BIOCHEMISTRY OF SALIVA. REGULATION AND PATHOLOGY OF SALIVA
LABORATORY DIAGNOSTICS OF THE DISEASES OF ORAL CAVITY AND
There are three major salivary glands: parotid, submandibular, and sublingual. These are paired glands that secrete a highly modified saliva through a branching duct system. Parotid saliva is released through Stenson’s duct, the orifice of which is visible on the buccal mucosa adjacent to the maxillary first molars. Sublingual saliva may enter the floor of the mouth via a series of short independent ducts, but will empty into the submandibular (Wharton’s) duct about half of the time. The orifice of Wharton’s duct is located sublingually on either side of the lingual frenum. There are also thousands of minor salivary glands throughout the mouth, most of which are named for their anatomic location (labial, palatal, buccal, etc). These minor glands are located just below the mucosal surface and communicate with the oral cavity with short ducts. Saliva is the product of the major and minor salivary glands dispersed throughout the oral cavity. It is a highly complex mixture of water and organic and non-organic components. Most of the constituents are produced locally within the glands; others are transported from the circulation. The three major salivary glands share a basic anatomic structure. They are composed of acinar and ductal cells arranged much like a cluster of grapes on stems. The acinar cells (the “grapes” in this analogy) make up the secretory end piece and are the sole sites of fluid transport into the glands. The acinar cells of the parotid gland are serous, those of the sublingual gland are mucous, and those of the submandibular gland are of a mixed mucous and serous type. The duct cells (the “stems”) form a
branching system that carries the saliva from the acini into the oral cavity. The duct cell morphology changes as it progresses from the acinar junction toward the mouth, and different distinct regions can be identified.
While fluid secretion occurs only through the acini, proteins are produced and transported into the saliva through both acinar and ductal cells. The primary saliva within the acinar end piece is isotonic with serum but undergoes extensive modification within the duct system, with resorption of sodium and chloride and secretion of potassium. The saliva, as it enters the oral cavity, is a protein-rich hypotonic fluid. The secretion of saliva is controlled by sympathetic and parasympathetic neural input. The stimulus for fluid secretion is primarily via muscarinic cholinergic receptors, and the stimulus for protein release occurs through β-adrenergic receptors. Ligation of these receptors induces a complex signaling and signal transduction pathway within the cells, involving numerous
transport systems.1 An important point to consider is that loss of acini, as occurs in a number of clinical conditions, will limit the ability of the gland to transport fluid and to produce saliva. Also, muscarinic agonists will have the greatest effect in increasing saliva output as they are primarily responsible for the stimulus of fluid secretion. These points have implications for the treatment of salivary gland dysfunction.
Symptoms in the patient with salivary gland hypofunction are related to decreased fluid in the oral cavity. Patients complain of dryness of all the oral mucosal surfaces, including the throat, and also of difficulty chewing, swallowing, and speaking. Many patients report a need to drink fluids while eating to help swallowing or report an inability to swallow dry foods. Most will carry fluids at all times for oral comfort and to aid speaking and swallowing. Pain is a common complaint. The mucosa may be sensitive to spicy or coarse foods, which limits the
patient’s enjoyment of meals.
Salivary glands are nonexcitable effector organs in which a large amount of fluid and electrolytes is transferred from the interior of the body to the outside. The amount of fluid translocated each day through salivary glands approaches 750 ml, which represents approximately 20 % of total plasma volume.
Saliva is secreted to the mouth by three major paired salivary glands (submaxillary, parotid, and sublingual glands) and by numerous minor mucous glands, at a rate of approximately 0.025 ml.min-1. The relative contributions of each of these glands to the total amount of saliva secreted average 65 per cent from the submandibular, 23 per cent from the parotid, 8 per cent from the minor mucous, and 4 per cent from the sublingual.
Both of the two major parenchymal sites of the salivary glands, the acini and the striated ducti, participate in salivary secretion. Transport of water and electrolytes, and synthesis of enzymes, proteins, mucin and other organic components, occur in the acini, which secrete a fluid isotonic with plasma. This fluid is then modified in the ductus system, by both reabsorption and secretion of electrolytes.
The majority of oral secretions are contributed by the sub-mandibular and parotid glands, which equally provide 80 to 90 per cent of the saliva. The remainder is formed by sublingual and minor salivary glands. One thousand to 1500 ml of saliva is produced daily. Saliva contributes to the digestion of food and to the maintenance of oral hygiene. Without normal salivary function the frequency of dental caries , gum disease (gingivitis), and other oral problems increases significantly.
LIGHT MICROSCOPIC FEATURES; ACINAR CELLS
The acini of parotid glands is made of pure serous type cells. The acinar cells have abundant cytoplasm, a large number of basophilic, and PAS positive and diastase resistant zymogen granules.
On occasion, in the parotid gland, sero-mucous type cells are seen. In these glands, the serous type cells appear as demilunes around the mucous cells.
These glands may contain mucous secreting cells, serous cells or a mixture of both.
Serous cells produce a watery saliva that contains the enzymes amylase and lysozyme, IgA (immunoglobulin A), and lactoferrin (an iron binding compound).
Saliva has important functions :
The submandibular salivary gland secretes a mixed product containing both serous and mucous secretions although the serous component is the larger. They are roughly ovoid in shape and are situated below the mandible (jaw bone) to the left and right. Their ducts open into the floor of the mouth on either side of the tongue's frenulum.
The parotid salivary glands secrete a serous product only. They are situated on either side of the head in front of the ears. They have long ducts which open into the mouth opposite the second molar tooth on either side.
The sublingual glands produce a mainly mucous product. They are situated just uner the back of the tongue again in a left and right pair. Their ducts open close to those of the submandibular glands.
In addition there are numerous smaller groups of salivary gland tissue scattered diffusely in the submucosa.
The most important are:
Both the sympathetic and the parasympathetic nervous systems innervate the salivary glands. It is evident that the sympathetic nervous system, although its role in salivation is still controversial, influences the blood flow to the salivary glands and activates myoepithelial cells within the salivary ducts. These myoepithelial cells expedite the flow of saliva by squeezing saliva out of the salivary glands.
Saliva is characteristically a colorless dilute fluid, with a density ranging from 18 to 35. Its pH is usually around 6.64, and varies depending on the concentration of CO2 in the blood. When blood CO2 concentration is increased, a higher fraction of CO2 is transferred from the blood to the saliva, and salivary pH decreases. If CO2 is low in blood, on the other hand, salivary pH increases as a result of a low transfer of blood CO2 to salivary glands.
Although a variety of components is always present in saliva, the total concentration of inorganic and organic constituents is generally low when compared to serum. The fraction of saliva represented by water usually exceeds 0.99. Of the inorganic constituents, sodium and potassium (and perhaps calcium) are the cations of major osmotic importance in saliva; the major osmotically active anions are chloride and bicarbonate. Although the percentage of total proteins in saliva is low in comparison to serum, specific proteins, such as the enzyme amylase, are synthesized in the salivary glands and may be present in saliva in concentrations exceeding those of serum. Other organic components existing in saliva include: maltase, serum albumin, urea, uric acid, creatinine, mucine, vitamin C, several amino acids, lysozime, lactate, and some hormones such as testosterone and cortisol. Some gases (CO2, O2, and N2) are also present in saliva. Saliva contains immunoglobins such as Ig A and Ig G, at an average concentration of 9.4 and 0.32 mg%, respectively. The concentration of potassium, calcium, urea, uric acid, and aldosterone are highly correlated to those existing in plasma. This high degree of correlation has not been shown, however, between salivary and plasma concentrations of phosphate. The physiological significance of other constituents of saliva, such as trace minerals, epithelial growth factor, neural growth factor, several enzymes and some proteins (kallikreins, calmodulin) remains unknown.
In animals, saliva is produced in and secreted from the salivary glands. It is a fluid containing
• Electrolytes: (2-21 mmol/L sodium, 10-36 mmol/L potassium, 1.2-2.8 mmol/L calcium, 0.08-0.5 mmol/L magnesium, 5-40 mmol/L cloride, 2-13 mmol/L bicarbonate, 1.4-39 mmol/L phosphate)
• Mucus. Mucus in saliva mainly consists of mucopolysaccharides and glycoproteins;
• Antibacterial compounds (thiocyanate, hydrogen peroxide, and secretory immunoglobulin A)
• Various enzymes. The major enzymes found in human saliva are alpha-amylase, lysozyme, and lingual lipase. Amylase starts the digestion of starch before the food is even swallowed. It has pH optima of 6.7-7.4. Human saliva contains also salivary acid phosphatases A+B, N-acetylmuramyl-L-alanine amidase, NAD(P)H dehydrogenase-quinone, salivary lactoperoxidase, superoxide dismutase, glutathione transferase, glucose-6-phosphate isomerase, and tissue protein. The presence of these things causes saliva to sometimes have a foul odor.
Healthy people produce about 1.5 L of saliva per day.
Amylase:- found in two forms
1- α-amylase (in saliva and pancreatic juice) which is endoglycosidase that attack starch randomly. Inactivated by the acidity of the stomach.
2- β-amylase (from plant origin) which is exoglycosidase cleaves maltose from the non-reducing end to produce β-maltose.
Regulation of saliva secretion
Secretion of saliva is usually elicited in response to stimulation of the autonomic innervation to the glands. Although no direct evidence for modification of salivary flow by hormones has been demonstrated in humans, catecholamines might also be involved in the control of saliva electrolytes and protein concentrations. Both salivary output and composition depend on the activity of the autonomic nervous system, and any modification of this activity can be observed indirectly by alterations in the salivary excretion. Although normal salivary secretion is dependent on the cooperation of sympathetic and parasympathetic nerves, the nervous control of saliva secretion is not identical in all salivary glands:
secretion of saliva from sublingual and minor mucous glands is mainly elicited in response to cholinergic stimulation, whereas secretion from the other glands is evoked mainly by adrenergic innervation. In any case, it is generally acknowledged that parasympathetic nerve impulses create the main stimulus for salivary control in general. Parasympathetic stimulation results in a copious flow of saliva low in organic and inorganic compounds concentrations. Sympathetic stimulation, on the other hand, produces a saliva low in volume. In addition, saliva evoked by action of adrenergic mediators is generally higher in organic content and its concentration of certain inorganic salts is also higher than saliva evoked by cholinergic stimulation. The higher organic content of saliva evoked by adrenergic stimulation trough the activity of adenyl-cyclase, includes elevated levels of total protein, especially the digestive enzyme alpha-amilase. High concentrations of alpha-amilase in saliva are indeed considered to be the best indicator of adrenergic evoked secretion of saliva. The levels of inorganic compounds, i.e., Ca++, K+ and HCO3-, are usually higher with sympathetic stimulation.
Besides the type of autonomic receptor being activated, the two other parameters that can affect salivary composition are the intensity and the duration of stimulation to the glands. The differences in composition between saliva collected after a change in the intensity or the duration of stimulation appear to be due to alterations in membrane permeability of secretory cells leading to changes in the rate at which electrolytes are lost from these cells.
