Objective hyposalivation measuring methods

Measuring salivary output can be easily done in an office setting by determining the total unstimulated output of saliva, termed the whole saliva flow rate (WSR). Salivary flow is classified as unstimulated, or resting, and stimulated, as it arises when an exogenous factor is acting on the secretory mechanisms (Dawes 1987). Whole saliva is basically the mixed fluid contents of the mouth. In 1992, Navazesh and colleagues suggested that salivary output can be measured and collected, less than 0.12 to 0.16 mL/min (unstimulated) is considered a criteria for hypofunction (Navazesh, Christensen and Brightman 1992; Pesce and Spitalnik 2007; Fox 2008). Another study indicated that unstimulated whole saliva flow rates less than 0.1 mL/min and stimulated whole saliva flow rates less than 0.7 mL/min are considered abnormally low (Jensen and Vissink 2014).

Some different methods of assessing salivary gland function are presented in Table 10, including sialochemistry which analyses the saliva composition (Kalk et al. 2001; Kalk et al. 2002), impression cytology of the buccal mucosa (Aguilar, Fonseca, and Croxatto 1991; Maragou et al. 1996), salivary electrophoresis (Al-Hashimi, Haghighat, and Fox 1998), saliva ferning (Aguilar, Fonseca, and Croxatto 1991; Maragou et al. 1996; el-Miedany, el-Hady, and el-Baddin 1999), and the use of iodine–starch reaction to identify the number of lip salivary gland ostia (Inamura et al. 2001). Salivary function assessment can also be performed using the following methods: the wafer test (Sánchez-Guerrero et al. 2002), the Saxon test (Kohler and Winter 1985), the oral Schirmer test (López-Jornet, Camacho-Alonso, and Bermejo- Fenoll 2006), the candy weight loss test (Sreebny and Valdini 1988), the palatal (Márton et al. 2006), and parotid gland saliva flow (Skopouli et al. 1989), the capsaicin-stimulated salivary flow using filter paper (Kanehira et al. 2011)and more

commonly the whole saliva collection, with or without stimulus (Speight, Kaul, and Melsom 1992; Vitali, Moutsopoulos, and Bombardieri 1994) see Table 10. Some of the most frequently used methods are explained in the next section with further details.

1.2.3.1 Resting whole saliva

Four techniques have been described to estimate resting whole saliva flow rate: the draining method, the spitting method, the suction method, and the swab technique. In the draining method, collections should be performed after an overnight fast, between 8 and 11 a.m., or at least at a regular time. Patients are instructed not to brush, use mouthwashes, drink, chew (e.g., food, gum) or smoke at least 90 minutes before the collection time. The test should be carried out in a quiet area. The patient is then seated in a chair, in an upright position with the head tilted down, given a funnel and a test tube, and is asked to swallow. Following this, they are asked to sit quietly for a period of 5 minutes and to allow the saliva to accumulate in the mouth and passively drain into the funnel. The volume of saliva is measured and the rate of flow is recorded as mL/min. Alternatively, the saliva may be collected into a weighing boat. In such cases, the boat is tared (zeroed) on a precision balance, the saliva is allowed to drool into the boat, and the boat is then weighed again after the test period. Results may be expressed as g/min or as mL/min. The spitting technique is similar to the draining method. The difference is that the patient allows the saliva to accumulate in the mouth and then spits it into the collecting vessel, 1–2 times per minute. The saliva may be collected either into the weighing boats, into test tubes, or into the sialometer. The suction method involves the use of the standard, plastic, dental saliva ejector. The swab method is conducted by placing preweighed cotton rolls or gauze sponges into the mouth, leaving them for a fixed period, and then

reweighing them after the test. However, regardless of the method used, the conditions of the test should be the same for each patient each time that saliva is collected (Sreebny and Vissink 2010).

1.2.3.2 Stimulated whole saliva

Whole saliva is generally stimulated by either chewing or tasting citric acid. Both methods are reliable. Flow rates using citric acid are generally greater than those induced by wax. When applying the masticatory method, the patient is either given a piece of paraffin wax, a piece of gum base, or a piece of Parafilm to chew for 5 minutes. The accumulated saliva is then actively spat into the collecting vessel every minute. The gustatory method uses a 2% solution of citric acid to stimulate flow. The solution is applied to the lateral borders of the tongue with a cotton applicator every 30 seconds for 5 minutes. As with the chewing method, the saliva is expectorated into the collecting vessel every minute (Vissink et al. 1983).

1.2.3.3 Parotid saliva

The orifice of the parotid gland is accessible for cannulation, but usually a (modified) Lashley or Carlsson-Crittenden cup is used. The Lashley cup is a bi-chambered device, which measures about 2 cm in diameter. The inner chamber is placed directly over the orifice of the Stensen duct and connected, via plastic tubing, to a (graduated) test tube. The outer chamber is attached to a rubber bulb or a suction device via plastic tubing and is secured to the mucosa by vacuum. Parotid saliva is usually collected under stimulated conditions because the flow rate of unstimulated parotid saliva is usually very low or even absent in healthy individuals. The most commonly applied stimulus is a 2% to 4% citric acid solution. This stimulus is applied to the lateral borders of the tongue at 30-second or 60-second intervals with a cotton swab. It is usually collected for 10 minutes (Navazesh 1993; Burlage et al. 2005).

