensive equipment and can only be done in specialized centers. Additionally, the latter technique is associated with radiation exposure and, therefore, alternative techniques are nowadays recommended.

Testing of Transport in Vitro

Bioptic or brushed material can be checked for the presence of cilia under phase-contrast microscopy. Real movements, such as displacements and rotations of cell clusters or cell sheets within the fluid and movement of particles within the fluid lining the cilia, are criteria for the presence of coordinated ciliary activity. Absence of these elements in the presence of ciliary activity is recorded as “uncoordinated ciliary activity.” Checking for the presence of cilia should always be done.

Testing of Ciliary Activity

Ex Vivo/In Vitro

In vitro brushings from the nasal cavity or biopsy samples taken from the inferior border of the middle turbinate or from the inferior turbinate can be evaluated for coordinated ciliary beating and ciliary beat frequency (CBF). Using microscope photometry, CBF can be deduced from fast Fourier transform analysis of the light scattering. Normal CBF values depend on the temperature, and normal values are around 8 Hz at room temperature and 12 Hz at 37°C.

The introduction of high-speed cameras (≤500 Hz) has created new possibilities. With high-speed video microscopy analysis (HVMA), ciliary beat pattern analysis can be done, and the amplitude, degree, and speed of the ciliary beat cycle can also be measured. In addition, field analysis with measurement of ciliary coordination on a whole area of ciliated cells has become available. However, HVMA protocols differ among centers in many respects (microscopes, cameras, sampling techniques, temperature during analysis, software, and evaluation criteria) and HVMA is not sufficiently standardized to rule in or rule out PCD. A CBF of less than 11 beats per second (<11 Hz) has been suggested as a cutoff value, with those with lower CBF proceeding to electron microscopy (EM). However, examination of the ciliary ultrastructure by EM remains a definitive diagnostic test for PCD if abnormal. Normal EM findings, however, cannot rule out PCD.

High-resolution immunofluorescence (IF) analysis is an emerging tool to investigate the subcellular localization of ciliary proteins in respiratory epithelium. It is likely that further development will help recognize an increasing number of PCD variants.

Cell Culture

Finally, epithelial cells from biopsies can be cultured in vitro. During the growth phase, epithelial cells will dedifferentiate, and cilia will get lost completely. A sequential monolayer-suspension culture system can then be used to let epithelial cells redifferentiate into ciliated cells. These newly formed cilia do not express the acquired abnormalities, but the inherited abnormalities (PCD) are expressed in the culture system. Because the functional abnormalities are also clearly present, this culture can be used for the diagnosis of PCD. With classic transmission EM on bioptic material, PCD with normal ultrastructure would easily be missed. Ciliogenesis in vitro has also been achieved by placing the cells in an air-liquid interface culture system.

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