The secretory cells are not the only glandular elements that respond to stimulation of the sympathetic innervation. Myoepithelial cells and blood vessels of the glands also respond to such innervation, and these responses can in turn modify the quantity and composition of the elaborated saliva. It has been shown, for example, that sympathetic stimulation to salivary glands can produce a markedly increased degree of vasoconstriction. Finally, other factors such as circannual rhythms and reflexly induced secretomotor responses might also influence salivary secretion.
Effects Of Exercise On Saliva Secretion And Its Composition
Several studies have shown decreases in salivary levels of immunoglobin A (s-Ig A) in response to high-intensity exercise. Lower resting levels of s-IgA have indeed been reported in cross-country skiers and in elite swimmers, when compared to matched controls of sedentary individuals. The levels of s-IgA decrease following intense exercise, and return to normal levels after 60 minutes from cessation of activity. Since Ig A represents the first line of defense against potentially pathogenic viruses, the exercise-induced decrease in s-IgA could contribute to the higher incidence of upper respiratory infections associated to strenuous athletic training. However, endurance exercise performed at lower intensities (i.e., training protocols within the guidelines recommended by the American College of Sports Medicine), does not seem to alter normal s-IgA levels.
Salivary flow rate appears to be modified during physical activity, according to most studies. Nevertheless, interpretation of the results obtained in these studies is sometimes difficult due to some methodological limitations, concerning mainly exercise protocols and saliva collection procedures. During exercise, salivary levels of total protein can be increased, since saliva secretion is then mainly evoked by action of adrenergic mediators. Exercise is indeed known to increase sympathetic activity and the high protein concentration following exercise may be due to increased ß-sympathetic activity in salivary glands. This elevated levels of protein could also be caused by the increase in blood catecholamines associated to exercise. During prolonged exercise at low to moderate intensities (lower than 60% of O2max), salivary secretion does not seem to be significantly modified. At higher intensities, however, salivary secretion decreases. Factors associated to high-intensity exercise such as an increased ß-adrenergic activity, dehydration, or evaporation of saliva through hyperventilation (although less probable) have been proposed to explain this lower secretion of saliva at high workloads.
Salivary levels of cortisol are considered to be a good indicator of the adrenocortical response to exercise by some authors, since salivary cortisol closely reflects plasma free cortisol levels, presenting advantage over total cortisol measurements. During exercise, salivary and serum concentrations of cortisol are indeed very similar. In addition, both salivary and blood levels of cortisol increase with exercise intensity until a certain exercise level, at which such increase loses it linearity. This inflection point in the increase of salivary and blood levels of cortisol coincides in most of the cases with the onset of blood lactate accumulation. It has been suggested that this lactate accumulation might activate chemoreceptors within the working muscles, which in turn could stimulate the hypothalamic-pituitary axis. However, a true cause-to-effect-relationship between these variables remains to be proven. Both increases of cortisol and lactate levels could occur as a result of a marked sympathetic activity or an increase in blood catecholamines which take place at exercise intensities above anaerobic threshold.
The effects of exercise on the salivary and serum levels of Na+ and K+ have also been studied. Prolonged exercise does not appear to have a significant effect on the serum Na+ and K+. On the other hand, the salivary Na+ concentration markedly increases whereas no noteworthy changes seem to occur in salivary K+, in response to prolonged exercise. In addition, this increase in the salivary Na+/K+ ratio is positively correlated to the exercise-induced increase in salivary protein concentration.
In our laboratory, we have studied the relationship between anaerobic threshold and variations in salivary electrolytes (Na+, K+, Cl-) in response to incremental exercise. Our results evidenced that salivary Na+ and Cl- showed a dual response to exercise: their levels decreased or remained stable during early phases of exercise, until a certain exercise level, at which they began to show a systematic increase. In contrast, K+ levels did not significantly vary during physical activity. The inflection point in the salivary Na+ and Cl- was highly correlated (r= 0.82; p<0.01) with lactate threshold, suggesting the possibility of determining anaerobic threshold with a noninvasive method involving saliva analysis.
These changes in the concentration of salivary electrolytes which occur at a certain exercise intensity might be elicited in response to sympathetic stimulation. This sympathetic stimulation might induce changes in salivary flow and in both reabsorption and secretion of electrolytes in secretory cells. The decreased in saliva secretion associated to exercise could also be the result of a reduction of blood flow to salivary glands caused by elevated adrenal-sympathetic activity. The results of our investigations demonstrate the existence of a catecholamine threshold highly correlated with blood lactate increases (r= 0.84, p<0.01) during incremental exercise. This catecholamine response which occurred at or close to lactate threshold was in turn well correlated (r=0.75, p<0.05) to the point ("saliva threshold") at which salivary electrolytes (especially Na+) showed an inflection point. Although further research in this field is necessary, our experiments suggest that saliva composition analysis might be a good estimate of the adrenal-sympathetic response during exercise. We therefore propose this new noninvasive method for anaerobic threshold determination. We believe that its potential applications in both clinical and exercise physiology areas are numerous.
Hyper-salivation may be associated with many disorders such as herpetic stomatitis, irritation by dentures and pregnancy, but drooling does not occur in these cases unless the ability to hold secretions within the mouth or the ability to swallow secretions is impaired. Patients with hyper-salivation may expectorate repeatedly, but this is not drooling. It is the difference between salivary production and the ability to swallow saliva that results in drooling rather than the absolute production of saliva.
Difficulty in swallowing saliva is encountered at three levels of function: the oral, pharyngeal and oesophageal components of deglutition. Some of the common disorders associated with drooling, classified according to the presumable level of malfunction, are as follows: oral (cerebral palsy, Parkinson's disease, motor-neurone disease, seventh-nerve palsy, facial disfigurement and radical cancer surgery); pharyngeal (motor-neurone disease, myasthenia gravis and polymyositis); and oesophageal (carcinoma or stricture).
Too much or too little saliva can affect oral health and quality of life. Lack of saliva leads to dental decay, oral yeast infections, taste problems, bad breath, difficulty speaking and swallowing, and recurrent salivary glands infections. Too much saliva can cause social problems and may be a sign of an underlying medical problem. We evaluate salivary problems by medical history review, head, neck and oral examination, diagnostic imaging, and salivary function measurements. Management strategies for improving salivary function and preserving oral health are developed for each individual.
Dental or tooth erosion is defined as a dissolving of tooth surfaces caused by acidic substances. (This is different than tooth surface loss caused by caries-producing bacteria.) Generally, in a given individual, all or most tooth surfaces are affected. Sources of acid may be from outside of one's own body (dietary or environmental) or from inside the body (e.g. acids from the stomach). Erosion may also be related to salivary function. Evaluation of this condition includes a medical history review, head, neck, and oral examination, and salivary function measurements. Treatment depends on the cause of the erosion.
The following taste disorders are evaluated and treated:
Evaluation includes medical history review, head, neck and oral examination, diagnostic imaging, salivary function assessment, and testing of ability to taste and smell.
Good mouth care is important to maintain quality of life. Speaking, the pleasure of eating, and the normal handling of saliva are taken for granted by most of us. It may be difficult to imagine the impact mouth disorders have on patients. As the mouth is largely hidden, the patient, family, and caregivers may not recognize problems when they occur. As an exercise, the reader is encouraged to consciously hold his or her mouth open for several minutes. Saliva will begin to pool about the lower teeth. At the same time the tongue will dry. Drooling eventually will occur. Suddenly, what we have taken for granted, swallowing spit, becomes precious.
Palliative Care Note
In end-of-life care examination and reexamination of the mouth is one of the most important tasks.
Xerostomia (Dry Mouth)
Common causes of xerostomia are
Dry mouth is very prevalent and troublesome. As this list suggests, treatable causes are common. Taking patients off of unnecessary anticholinergic medications, for example, can be of great help. Other causes, such as dehydration, radiation-related xerostomia, and mouth breathing may be harder to address directly.
The relationships between dehydration, thirst, and dry mouth are complex and frequently misunderstood. They are discussed in more detail in the chapter 6. While systemic dehydration undoubtedly contributes to decreased saliva production, rehydration with IV fluids, for example, does not necessarily correct the problem and may be associated with undesired side effects, such as worsening respiratory secretions. Side effects of medications, especially anticholinergic agents and opioids, and mouth breathing may significantly contribute to this symptom
Difficulty Handling Saliva, Drooling, and Sialorrhea
In rare cases patients may produce excess saliva. More commonly, they have difficulty handling normally produced saliva because of alterations in mouth anatomy or because of impaired neurologic control of the swallowing reflex. The latter, often manifested by drooling, is the more common. Drooling carries a great social stigma and can be very disturbing to patients and families. Patients with Parkinson's disease, amyotrophic lateral sclerosis, cerebral vascular accidents, dementia, and developmental disorders are prone to this. Patients in the very advanced stages of dying may also experience difficulties as they loose their swallowing and cough reflexes.
Usually, the underlying cause is untreatable. However, anticholinergic agents can be of some help in decreasing salivary flow. Care should be taken in using systemically absorbable agents, as they can produce troubling side effects. In addition, for some patients the dry mouth that results from medication may be as troubling as the earlier drooling. Studies in developmentally delayed children and more recent studies of adults who drool suggest that glycopyrrolate may be effective in decreasing salivary production with little, if any, systemic toxicity. Glycopyrrolate is an anticholinergic agent that is poorly absorbed from the GI tract and that minimally crosses the blood-brain barrier if given systemically (as it often is in anesthesia). I have had some success with this agent. Tablets of 1 mg can be dissolved in a small amount of water and held in the mouth (or swabbed onto mucosa if unable to be held) and then spit out. This is usually given BID or TID. If swallowed, glycopyrrolate will have a strong local anticholinergic effect on the GI tract and decrease motility and secretion into the gut. This will worsen constipation or treat diarrhea but decrease the systemic effect, as only 5% of the drug is absorbed. As the goal of therapy is to reduce the production of saliva, not to dry the mouth completely, the mouth should be moistened with artificial saliva if secondary xerostomia results.
Candidal infections of the mouth occur frequently, especially in patients who are on steroids and in diabetics. Thrush is relatively easy to recognize. White cottage cheese-like plaques are found, often associated with tenderness, dysphagia, and altered taste (dysgeusia).
More difficult to recognize are the atrophic forms, both acute and chronic. Acute atrophic candidiasis usually presents as a reddened tongue with depapillation, which is also associated with dysgeusia. It is my impression that this form may be more common in patients with xerostomia, as inadequate moisture exists to create classic thrush. Vitamin deficiencies, poor nutrition, and xerostomia itself may all create a similar picture, making definitive diagnosis difficult on exam alone. Chronic atrophic candidiasis is similar to acute (reddened mucosa, especially in the area where upper dentures are in contact with the palate) and is most common in elderly patients with dentures. It is often associated with angular cheilitis, which is painful.