1.2.3.4 Submandibular/sublingual (SM/SL) saliva

About 70% of the oral secretions stem from the combined SM/SL glands. Therefore, most studies consider that the flow and composition of saliva obtained from the SM/SL glands are similar to that gained with whole saliva. The suction method is mostly used to collect these secretions. In this technique, the Stenson ducts are blocked with either Lashley cups or cotton roles. This strategy allows SM/SL saliva to flow from the Warthin the Bartholin ducts. Saliva which accumulates on the floor of the mouth, can be aspirated with a syringe, micropipette, or with gentle suction. The SM/SL saliva can be collected in the resting or stimulated state (Sreebny and Vissink 2010).

1.2.3.5 Minor salivary gland secretions

The advent of the Periotron®, has allowed the development of a simple to measure the volume of saliva from the minor salivary glands and to calculate the thickness of the salivary film on the oral mucosa. In practice, a small piece of pan-shaped filter paper (Sialopaper TM) is placed at a selected site on the mucosa and held there for 5 seconds. The Sialopaper TM is then removed, placed between the ‘jaws’ (electronic sensors) of the Periotron®, and the reading is shown on the screen. The Periotron® is a micro moisture meter that reads volumes up to 3 μL. The Sialopaper TM strips collect 0–3μL of fluid. To calculate the thickness of the mucosal film (in μm), one divides the volume of the collected saliva by the area of the Sialopaper TM test strip (31.7 mm2).

Several papers have now recorded the normal values for various sites in the mouth (DiSabato-Mordarski and Kleinberg 1996; Wolff and Kleinberg 1998a; Won et al. 2001; Lee et al. 2002). Of particular interest is the one that is located on the hard palate. It is the driest site in the oral cavity. The thickness of the salivary film at this

site may well be a valid sialometric indicator of the subjective feeling of oral dryness (Sreebny and Vissink 2010).

Table 10. Objective salivary hypofunction tests (Hernández-Molina and Sánchez-Hernández 2013)

Test Method Abnormal test Disadvantage

Whole saliva

flow collection Un-stimulated: saliva collection is performed during 5 min to 15 min by the spitting method.

Stimulated: after chewing wax or gum for 1 min, the volume of saliva expectorated during that time is measured.

Non-stimulated ≤1.5 ml/15 min

Stimulated ≤0.6 ml in 1 min

Clinical practice test. Affected by age, time of the day and drugs.

Palatal saliva

flow Dry weighed 8 mm disks are placed on both sites of the palate, at the level of the upper first molars. Collection is carried out for 30 s and then the disk is weighted.

1.35 ±

2.5 μcm-2min-1 Clinical practice test. Affected by age, time of the day and drugs.

Stimulated parotid saliva flow

Saliva is collected under a stimulus with a cannula or other collection device.

1.5 ml/5 min Clinical practice test. Special collection material needed.

Wafer test After swallowing any residual saliva, the wafer is put on the centre of the subject’s tongue, and wafer dissolution time is measured.

>4 min Screening/clinical practice test. Affected by age, time of the day and drugs.

Oral Shirmer

test After placing a filter paper on the floor of the mouth, the wetted length after 5 min is measured.

≤30 mm/5 min Screening/clinical practice test. Affected by age, time of the day and drugs.

Candy weight loss test

The weight loss of a standard hard sugar candy after 3 min of passive incubation between tongue dorsum and palate is tested.

<0.23 g Screening/clinical practice test. Affected by age, time of the day and drugs.

Impression

cytology Cellulose acetate paper is applied on the internal surface of the inferior lip and the obtained sample is stained with hematoxylin and PAS.

Keratinized epithelium

is abnormal Research test. Lightmicroscopy needed. Age dependent.

Iodine–starch reaction

A test tape of 1×1 cm2 _containing

iodine and starch is set on the labial mucosa anterior to the labial frenulum for 30 s. The number of blue spots corresponds to the number of salivary gland ostia.

Controls 9.4 ±2.5 spots Oral dryness 4.5 ± 3.1 spots Sjögren’s 2.1 ± 1.3 spots Screening/clinical practice test. Age dependent. Capsaicin- stimulated salivary flow

An assay system comprising 5 spots containing starch, potassium iodide and a colouring reagent with or without capsaicin is placed in the mouth.

In controls the capsaicin-stimulated salivary flow increased from 1.2 ± 1.4 to 2.9 ± 1.3 coloured spots No change in the hyposalivation group Screening/clinical practice test. Age dependent.

Sialochemistry Measurement of specific proteins (lactoferrin and lysozyme), carbohydrate, and electrolytes.

Laboratory kits Cut-offs Research test. No agreement about significant cut-offs. Variation between resting and stimulated saliva. Moderate amount of saliva is required.