A variety of antifungals can be employed in therapy. Nystatin suspension is often well tolerated, as it is a liquid. Because efficacy relates to drug contact time with the mucosa, some caregivers make small "popsicles" with toothpicks for patients to suck on. Some strains of candida are resistant and may respond better to other agents. Mycelex troches are typically given five times a day, although less frequent administration can be given to the dying. Patients with significant xerostomia may have trouble dissolving troches. Systemic agents, such as fluconazole are rarely required and are expensive. Fluconazole may be indicated for resistant strains and when candida is suspected beyond the GI tract, such as when a patient has new-onset hoarseness with a sore throat in association with oral candida (often indicative of laryngeal involvement).
Viral and Bacterial Infections
Immuno-suppressed patients are at a higher risk for both viral (predominantly herpes simplex) and bacterial infections. Herpes infections should be suspected when such patients have new-onset pain or odynophagia (common with esophageal herpes); it is best treated with acyclovir. Patients with xerostomia appear to be at higher risk of bacterial parotitis and present with the sudden onset of a firm, warm, painful swelling under the angle of the jaw. They may be susceptible because of decreased salivary flow from the parotid gland. Broad-spectrum treatment with an antibiotic such as Augmentin is usually effective.
Salivary flow rates provide essential information for diagnostic and research purposes. Salivary gland function should be determined by objective measurement techniques. Salivary flow rates can be calculated from the individual major salivary glands or from a mixed sample of the oral fluids, termed “whole saliva.”
Whole saliva is the mixed fluid contents of the mouth. The main methods of whole saliva collection include the draining, spitting, suction, and absorbent (swab) methods. The draining method is passive and requires the patient to allow saliva
to flow from the mouth into a pre weighed test tube or graduated cylinder for a timed period. In the spitting method, the patient allows saliva to accumulate in the mouth and then expectorates into a pre weighed graduated cylinder, usually every 60 seconds for 2 to 5 minutes. The suction method uses an aspirator or saliva ejector to draw saliva from the mouth into a test tube for a defined time period. The absorbent method uses a preweighed gauze sponge that is placed in the patient’s mouth for a set amount of time. After collection, the sponge is weighed again, and the volume of saliva is determined gravimetrically.
The suction and absorbent (swab) methods give a variable degree of stimulation of secretion and are therefore less reproducible. The draining and the spitting methods are more reliable and reproducible for unstimulated whole saliva collection. If a stimulated whole saliva collection is desired, a standardized method of stimulation should be used. Chewing unflavored gum base or an inert material such as paraffin wax or a rubber band at a controlled rate is a reliable and reproducible means of inducing saliva secretion. One can also apply 2 % citric acid to the tongue at regular intervals. It is difficult to determine a “normal” value for salivary output as there is a large amount of interindividual variability and consequently a large range of normal values. However, with the collection methods described above, most experts do agree on the minimal values necessary to consider salivary output normal. Unstimulated whole saliva flow rates of < 0.1 mL/min and stimulated whole saliva flow rate’s of < 1.0 mL/min are considered abnormally low and indicative of marked salivary hypofunction. It is important to recognize that greater levels of output do not guarantee that function is normal. Indeed, they may represent marked hypofunction for some individuals. These values represent a lower limit of normal and a guide for the clinician.
Individual parotid gland saliva collection is performed by using Carlson-Crittenden collectors.
The collectors are placed over the Stensen duct orifices and are held in place with gentle suction. Saliva from individual submandibular and sublingual
glands is collected with an aspirating device or an alginate-held collector called a segregator. When using the suction device, gauze is placed sublingually to dry and isolate the sublingual region. The gauze and tongue are gently retracted away from
the duct orifice.Gentle
suction is used to collect the saliva as itis produced.The segregator is
positioned over Wharton’s ducts and is then held in place by alginate. As
saliva is produced, it flows through tubing and is collected in a preweighed
vesselStimulated saliva from individual glands is obtained by applying a
sialagogue such as citric acid to the dorsal surface of the tongue. Preweighed
tubes are used for individual salivary gland collections and for some of the
whole saliva collection techniques, and flow rates are determined
gravimetrically in milliliters per minute per gland, assuming that the specific
gravity of saliva is 1 (ie,
Flow rates are affected by many factors. Patient position, hydration, diurnal variation, and time since stimulation can all affect salivary flow. Whichever technique is chosen for saliva collection, it is critical to use a well-defined, standardized, and clearly documented procedure. This allows meaningful comparisons to be made with other studies and with repeat measures in an individual over time. It is best to collect saliva in the morning. To insure an unstimulated sample, patients should refrain from eating, drinking, or smoking for 90 minutes prior to the collection.
For a general assessment of salivary function, unstimulated whole saliva collection is the most valuable method of collection. It is easy to accomplish and is accurate and reproducible if carried out with a consistent and careful technique. Ideally, dentists would determine baseline values for unstimulated whole saliva output at an initial examination. This would allow later comparisons if patients begin to complain of oral dryness or present with other signs and symptoms of salivary
dysfunction. For research purposes, or if more specific functional information is required for one particular gland, individual gland collection techniques should be used. These are not difficult but require specialized equipment and more time to accomplish.
SALIVARY GLAND BIOPSY
Definitive diagnosis of salivary pathology may require tissue examination.When Sjögren’s syndrome is suspected, the labial minor salivary gland is the most frequently sampled site. This procedure is considered to be the most accurate sole criterion for diagnosis of the salivary component of this disorder. Standardized histopathologic grading systems are used to assess the extent of changes (this is described in greater detail in the section of this chapter detailing Sjögren’s syndrome). Biopsy of minor glands can also be used to diagnose amyloidosis.
Biopsy of a minor gland of the lower lip is a minimal operative procedure that can be done with limited morbidity, using appropriate techniques. The incision is made on the inner aspect of the lower lip so that it is not externally visible. Six to ten minor gland lobules from just below the mucosal surface are removed and submitted for examination. The incision should be made through normal-appearing tissue, avoiding areas of trauma or inflammation of the lip that could influence the appearance of the underlying minor glands. Biopsy of the major salivary glands requires an extraoral approach. There is increased morbidity, and major gland biopsy has not been shown to offer diagnostic superiority to the minor gland procedure in patients with Sjögren’s syndrome. When major gland biopsy is indicated, such as for the evaluation of a distinct salivary mass, fine-needle aspiration can be attempted. If this does not yield an adequate sample for diagnosis, an open biopsy procedure should be done. In cases of suspected lymphoma, immunophenotyping of the tissue is essential for diagnosis.
Laboratory blood studies are helpful in the evaluation of dry mouth, particularly in suspected cases of Sjögren’s syndrome. The presence of nonspecific markers of autoimmunity, such as antinuclear antibodies, rheumatoid factors, elevated immunoglobulins (particularly immunoglobulin G [IgG]), and erythrocyte sedimentation rate, or the presence of antibodies directed against the more specific extractable nuclear antigens SS-A/Ro or SS-B/La are important contributors to the definitive diagnosis of Sjögren’s syndrome. Approximately 80 % of patients with Sjögren’s syndrome will display antinuclear antibodies, and about 60% will have antibodies against anti-SSA/ Ro. This latter autoantibody is considered the most specific marker for Sjögren’s syndrome although it may be found in a small percentage of patients with systemic lupus erythematosus or other autoimmune connective-tissue disorders. Another serologic marker that may prove useful for the diagnosis of salivary gland disorders is serum amylase. This is frequently
elevated in cases of salivary gland inflammation. Determination of amylase isoenzymes (pancreatic and salivary) will allow the recognition of salivary contributions to the total serum amylase concentration.
Medication-Induced Salivary Dysfunction
There are over 400 medications that are listed as having dry mouth as an adverse event. However, there are relatively few drugs that have been shown objectively to reduce salivary function. The reason for this disparity is unclear. It may be due to
unrecognized alterations in saliva composition that lead to the perception of oral dryness in spite of an apparently unchanged volume of saliva. Drugs that have been shown to result in salivary dysfunction include anticholinergics, antidepressants (particularly tricyclics), antihypertensives, and antihistaminics. Medication-induced salivary hypofunction usually affects the unstimulated output, leaving stimulated function intact. When the causative drug is withdrawn, function often returns to normal.
Some systemic diseases affect salivary glands directly or indirectly, and may influence the quantity of saliva that is produced, as well as the composition of the fluid. These characteristic changes may contribute to the diagnosis and early detection of these diseases.
Alcohol Screening Test
Cystic fibrosis (CF) is a genetically transmitted disease of children and young adults, which is considered a generalized exocrinopathy. CF is the most common lethal autosomal-recessive disorder in Caucasians in North America, with an incidence of 1 in 2500 and a carrier frequency of 1 in 25-30 of the population. The gene defect causing CF is present on chromosome 7 and codes for a transmembrane-regulating protein called the cystic fibrosis transmembrane conductance regulator (CFTR; Riordan et al., 1989; Dinwiddie, 2000). A defective electrolyte transport in epithelial cells and viscous mucus secretions from glands and epithelia characterize this disorder (Grody, 1999). The CFTR is also important for plasma membrane recycling (Bradbury et al., 1992). The organs mostly affected in CF are: sweat glands, which produce a secretion with elevated concentrations of sodium and chloride; the lungs, which develop chronic obstructive pulmonary disease; and the pancreas, resulting in pancreatic insufficiency (Davis, 1987). Since a large number of identified mutations in the CF gene exist, DNA analysis is not used for diagnosis of the disease. The diagnosis is derived from the characteristic clinical signs and symptoms and analysis of elevated sweat chloride values.
The abnormal secretions present in CF caused clinicians to explore the usefulness of saliva for the diagnosis of the disease. Most studies agree that saliva of CF patients contains increased calcium levels (Mandel et al., 1967; Blomfield et al., 1976; Mangos and Donnelly, 1981). Elevated levels of calcium and proteins in submandibular saliva from CF patients were found, and resulted in a calcium-protein aggregation which caused turbidity of saliva (Boat et al., 1974). The elevated calcium and phosphate levels in the saliva of children diagnosed with CF may explain the fact that these children demonstrate a higher occurrence of calculus as compared with healthy controls (Wotman et al., 1973). The submandibular saliva of CF patients was also found to contain more lipid than saliva of non-affected individuals, and the levels of neutral lipids, phospholipids, and glycolipids are elevated. These alterations in salivary lipids in CF patients may account, in part, for the altered physico-chemical properties of saliva in this disease (Slomiany et al., 1982). Apparently, salivary alterations in CF patients are to a large extent due to alterations in submandibular saliva. Elevations in electrolytes (sodium, chloride, calcium, and phosphorus), urea and uric acid, and total protein were observed in the submandibuar saliva of CF patients (Mandel et al., 1967). Minor salivary glands are also affected. Elevated levels of sodium and a decrease in flow rate were reported for these glands in CF patients (Wiesman et al., 1972). However, the parotid saliva of CF patients does not demonstrate qualitative changes as compared with that of healthy individuals. Amylase and lysozyme activity in the parotid saliva of CF patients was reported to be similar to that in healthy controls, and therefore parotid saliva cannot provide diagnostically relevant information for this disease (Blomfield et al., 1976).
Decreased protease activity in saliva from CF patients was observed relative to healthy controls; however, significant overlap between the protease activity values in the two groups was detected, which makes the diagnostic significance of these findings questionable (Kittang et al., 1986). Saliva from CF patients was found to contain an unusual form of epidermal growth factor (EGF). The EGF from these patients demonstrated poor biological activity compared with EGF from healthy controls. It was suggested that this EGF anomaly might contribute to the pathology of CF (Aubert et al., 1990). Further, abnormally elevated levels of prostglandins E2 (PGE2) were detected in the saliva of CF patients as compared with that of healthy controls (Rigas et al., 1989). However, the diagnostic and clinical importance of the EGF anomaly and elevated salivary levels of PGE2 is difficult to interpret, since the role of EGF and PGE2 in the pathogenesis of CF is not defined.
Most of the studies concerning the diagnostic application of saliva for CF are relatively old, and saliva is not currently used for the diagnosis of this disorder. More important perhaps than the identification of diseased individuals is the detection of carriers (heterozygotes) for the disease, which are asymptomatic and cannot be detected by salivary or other biochemical diagnostic tests. Detection of carriers will help to reduce the incidence of CF. Screening for these carriers can be performed only at the DNA level. Due to the high number of possible mutations detected in the CF gene, the utilization of DNA diagnostic techniques for the identification of carriers is difficult, and research will most likely focus on this aspect of diagnosis.
Coeliac disease is a congenital disorder of the small intestine that involves malabsorption of gluten. Gliadin is a major component of gluten. Serum IgA antigliadin antibodies (AGA) are increased in patients with coeliac disease and dermatitis herpetiformis. Measurement of salivary IgA-AGA has been reported to be a sensitive and specific method for the screening of coeliac disease, and for monitoring compliance with the required gluten-free diet (al-Bayaty et al., 1989; Hakeem et al., 1992). However, contradictory results were also reported. While elevated levels of serum IgA-AGA were detected in serum, this elevation was not detected in saliva (Patinen et al.,1995). No obvious explanation for the difference between the two studies is apparent, since both reports were similar in both methods of patient evaluation and salivary analysis. In a more recent study, salivary IgA-AGA produced sensitivity of 60% and specificity of 93.3% in the detection of coeliac disease. In comparison, serum IgG-AGA produced excellent sensitivity (100%) but lower specificity (63.3%). Because of the relative lower sensitivity, the authors did not recommend the use of salivary IgA-AGA for screening for coeliac disease (Rujner et al., 1996).
21-Hydroxylase deficiency is an inherited disorder of steroidogenesis which leads to congenital adrenal hyperplasia. In non-classic 21-hydroxylase deficiency, a partial deficiency of the enzyme is present (Carlson et al., 1999). Early morning salivary levels of 17-hydroxyprogesterone (17-OHP) were reported to be an excellent screening test for the diagnosis of non-classic 21-hydroxylase deficiency, since the salivary levels accurately reflected serum levels of 17-OHP. A high correlation (r = 0.93) between salivary and serum concentrations of 17-OHP was observed in both affected and healthy individuals (Zerah et al., 1987).
Autoimmune diseases—Sjögren's syndrome
Sjögren's syndrome (SS) is an autoimmune exocrinopathy of unknown etiology. The majority of patients are women, and the estimated prevalence of the disease in the United States is more than 1 million. A reduction in lacrimal and salivary secretions is observed, associated with keratoconjunctivitis sicca and xerostomia. The presence of these two phenomena leads to a diagnosis of primary SS. In secondary SS, a well-defined connective tissue disease (most commonly rheumatoid arthritis or systemic lupus erythematosus) is present in addition to the xerostomia and/or the keratoconjunctivitis (Schiødt and Thorn, 1989; Thorn et al., 1989). In addition to involvement of the salivary and lacrimal glands, SS may also affect the skin, lungs, liver, kidneys, thyroid, and nervous system (Talal, 1992). The diagnostic criteria for SS are still uncertain, and a single marker that is associated with all cases does not exist. The accepted procedure for the diagnosis of the salivary involvement of SS is a biopsy of the minor salivary glands of the lip. SS is characterized by the presence of a lymphocytic infiltrate (predominantly CD4+ T-cells) in the salivary gland parenchyma (Daniels, 1984; Daniels and Fox, 1992). A low resting flow rate and abnormally low stimulated flow rate of whole saliva are also indicators of SS (Sreebny and Zhu, 1996a). Serum chemistry can demonstrate polyclonal hypergammaglobulinemia and elevated levels of rheumatoid factor, antinuclear antibody, anti-SS-A, and anti-SS-B antibody (Atkinson et al., 1990; Fox and Kang, 1992). The immunologic mechanisms involved in the pathogenesis of the disease appear also to involve B-cells (the majority of lymphomas associated with SS are of the B-cell type), salivary epithelial cells, an activated mononuclear cell infiltrate, cytokines, and adhesion molecules (Fox and Speight, 1996).
Sialochemistry may also be used to assist in the diagnosis of SS. A consistent finding is increased concentrations of sodium and chloride. This increase is evident in both whole and gland-specific saliva (Tishler et al., 1997). In addition, elevated levels of IgA, IgG, lactoferrin, and albumin, and a decreased concentration of phosphate were reported in saliva of patients with SS (Ben-Aryeh et al., 1981; Stuchell et al., 1984). Analysis of unstimulated whole saliva was more sensitive than analysis of stimulated whole saliva for detection of these changes, since stimulation caused the elevated levels of sodium and IgA seen in SS patients to decline to the levels observed in healthy controls (Nahir et al., 1987). In contrast, normal concentrations of potassium and calcium are usually found in the saliva of SS patients. Although the amylase concentration in saliva is also normal, the production of amylase is reduced, but so is the amount of fluid. Therefore, measurement of amylase is not useful for the evaluation of salivary gland function in SS patients (Mandel, 1980). Other salivary changes associated with SS include an elevated concentration of β2 microglobulin, although differences exist between patients (Michalski et al., 1975; Swaak et al., 1988). In addition, elevated lipid levels (Slomiany et al., 1986) and increased concentrations of cystatin C and cystatin S have been observed (van der Reijden et al., 1996). Increased salivary concentrations of inflammatory mediators—i.e., eicosanoids, PGE2, thromboxane B2, and interleukin-6—have been reported (Tishler et al., 1996a,b). Elevated levels of salivary soluble interleukin-2 receptor were also found in SS patients; however, no correlation was detected between clinical, serological, or histopathological variables and the salivary or serum levels of this receptor (Tishler et al., 1999). Furthermore, elevated levels of salivary kallikrein have been found in association with SS. Again, no correlation was observed between kallikrein levels and the extent of inflammation in the labial salivary glands or the salivary flow rate (Friberg et al., 1988).
SS is characterized by autoantibodies to the La and Ro ribonucleoprotein antigens. These autoantibodies have been shown to target intracellular proteins which may be involved in the regulation of RNA polymerase function (Tan, 1989). Autoantibody, especially of the IgA class, can be synthesized in salivary glands and can be detected in the saliva of SS patients prior to detection in the serum (Horsfall et al., 1989). In addition to IgA, saliva has also been reported to contain IgG autoantibody, while serum contained primarily IgG and IgM autoantibody (Ben-Chetrit et al., 1993). SS anti-La antibodies were primarily found in the saliva of patients whose resting and stimulated whole saliva flow rates were abnormally low. Furthermore, a strong correlation was observed between the presence of this autoantibody in serum and that in saliva. However, in some patients, the antibody was detected in whole saliva but not in serum, which suggested that the antibody is produced in the salivary glands (Sreebny and Zhu, 1996b). The deposition of this antibody within salivary gland tissue may contribute to the pathogenesis of SS. The diagnostic value of these salivary antibodies has not been determined by comparison with serum levels.
The diagnosis and early detection of SS present a serious challenge that has still not been met. Since no single salivary or serum constituent can accurately serve as a diagnostic marker for SS, the most important aspect of salivary diagnosis for this disease is evaluation of the reduced quantity of saliva. Cut-off values of 0.1 mL/min for resting whole saliva and 0.5 mL/min for stimulated saliva may be considered as indicative of salivary gland hypofunction (Sreebny and Zhu, 1996a). Nevertheless, general agreement about these cut-off values does not exist. Although variations in these cut-off values between clinicians may lead to differences in sensitivity and specificity in the diagnosis of SS, the quantitative evaluation of resting and stimulated saliva is a simple, non-invasive method of screening for patients who may have SS. Reduced salivary flow, although not pathognomonic for SS, is of clinical importance and can lead to a variety of oral signs and symptoms, such as progressive dental caries, fungal infections, oral pain, and dysphagia (Daniels and Fox, 1992). Dentists are normally the first to encounter these patients. Affected individuals should be referred for a comprehensive evaluation of the cause for the reduced salivary flow.
Basal cell adenoma
Basal cell adenoma is a neoplasm of a uniform population of basaloid epithelial cells arranged in solid, trabecular, tubular, membranous or dermal analogue patterns. For this reason, this tumor has been called tubular adenoma, trabecular adenoma, dermal analogue tumor, canalicular adenoma, basaloid adenoma, clear cell adenoma and monomorphic adenoma.
Salivary glands tumor
Salivary analysis may aid in the early detection of certain malignant tumors. p53 is a tumor suppressor protein which is produced in cells exposed to various types of DNA-damaging stress. Inactivation of this suppressor through mutations and gene deletion is considered a frequent occurrence in the development of human cancer (Hainaut and Vahakangas, 1997; Tarapore and Fukasawa, 2000). As a result, accumulation of inactive p53 protein is observed, which in turn may lead to the production of antibodies directed against this protein (Bourhis et al., 1996). These antibodies can be detected in sera of patients with different types of malignancies (Lubin et al., 1995). p53 antibody can also be detected in the saliva of patients diagnosed with oral squamous cell carcinoma (SCC), and can thus assist in the early detection of, and screening for, this tumor (Tavassoli et al., 1998).
Defensins are peptides which possess antimicrobial and cytotoxic properties. They are found in the azurophil granules of polymorphonuclear leukocytes (PMNs; Lichtenstein et al., 1986; Lehrer et al., 1991). Elevated levels of salivary defensin-1 were found to be indicative of the presence of oral SCC. Higher concentrations of salivary defensin-1 were detected in patients with oral SCC in comparison with the defensin-1 concentration in the saliva of patients with adenocarcinoma and in healthy controls. A high-positive correlation was observed between salivary defensin-1 levels and serum levels of SCC-related antigen (r = 0.879; Mizukawa et al., 1998).
In a recent preliminary study, elevated levels of recognized tumor markers c-erbB-2 (erb) and cancer antigen 15-3 (CA15-3) were found in the saliva of women diagnosed with breast carcinoma, as compared with patients with benign lesions and healthy controls. However, while low levels of CA15-3 were also detected in the saliva and serum of healthy individuals, erb was not detected in healthy subjects and thus appears to hold greater promise for the early screening and detection of breast cancer (Streckfus et al., 2000).
CA 125 is a tumor marker for epithelial ovarian cancer. Elevated salivary levels of CA 125 were detected in patients with epithelial ovarian cancer as compared with patients with benign pelvic masses and healthy controls. A positive correlation was found between salivary and serum levels of CA 125. A further analysis of this relationship revealed that saliva demonstrated a somewhat lower sensitivity than serum (81.3% vs. 93.8%, respectively); however, the specificity and positive predictive value were higher for saliva vs. serum (88.0% vs. 59.8% and 54.2% vs. 28.8%, respectively; Chien and Schwartz, 1990).
Tumor markers that can be identified in saliva may be potentially useful for screening for malignant diseases. Salivary diagnosis may be part of a comprehensive diagnostic panel that will provide improved sensitivity and specificity in the detection of malignant diseases and will assist in monitoring the efficacy of treatment. Additional studies are certainly required to determine which salivary markers can be used for these diagnostic purposes, and to determine their diagnostic value in comparison with other, more established, diagnostic tests.
Helicobacter pylori infection is associated with peptic ulcer disease and chronic gastritis. Infection with this bacterium stimulates the production of specific IgG antibody. An ELISA test for the detection of IgG antibody in serum produced 97% sensitivity and 94% specificity in detection of the disease. In parallel, saliva samples were tested for the presence of H. pylori DNA by polymerase chain-reaction (PCR) assay, and sensitivity of 84% was reported. The results also indicated that H. pylori exists in higher prevalence in saliva than in feces, and the oral-oral route may be an important means of transmission of this infection in developed countries (Li et al., 1996). In another study, testing for salivary antibodies against H. pylori yielded sensitivity of 85%, specificity of 55%, positive predictive value of 45%, and negative predictive value of 90% (Loeb et al., 1997).
A variety of other infections has also been monitored by the detection of specific antibodies in saliva. Evaluation of the secretory immune response in the saliva of children infected with Shigella revealed higher titers of anti-lipopolysaccharide and anti-Shiga toxin antibody in comparison with healthy controls. It was suggested that salivary levels of these immunoglobulins could be used for monitoring of the immune response in shigellosis (Schultsz et al., 1992).
Pigeon breeder's disease (PBD) is an interstitial lung disease induced by exposure to antigens derived from pigeons. Measurement of salivary IgG against these antigens may assist in the evaluation of patients with this disease. A correlation coefficient of 0.58 was observed between IgG antibody levels in serum and saliva (Mendoza et al., 1996). A similar correlation (r = 0.52) between IgG levels in saliva and serum was also reported in a more recent study (McSharry et al., 1999). Furthermore, the detection of pneumococcal C polysaccharide in saliva by ELISA may offer a valuable complement to conventional diagnostic methods for pneumococcal pneumonia. Detection of this antigen in saliva demonstrated a sensitivity of 55% and specificity of 97%. The positive and negative predictive values were 0.94 and 0.73, respectively (Krook et al., 1986).
Lyme disease is caused by the spirochete Borrelia burgdorferi and is transmitted to humans by blood-feeding ticks. The detection of anti-tick antibody in saliva has potential as a biologic marker of exposure to tick bites, which in turn may serve as a screening mechanism for individuals at risk for Lyme disease (Schwartz et al., 1991).
Specific antibody to Taenia solium larvae in serum demonstrated greater sensitivity than antibody in saliva for identification of neurocysticercosis (100% vs. 70.4%, respectively). However, considering the simple and non-invasive nature of saliva sampling, it was suggested that saliva could be used in epidemiologic studies of this disease (Feldman et al., 1990).
Viral Diseases (exclusive of HIV)
The antibody response to infection is the basis for many diagnostic tests in virology. Saliva contains immunoglobulins that originate from two sources: the salivary glands and serum. The predominant immunoglobulin in saliva is secretory IgA (sIgA), which is derived from plasma cells in the salivary glands, and constitutes the main specific immune defense mechanism in saliva. Although the minor salivary glands play an important role in sIgA-mediated immunity of the oral cavity, cells in the parotid and submandibular glands are responsible for the majority of the IgA found in saliva (Bienenstock et al., 1980; Korsrud and Brandtzaeg, 1980; Nair and Schroeder, 1986). In contrast, salivary IgM and IgG are primarily derived from serum via GCF, and are present in lower concentrations in saliva than is IgA. Antibodies against viruses and viral components can be detected in saliva and can aid in the diagnosis of acute viral infections, congenital infections, and reactivation of infection (Mortimer and Parry, 1988).
Saliva was found to be a useful alternative to serum for the diagnosis of viral hepatitis. Acute hepatitis A (HAV) and hepatitis B (HBV) were diagnosed based on the presence of IgM antibodies in saliva. The ratio of IgM to IgG anti-HAV antibody correlated with the time interval from onset of infection (Parry et al., 1989). Further, salivary antibody levels were used for the detection of infected individuals in a school outbreak of HAV (Bull et al., 1989; Stuart et al., 1992). Saliva has also been utilized to detect very low levels of antibodies to HAV, which, for example, are associated with vaccine-induced immunity. Comparison of serum and saliva levels of antibody to HAV revealed excellent agreement (sensitivity = 98.7% and specificity = 99.6%; Ochnio et al., 1997). Similarly, analysis of saliva provided a highly sensitive and specific method for the diagnosis of viral hepatitis B and C (El-Medany et al., 1999). Analysis of oral fluid samples collected with Orasure® provided an excellent method for the diagnosis of viral hepatitis B and C. Sensitivity and specificity of 100% for the detection of antibodies for both diseases in oral fluid in comparison with serum antibodies were reported (Thieme et al., 1992). Saliva has also been used for screening for hepatitis B surface antigen (HbsAg) in epidemiological studies. Comparing the detection of HbsAg in saliva with that in serum by means of a commercially available serological kit yielded a sensitivity of 92% and specificity of 86.8% (Chaita et al., 1995).
Saliva may also be used for determining immunization and detecting infection with measles, mumps, and rubella (Friedman, 1982; Perry et al., 1993; Brown et al., 1994). The detection of antibodies in oral fluid samples produced sensitivity and specificity of 97% and 100% for measles, 94% and 94% for mumps, and 98% and 98% for rubella, respectively, in comparison with detection of serum antibodies for these viruses (Thieme et al., 1994).
For newborn infants, the salivary IgA response was found to be a better marker of rotavirus (RV) infection than the serum antibody response. Neonatal RV infection elicited specific mucosal antibody response which persisted for at least 3 months. However, a similar systemic immune response could not be observed, possibly due to interference by maternal antibody. The authors proposed that saliva, rather than serum, can be used to monitor the immune response to vaccination and infection with RV (Jayashree et al., 1988).
The shedding of herpesviruses (human herpesvirus –8, cytomegalovirus, and Epstein-Barr virus) in nasal secretions and saliva of infected patients has been reported (Blackbourn et al., 1998). Other investigators suggested that reactivation of herpes simplex virus type-1 (HSV-1) is involved in the pathogenesis of Bell's palsy and reported that PCR-based identification of virus in saliva is a useful method for the early detection of HSV-1 reactivation in patients with Bell's palsy. The shed HSV-1 virus was detected in 50% of patients with Bell's palsy in comparison with 19% in healthy controls (Furuta et al., 1998).
Dengue is a mosquito-transmitted viral disease. Primary infection of the virus may lead to a self-limiting febrile disease, and secondary infection may cause serious complications like dengue hemorrhagic fever or dengue shock syndrome (Burke et al., 1988). Salivary levels of anti-dengue IgM and IgG demonstrated sensitivity of 92% and specificity of 100% in the diagnosis of primary and secondary infection, and salivary levels of IgG proved useful in differentiating between primary and secondary infection (Cuzzubbo et al., 1998). Saliva was also found to be a reliable alternative to serum for identification of the antibody to parvovirus B 19. Sensitivity of 100% and specificity of 95% were observed for the detection of infected individuals at a primary school (Rice and Cohen, 1996).
Studies have demonstrated that the diagnosis of infection with the human immunodeficiency virus (HIV) based on specific antibody in saliva is equivalent to serum in accuracy, and therefore applicable for both clinical use and epidemiological surveillance (Malamud, 1992). Antibody to HIV in whole saliva of infected individuals, which was detected by ELISA and Western blot assay, correlated with serum antibody levels (Holmstrom et al., 1990; Frerichs et al., 1994). As compared with serum, the sensitivity and specificity of antibody to HIV in saliva for detection of infection are between 95% and 100% (Tamashiro and Constantine, 1994; Tess et al., 1996; Emmons, 1997; Malamud, 1997). Salivary IgA levels to HIV decline as infected patients become symptomatic. It was suggested that detection of IgA antibody to HIV in saliva may, therefore, be a prognostic indicator for the progression of HIV infection (Matsuda et al., 1993).
Analysis of antibody in saliva as a diagnostic test for HIV (or other infections) offers several distinctive advantages when compared with serum. Saliva can be collected non-invasively, which eliminates the risk of infection for the health care worker who collects the blood sample. Furthermore, viral transmission via saliva is unlikely, since infectious virus is rarely isolated from saliva (Ho et al., 1985). Saliva collection also simplifies the diagnostic process in special populations in whom blood drawing is difficult, i.e., individuals with compromised venous access (e.g., injecting drug users), patients with hemophilia, and children (Archibald et al., 1993).
Several salivary and oral fluid tests have been developed for HIV diagnosis. Orasure® is a testing system that is commercially available in the United States and can be used for the diagnosis of HIV. The test relies on the collection of an oral mucosal transudate (and therefore IgG antibody). IgG antibody to the virus is the predominant type of anti-HIV immunoglobulin (Cordeiro et al., 1993; Gaudette et al., 1994). Different oral pathologic lesions, which are relatively common in HIV-infected individuals, do not appear to influence the results (Emmons et al., 1995; Gallo et al., 1997). In conclusion, collection and analysis of saliva offer a simple, safe, well-tolerated, and accurate method for the diagnosis of HIV infection.
Similar to other body fluids (i.e., serum, urine, and sweat), saliva has been proposed for the monitoring of systemic levels of drugs (Danhof and Breimer, 1978; Drobitch and Svensson, 1992). A fundamental prerequisite for this diagnostic application of saliva is a definable relationship between the concentration of a therapeutic drug in blood (serum) and the concentration in saliva. For a drug to appear in saliva, drug molecules in serum must pass through the salivary glands and into the oral cavity. Therefore, the presence of a drug in saliva is influenced by the physicochemical characteristics of the drug molecule and its interaction with the cells and tissues of the salivary glands, as well as by extravascular drug metabolism. Factors such as molecular size, lipid solubility, and the degree of ionization of the drug molecule, as well as the effect of salivary pH and the degree of protein binding of the drug, are important determinants of drug availability in saliva (Drobitch and Svensson, 1992; Siegel, 1993).
Passive diffusion across a concentration gradient is thought to be the major mechanism to account for the appearance of a drug in saliva. Generally, smaller molecules diffuse more easily than larger ones. Due to the presence of the phospholipid layer of the cell membrane, lipophilic molecules diffuse more easily than lipophobic molecules. For similar reasons, non-ionized molecules diffuse more readily through lipid membranes than do ionized molecules. The pKa of the drug (the pH at which 50% of the drug molecules are ionized) and the pH gradient between plasma and saliva determine the concentration gradient on both sides of the membrane, and influence the availability of a drug in saliva (Haeckel and Hanecke, 1996). Therefore, drugs which are not ionizable, or are not ionized within the pH range of saliva, are the most suited to salivary drug monitoring. Due to their size, serum-binding proteins do not cross the membrane. Therefore, only the unbound fraction of the drug in serum is available for diffusion into saliva (Haeckel, 1993). The unbound fraction of a drug is usually the pharmacologically active fraction. This may represent an advantage of drug monitoring in saliva in comparison with drug monitoring in serum, where both bound and unbound fractions of a drug can be detected (Gorodischer and Koren, 1992). Other parameters which may influence the availability of drugs in saliva are the mechanism of drug transfer into saliva (since some drugs reach saliva in ways other than passive diffusion), salivary flow rate (increased flow rate affects salivary pH by increasing bicarbonate secretion), and drug stability in saliva.
The application of saliva for monitoring drug levels has been the subject of considerable investigation (Table 2). Saliva may be used for monitoring patient compliance with psychiatric medications (El-Guebaly et al., 1981). A significant correlation (r = 0.87) exists between the salivary and serum lithium levels in patients receiving lithium therapy (Ben-Aryeh et al., 1980, 1984). Saliva is also useful for the monitoring of anti-epileptic drugs. Salivary carbamazepine levels were found to be 38% of serum carbamazepine levels, and a positive correlation (r = 0.89) between salivary and serum carbamazepine levels was observed. Stimulation of salivary flow and storage of saliva for several days did not affect this correlation (Rosenthal et al., 1995). In another study, salivary levels of phenobarbital and phenytoin demonstrated excellent correlations (r = 0.98 and 0.97, respectively) with serum levels of these medications (Kankirawatana, 1999). A lower correlation (r = 0.68) was found between salivary and total serum levels of cyclosporine. Cyclosporine is a neutral lipophilic molecule that enters saliva mostly by passive diffusion, and salivary levels of this drug reflect the serum levels of free cyclosporine. Therefore, salivary cyclosporine levels may correlate better with serum levels of free, rather than total, cyclosporine (Coates et al., 1988). Similarly, salivary theophylline concentration demonstrated a better correlation with serum concentration of free theophylline (r = 0.85) than with serum concentration of total theophylline (r = 0.85; Kirk et al., 1994).
Drug Monitoring in Saliva
Saliva may also be used for monitoring levels of anti-cancer drugs. Saliva was found to be a reliable alternative to serum for the monitoring of irinotecan levels. A correlation of r = 0.73 between salivary and serum levels was reported (Takahashi et al., 1997). Salivary analysis may be used to evaluate the cisplatin concentration in serum; however, a defined correlation between salivary and serum levels was not reported (Holding et al., 1999). Conversely, serum carboplatin concentration demonstrated considerable variations and was found to be unreliable in measurements of serum carboplatin (van Warmerdam et al., 1995).
Of particular interest is the use of saliva for the evaluation of illicit drug use. Following drug use, the appearance of the drug in saliva follows a time course that is similar to that of serum. In contrast, drugs appear at a later time point in urine. Nevertheless, as opposed to what is needed for the monitoring of therapeutic drugs, the presence of illicit drugs, and not their concentration, is usually sufficient for forensic purposes. One important exception is ethanol. Ethanol is not ionized in serum, is not protein-bound, and, due to its low molecular weight and lipid solubility, diffuses rapidly into saliva. Consequently, the saliva-to-serum ratio is generally about 1. A significant correlation between salivary and serum alcohol levels was reported (Penttila et al., 1990). Salivary ethanol concentration may be used as an index of the blood ethanol concentration, provided that the salivary sample is obtained at least 20 min following ingestion. This will allow for absorption and distribution of alcohol, and prevent a falsely elevated reading due to the oral route of consumption (McColl et al., 1979).
Other recreational drugs that can be identified in saliva are amphetamines, barbiturates, benzodiazepines, cocaine, phencyclidine (PCP), and opioids (Cone, 1993; Kidwell et al., 1998; Table 2). Saliva can also be used to detect recent marijuana use by means of radiommunoassay (Gross et al., 1985). ▵9-Tetrahydrocannabinol (▵9-THC), a major psychoactive component of marijuana, can be detected in saliva for at least 4 hours after marijuana is smoked (Maseda et al., 1986). Furthermore, saliva can be used to monitor tobacco smoking and exposure to tobacco smoke. The major nicotine metabolite cotinine was investigated as an indicator of exposure to tobacco smoking. Cotinine is tobacco-specific and has a relatively long half-life compared with nicotine (Benowitz, 1983). Salivary cotinine levels were found to be indicative of active and passive smoking (Istvan et al., 1994; Repace et al., 1998). Salivary thiocyanate was also found to be an indicator of cigarette smoking (Luepker et al., 1981); however, cotinine levels are considered the most reliable marker (Di Giusto and Eckhard, 1986).
The Monitoring of Hormone Levels
Saliva can be analyzed as part of the evaluation of endocrine function. The factors that affect drug availability in saliva are generally true also for salivary hormones. The majority of hormones enter saliva by passive diffusion across the acinar cells. Most of these hormones are lipid-soluble (i.e., steroids). Small polar molecules do not readily diffuse across cells and instead enter saliva through the tight junctions between cells (ultrafiltration; Quissell, 1993; Read, 1993). The molecular-weight cut-off for ultrafiltration is 100-200. This relatively small molecular size prevents many hormones from entering saliva from serum by means of ultrafiltration. In addition, active transport does not appear to facilitate hormone transfer into saliva (Vining and McGinley, 1986). Measurements of salivary hormone levels are of clinical importance if they accurately reflect the serum hormone levels, or if a constant correlation exists between salivary and serum hormone levels. For neutral steroids which diffuse readily into saliva, salivary hormone levels represent the non-protein-bound (free) serum hormone levels. Conversely, due to their size, protein hormones do not enter saliva through passive diffusion, but primarily through contamination from serum as a result of outflow of GCF or from oral wounds. Furthermore, some steroid hormones can be metabolized in the salivary epithelial cells by intracellular enzymes during transcellular diffusion, which can affect the availability of these hormones in saliva (Quissell, 1993).
Due to their lipid solubility, steroid hormones can be detected in saliva. Salivary cortisol levels demonstrate excellent correlation with free serum cortisol levels (r = 0.97; Peters et al., 1982; Vining et al., 1983a). This high correlation is not affected by changes in concentrations of serum-binding proteins. However, the actual salivary cortisol levels are lower than the serum-free cortisol levels, possibly due to enzymatic degradation in the salivary epithelial cells during transcellular diffusion (Quissell, 1993). Salivary cortisol levels were found to be useful in identifying patients with Cushing's syndrome and Addison's disease (Hubl et al., 1984), and also for monitoring the hormone response to physical exercise (Lac et al., 1997) and the effect of acceleration stress (Tarui and Nakamura, 1987; Obminski et al., 1997). Contrary to cortisol, salivary cortisone levels do not accurately reflect serum cortisone levels. Cortisone is a neutral steroid and therefore readily diffuses into saliva; however, cortisol is converted to cortisone by an enzyme present in the salivary glands (11 β-hydroxysteroid dehydrogenase). Thus, cortisone levels in saliva are higher than in serum and do not bear any diagnostic significance (Vining and McGinley, 1986). Other corticosteroids, like prednisone and prednisolone, also do not show a consistant correlation between serum and salivary levels, possibly due to the effect of the same enzyme (Lowe and Dixon, 1983).
Salivary aldosterone levels demonstrated a high correlation with serum aldosterone levels (r = 0.96), and increased aldosterone levels were found in both the serum and saliva of patients with primary aldosteronism (Conn's syndrome; McVie et al., 1979). A similar high correlation (r = 0.92) between salivary and serum aldosterone levels was observed with the use of a solid-phase enzyme immunoassay (Hubl et al., 1983). These findings were supported by an additional study (r = 0.93), and salivary aldosterone levels were found to be approximately one-third of serum levels (Atherden et al., 1985). Testosterone and dehydroepiandrosterone have also been identified in saliva. Salivary concentrations were found to be 1.5-7.5% of the serum concentrations of these hormones (Gaskell et al., 1980). Similarly, salivary testosterone levels were detected in an additional study which proposed the use of salivary testosterone levels for the assessment of testicular function (Walker et al., 1980). By a direct radioimmunoassay technique, a high correlation between salivary and serum-free testosterone concentration (r = 0.97) and salivary and serum total testosterone concentration (r = 0.7-0.87) was reported (Vittek et al., 1985). A significant correlation (r = 0.79) between the concentration of unbound salivary and serum testosterone was observed when hormone levels in normal and hyperandrogenic women were evaluated (Baxendale et al., 1982). Monitoring salivary testosterone levels may also be useful in behavioral studies of aggression, depression, abuse, and violent and antisocial behavior (Dabbs, 1993; Granger et al., 1999). However, variability in results between laboratories has been reported (Dabbs et al., 1995). A high correlation between the salivary concentration of androstenedione and dihydrotestosterone and the unbound serum concentration of these hormones has also been reported (r = 0.92 and 0.82, respectively; Baxendale et al., 1983).
Estradiol can be detected in saliva in concentrations that are only 1-2% of serum concentrations. These concentrations are similar to the serum concentrations of free estradiol, which can diffuse into saliva. A significant correlation (r = 0.78) between salivary estradiol levels and serum levels of free estradiol was reported (Wang et al., 1986). Salivary estradiol levels followed the same trends as serum estradiol levels during a menstrual cycle (Evans et al., 1980). Furthermore, salivary estriol levels showed a very high correlation (r = 0.98) with serum levels of free estriol in pregnant women, and salivary estriol levels were suggested as a means for the assessment of feto-placental function (Kundu et al., 1983; Vining et al., 1983b). Salivary progesterone levels showed good correlation (r = 0.47-0.58) with serum levels during the menstrual cycle and reflected the free serum progesterone levels (Luisi et al., 1981; Choe et al., 1983). More recent studies supported the use of salivary diagnosis for the evaluation of clinical problems associated with these hormones. Salivary progesterone levels can be useful for the prediction of ovulation, demonstrating a correlation of 0.75 with serum progesterone levels, and salivary estradiol and progesterone levels can be used for the evaluation of ovarian function (Lu et al., 1997, 1999). Decreased salivary estriol was suggested as a marker of fetal growth retardation (Lechner et al., 1987). Furthermore, an increased salivary estriol-to-progesterone ratio may be a predictor of pre-term delivery (Darne et al., 1987).
Insulin can be detected in saliva, and salivary insulin levels have been evaluated as a means of monitoring serum insulin levels. A positive correlation between saliva and serum insulin levels following a glucose tolerance test was reported for healthy subjects (r = 0.52), non-insulin-dependent diabetic patients (r = 0.50), and obese non-diabetic patients (r = 0.69; Marchetti et al., 1986). Additional work by the same authors utilizing similar methods reported a better correlation between salivary and serum insulin levels in 93 healthy subjects (r = 0.75 in males and r = 0.72 in females; Marchetti et al., 1988). As assessed by radioimmunoassay, a glucose tolerance test performed on nine healthy patients produced a positive correlation between salivary and serum insulin levels (r = 0.74). Salivary insulin levels reached maximal values approximately 30 minutes after the serum levels (90 min vs. 60 min; Fekete et al., 1993). Other investigators also reported a similarly high correlation between salivary and serum insulin levels in healthy individuals and insulin-dependent diabetic patients (0.81 and 0.91, respectively), but proposed that the use of salivary insulin levels for the evaluation of serum insulin levels could be misleading, since significant discrepancies between salivary and serum insulin levels were detected for several individuals (Pasic and Pickup, 1988). Additional studies are required to determine if salivary insulin levels should be used for the evaluation of serum insulin levels.
In general, serum and salivary levels of protein hormones are not well-correlated. These hormones are too large to reach saliva by means of passive diffusion across cells or by ultrafiltration, and the detection of these hormones in saliva is primarily due to contamination from serum through GCF or oral wounds. Therefore, serum levels of protein hormones such as gonadotrophins, prolactin, and thyrotropin cannot be accurately monitored by means of salivary analysis (Vining and McGinley, 1986, 1987).
Salivary monitoring of hormone levels has many advantages over the more conventional serum analysis. In addition to the other advantages of salivary diagnosis presented in this article, hormone evaluation often necessitates multiple sample collection in a relatively short time interval, which makes the non-invasive collection of saliva ideal for this purpose (Ellison, 1993). However, it is important to consider the possible limitations of salivary analysis for hormone evaluation. Hormones enter saliva by passive diffusion and ultrafiltration, and active transport of hormones into saliva does not exist. Therefore, mostly lipid-soluble and hormones with small molecular weight can be detected in saliva. Most hormones are protein-bound in serum, and thus salivary hormone levels represent the free hormone levels which are available for diffusion into saliva. This may provide more clinically useful information, since free serum hormone levels are the biologically active fraction of hormone in serum. For accurate results, a constant and predictable correlation must exist between salivary and serum hormone levels. However, different hormones are bound to similar serum carrier proteins, and thus changes in levels of one hormone may affect the free levels of others. For hormones that demonstrate a constant but low salivary-to-serum ratio, a sufficiently large sample volume or a more sensitive analysis method is required. In addition, many hormones exhibit marked circadian variations. Therefore, timing of saliva collection may affect the results. The salivary flow rate can also affect the concentrations of certain hormones. An increase in salivary flow rate will usually result in reduced concentrations of molecules that reach saliva by diffusion. However, the rate of diffusion of steroid hormones, particularly cortisol, is usually high enough to maintain a constant relationship between salivary and serum levels of the hormone regardless of the salivary flow rate. The concentrations of hormones that reach saliva by ultrafiltration, such as dehydroepiandrosterone sulphate, are more affected by changes in salivary flow rate. Changes in salivary flow rate may lead to changes in salivary pH. This may affect the entry into saliva of molecules according to their pka. The stability of hormones in saliva is important as well for accurate evaluation. Hormones in saliva can be degraded, among other ways, by enzymes native to saliva, enzymes derived from oral micro-organisms, and enzymes derived from leukocytes that enter the oral cavity from the gingival sulcus. In addition, molecules that reach saliva by passive diffusion across cells, like unconjugated steroids, may be subjected to enzymatic degradation within the salivary glands, prior to entering saliva (Vining and McGinley, 1986; Quissell, 1993; Read, 1993). These factors have to be considered when saliva is evaluated as an alternative for the evaluation of serum hormone levels.
Diagnosis of Oral Disease with Relevance for Systemic Diseases
The monitoring of gland-specific secretions is important for the differential diagnosis of diseases that may have an effect on specific salivary glands, like obstruction or infection (Mandel, 1989). However, monitoring gland-specific saliva can be complicated and time-consuming. Evaluation of the quantity of whole saliva is simple and may provide information which has systemic relevance. Quantitative alterations in saliva may be a result of medications. At least 400 drugs may induce xerostomia. Diuretics, antihypertensives, antipsychotics, antihistamines, antidepressants, anticholinergics, antineoplastics, and recreational drugs such as opiates, amphetamines, barbiturates, hallucinogens, cannabis, and alcohol have been associated with a reduction in salivary flow (Sreebny and Schwartz, 1997; Rees, 1998). Reduced salivary flow may lead to oral problems like progressive dental caries, fungal infection, oral pain, and dysphagia. The reasons for such clinical findings should be thoroughly investigated, since they may be signs of an underlying systemic problem. Systemic disorders that may affect salivary glands and saliva are presented in Table 3.
Systemic Diseases Affecting Salivary Glands and Saliva
Qualitative changes in salivary composition can also provide diagnostic information concerning oral problems. Increased levels of albumin in whole saliva were detected in patients who received chemotherapy as treatment for cancer and subsequently developed stomatitis. However, no difference in albumin levels in parotid saliva was observed, which implied that the salivary albumin originated from the mucosal lesions as a result of loss of epithelial barrier function. This was further supported by the fact that salivary levels of another serum constituent, IgG, showed changes similar to those in albumin levels. The increase in the concentration of albumin in whole saliva was always detected prior to the clinical appearance of stomatitis, suggesting that albumin in whole saliva may be a marker and predicter of this complication. Therefore, the monitoring of salivary albumin can assist in the identification of stomatitis at a pre-clinical stage and enable the chemotherapy dosage to be adjusted or treatment for the stomatitis to be initiated at an early stage (Izutsu et al., 1981). Furthermore, a significant negative correlation was found between normalized EGF (concentration of salivary EGF relative to total salivary protein concentration) and severity of mucositis in patients receiving radiation therapy to the head and neck. This negative correlation suggests that reduced salivary EGF levels may be important for the progression of radiation-induced mucositis (Dumbrigue et al., 2000).
It has been suggested that salivary nitrate, nitrite, and nitrosamine may be related to the development of oral and gastric cancer (Tenovuo, 1986). Increased consumption of dietary nitrate and nitrite is associated with elevated levels of salivary nitrite. Higher levels of salivary nitrate and nitrite, and increased activity of nitrate reductase, were found in oral cancer patients compared with healthy individuals, and were associated with an increased odds ratio for the risk of oral cancer (Badawi et al., 1998).
Saliva can be used for the detection of oral candidiasis, and salivary fungal counts may reflect mucosal colonization (Bergmann, 1996; Hicks et al., 1998). Saliva may also be used for the monitoring of oral bacteria. Bacteria (including anaerobic species) can survive in saliva, and can utilize salivary constituents as a growth medium (de Jong et al., 1984; Bowden, 1997). Furthermore, increased numbers of Streptococcus mutans and Lactobacilli in saliva were associated with increased caries prevalence (Klock et al., 1990; Kohler and Bjarnason, 1992) and with the presence of root caries (Van Houte et al., 1990). Saliva can serve as a vector for bacterial transmission, and also as a reservoir for bacterial colonization (Greenstein and Lamster, 1997). Detection of certain bacterial species in saliva can reflect their presence in dental plaque and periodontal pockets (Asikainen et al., 1991; Umeda et al., 1998). Saliva may also be used for periodontal diagnosis, due in large part to contributions from GCF. A comprehensive analysis of this topic is beyond the scope of this review and is covered elsewhere (Kaufman and Lamster, 2000). Nevertheless, the recent focus on the potential role of periodontal disease as a risk factor for cardiovascular and cerebrovascular diseases (Joshipura et al., 1998; Morrison et al., 1999) and the occurrence of pre-term low-birth-weight babies (Offenbacher et al., 1998) bring new importance to this aspect of salivary analysis.
Saliva offers an alternative to serum as a biologic fluid that can be analyzed for diagnostic purposes. Whole saliva contains locally produced as well as serum-derived markers that have been found to be useful in the diagnosis of a variety of systemic disorders. Whole saliva can be collected in a non-invasive manner by individuals with modest training, including patients. This facilitates the development and introduction of screening tests that can be performed by patients at home. Analysis of saliva can offer a cost-effective approach for the screening of large populations, and may represent an alternative for patients in whom blood drawing is difficult, or when compliance is a problem (Bailey et al., 1997).
This review suggests that certain diagnostic uses of saliva hold considerable promise. Monitoring of the immune responses to viral infections, including hepatitis and HIV, may prove valuable in the identification of infected individuals, non-symptomatic carriers, and immune individuals. Saliva can also be useful in the monitoring of therapeutic drug levels and the detection of illicit drug use. Further, analysis of saliva may provide valuable information regarding certain endocrine disorders.
Nevertheless, levels of certain markers in saliva are not always a reliable reflection of the levels of these markers in serum. The transfer of serum constituents which are not part of the normal salivary constituents into saliva is related to the physicochemical characteristics of these molecules. Lipophilic molecules diffuse more readily into saliva than do lipophobic molecules. Furthermore, different substances reach saliva by different mechanisms. Although passive diffusion is considered to be the most common mechanism for drugs and hormones, ultrafiltration and active transport have also been proposed for some substances. For accurate diagnosis, a defined relationship is required between the concentration of the biomarker in serum and the concentration in saliva. Normal salivary gland function is usually required for the detection of salivary molecules with diagnostic value. Salivary composition can be influenced by the method of collection and the degree of stimulation of salivary flow. Changes in salivary flow rate may affect the concentration of salivary markers and also their availability due to changes in salivary pH. Variability in salivary flow rate is expected between individuals and in the same individual under various conditions. In addition, many serum markers can reach whole saliva in an unpredictable way (i.e., GCF flow and through oral wounds). These parameters will affect the diagnostic usefulness of many salivary constituents (FDI Working Group 10, Core, 1992). Furthermore, certain systemic disorders, numerous medications, and radiation may affect salivary gland function and consequently the quantity and composition of saliva (Sreebny and Schwartz, 1997; Fox, 1998). Whole saliva also contains proteolytic enzymes derived from the host and from oral micro-organisms (Chauncey, 1961). These enzymes can affect the stability of certain diagnostic markers. Some molecules are also degraded during intracellular diffusion into saliva. Any condition or medication that affects the availability or concentration of a diagnostic marker in saliva may adversely affect the diagnostic usefulness of that marker.
Despite these limitations, the use of saliva for diagnostic purposes is increasing in popularity. Several diagnostic tests are commercially available and are currently used by patients, researchers, and clinicians. Saliva is particularly useful for qualitative (detection of the presence or absence of a marker) rather than quantitative diagnosis, which makes it an important means for the detection of viral infection (especially HIV due to the non-invasive method of collection), past exposure and immunity, and the detection of illicit drug use. Saliva is also useful for the monitoring of hormone levels, especially steroids, and facilitates repeated sampling in short time intervals, which may be particularly important for hormone monitoring and avoiding compliance problems.
Due to its many potential advantages, salivary diagnosis provides an attractive alternative to more invasive, time-consuming, complicated, and expensive diagnostic approaches. However, before a salivary diagnostic test can replace a more conventional one, the diagnostic value of a new salivary test has to be compared with accepted diagnostic methods. The usefulness of a new test has to be determined in terms of sensitivity, specificity, correlation with established disease diagnostic criteria, and reproducibility. This review has discussed many disease markers identified in saliva. It is difficult to interpret the significance of a single report that examines levels of any particular marker. However, due to the many potential limitations of salivary diagnosis, promising results from pilot studies must be confirmed in larger, well-controlled trials.
While many questions remain, the potential advantages of salivary analysis for the diagnosis of systemic disease suggest that further studies are warranted. Definition of specific disorders that can be identified or monitored by the analysis of saliva offers the possibility of improved patient management. Consequently, we are likely to see the increased utilization of saliva as a diagnostic fluid. As a result, dentists will have greater involvement in the identification and monitoring of certain non-oral disorders.
CLINICAL LABORATORY DIAGNOSTICS OF PATHOLOGICAL PROCESSESS IN LUNGS
Normal and pathologic biochemistry of lung
Only recently has the lung been recognized as an important metabolic organ rather than just a tissue for passive gas exchange. A major reason for the delay in appreciation of the metabolic role of the lung is related to its structure and ana-tomic relationships. The lung, although filling most of the thoracic cavity, actually comprises only 1% of the body weight, and approximately 30 % of that weight is due to contained blood. Further, the blood flow to the lung comprises the entire cardiac output making it the most richly perfused organ in the body. Because of the high blood flow in relation to metabolizing tissue mass, arteriovenous differences of most metabolites cannot be measure easured across the lung in situ. Consequently, it has been necessary to develop in vitro models for study of lung metabolism. One model that has been extensively used is the isolated perfused lung preparation. Perfusion of the lung with artificial media removes the red cells from the pulmonary capillaries and results in tissue with a completely white appearance. Additional models to study lung metabolism are tissue slices and preparations of subcellular organelles.
Measurements with the isolated perfused lung preparation or lung slices have shown oxygen up-take in the range of 30-150 jA/min-g dry weight, depending on species and preparation. There-fore, lung tissue has significant O2 consumption although values are low compared with the metabolically very active organs. For example, dog lung oxygen uptake per unit weight is only 10-20 % of the oxygen uptake of canine heart, kidney, thyroid, and brain. The oxygen uptake of the lung is greater, however, than that of resting skeletal muscle, intestine, and many other metabolically less active tissues. Actually, the lung can be considered an average organ in terms of O2 utilization, since its oxygen uptake of the organism, and this is approximately the contribution of the lung to total body weight. On the other hand, it should be noted that the lung represents a heterogeneous collection of cell types, and it is likely that some components of the lung, e.g., the type II granular pneumocytes, have considerably higher oxygen uptake than the mean for the whole lung.
What are the substrates utilized by the lung for its metabolic requirements? Although intact lungs and lung subcellular organelles can oxidize fatty acids, glucose probably serves as the major oxidizable substrate under usual conditions. Approximately half of the glucose was converted to lactate and pyruvate. The "reason" for the high rate of production of these three-carbon compounds has not been defined. One postulated explanation relates to the presence of numerous cells in the lung with relatively sparse mitochondria and, therefore, limited citric acid cycle activity. Additional possibilities include limited activity of mitochondrial H+ shuttle mechanisms or mitochondrial pyruvate dehydrogenase. In any case, the high lactate production under control conditions was probably not due to cellular hypoxia since the lung was being ventilated with 95 % 02, the perfusate L/P ratio was within a normal range (i.e., 5-10), and the lung responded briskly to inhibitors of oxidative metabolism with change in redox state. Approximately one fourth of the glucose carbons utilized by the perfused lungs are oxidized to CO2. There are also active pathways for incorporation of glucose carbons into tissue components including proteins, nucleic acids, polysaccharides (chiefly glycogen), and other unidentified components. Finally, a small but significant fraction of glucose carbons is used for synthesis of lipids, including the fatty acid as well as glyceride-glycerol moieties.
The next question to explore is whether oxidative metabolism is required in order to maintain normal energy stores of the lung tissue. In-sight into this problem can be obtained by measurement of changes in lung tissue adenine nucleotide content during inhibition of oxidative metabolism, or uncoupling of oxidative phosphoryla-tion. During control perfusion, ATP content of the lung per unit weight is comparable to values observed in other aerobic tissues able to values observed in other aerobic tissues and the ATP/ADP ratio is approximately 8.5.
What are the metabolic processes in the lung that are energy-dependent? Certainly the lung has no physiologic process that requires large expenditures of energy such as occurs with cardiac muscle contraction, renal transport, or maintenance of ionic gradients in nerve tissue. Energy utilization, however, is required for functioning of several lung systems. For example, lung clearance depends on bronchial ciliary activity and phagocytosis by alveolar macrophages, both of which are energy dependent. During anoxia, there is cessation of ciliary beating and inhibition of particle phagocytosis. Secretion by bronchial glands and constriction of tracheobronchial smooth muscle are other processes that presumably are energy-dependent. Synthesis of dipalmitoyl lecithin, a major component of the lung surfactant system, requires a supply of ATP.
The actual secretion of surfactant is also probably an energy-requiring process analogous to cellular secretion elsewhere. Finally, energy is required for cell transport processes.
The most dramatic metabolic function of the lung and the one that has been studied to the greatest extent relates to the synthesis of lung surfactant. The lung surfactant is contained in the extracellular alveolar lining layer that coats the epithelial surface of the lung alveoli. The presumed physiologic function of the surfactant system is to maintain the surface tension at the interface between the alveolar surface and the air spaces at low levels and thereby promote alveolar stability. The extracellular material can be obtained for study by alveolar lavage with saline or other physiologic solution. The material so obtained contains approximately 75° phospholipids, 15% neutral lipids, and 10 % protein. Dipalmitoyl lecithin (dipalmitoyl phosphatidyl-choline) accounts for approximately 40 % of the Dipalmitoyl lecithin (dipalmitoyl phosphatidyl-choline) accounts for approximately 40 % of the total solids in the surfactant fraction. Synthetic dipalmitoyl lecithin (DPL) manifests similar surface active properties to the crude surfactant fraction so that the physiologic properties of the surfactant are thought to be related chiefly to the presence of DPL. An additional 25 % of the surfactant material is comprised of lecithins containing unsaturated fatty acids. The physiologic role of this fraction of the surfactant is not known but may be important as an aid to spreading or other-wise influencing dispersion of the dipalmitoyl lecithin fraction. The role of the protein in surfactant is also incompletely understood. Current evidence suggests that the protein is specific to the lung and that surfactant is secreted as a lipoprotein complex.
What is the source of surfactant and what are the pathways involved in its production? It now seems clear that surfactant is synthesized in the lung and subsequently secreted onto the alveolar surface. Most studies that have investigated surfactant production have focused on pathways of dipalmitoyl lecithin synthesis although, as noted above, DPL is only one component of the surfactant fraction. These studies have shown that DPL is synthesized chiefly by the classical pathway involving phosphatidic acid (formed from glycerol-3-phosphate and palmitate) and CDP-choline (formed from CTP and choline). Considerable effort is currently being directed to determine the factors that are important in control of this path-way in the lung. Based on experiments, the current concept is that surfactant synthesis is a major metabolic activity of the lung. Substrates for surfactant synthesis are transferred from the circulation or alveolar space to type II epithelial cells where the lipid and protein components are synthesized, presumably in the endoplasmic reticulum and Golgi organelles. These components are then packaged in lamellar bodies for storage and subsequently released on-to the alveolar surface. Considerable work remains in order to define factors controlling synthesis, release, and turnover of surfactant.
Disorders of Surfactant Synthesis
Shortly after appreciation of the importance of surfactant in respiratory physiology, surfactant deficiency was found in lungs of infants with respiratory distress syndrome (RDS). Investigations into possible mechanisms have resulted in increased understanding of the development and maturation of the surfactant system. One key finding is that the lung surfactant system matures relatively late in the course of fetal development. In the monkey (gestation period 170 days), significant amounts of lecithin are not present in the alveolar lining material until gestation is approximately 80 % complete. Appearance of lecithin on the alveolar surface correlates with a sharp increase of lecithin in the lung hemogenate and the presence of lamellar bodies in the type II alveolar cells. Since respiratory distress syndrome in the newborn is associated with prematurity, the concept has arisen that RDS represents birth of the fetus before complete maturation of the lung surfactant system. Recently, several groups have investigated potential mechanisms to accelerate the normal rate of maturation of the surfactant system. One promising method is administration of adrenocorticosteroid hormones which do appear to accelerate maturation and protect infants delivered prematurely against development of RDA. Several of the enzymes in the pathways of phospholipid synthesis undergo induction with steroid treatment which might account for the accelerated maturation. This area is under active investigation at the moment and promises great potential benefit in terms of the prevention of a common cause of infant mortality.
Metabolism of Hormones and Xenobiotics
Another important role for the lung that has recently been extensively investigated is concerned with the metabolism of xenobiotics and endogenous hormones. Through these pathways, the lungs are able to exert a major influence on bodily homeostasis. As one example of hormone metabolism, lungs rapidly clear serotonin (5-HT) from blood or other media perfusing the pulmonary circulation. This amine can be taken up against a concentration gradient by a mechanism that shows saturation kinetics, is dependent on external sodium, and can be blocked by specific competitive inhibitors, inhibitors of oxidative metabolism. After uptake, serotonin is metabolized by a monoamine oxidase (MAO) to 5-hydroxy-indole-acetic acid. The rate of metabolism does not significantly influence the rate of uptake oxidase (MAO) to 5-hydroxy-indole-acetic acid. The rate of metabolism does not significantly influence the rate of uptake. These characteristics suggest that uptake of serotonin by the lung occurs by active transport and that transport is the rate limiting step in serotonin clearance. Autoradiographic studies have demonexclusively into the pulmonary endothelium. The mechanism for amine transport shows specificity, since histamine, another vasoactive compound, is not taken up nor metabolized by lung to any significant extent. As another example of
specificity, norepinephrine is taken up and metabolized whereas epinephrine is not.
Uptake and metabolism is only one mechanism by which the lung transforms vasoactive compounds. An additional pattern is operative with respect to conversion of angiotensin I to angiotensin II (resulting in formation of a potent vasoactive compound) or hydrolysis of peptide bonds in brandykinin (resulting in a loss of biological activity). The responsible peptidase enzyme (converting enzyme) is either the same for both reactions or represents a group of closely related enzymes. Converting enzyme activity is present on the luminal surface of the pulmonary endothelium, so that hydrolysis occurs at the membrane surface and active transport into the cell is not required. A more complex relationship can be seen with several of the prostaglandins since the lung is an important site for uptake and metabolism as well as synthesis, storage, and release.
In addition to contact with endogenous agents, the lung is exposed to a wide variety of potentially toxic substances delivered either through in-halation or via the circulation. A major pathway for detoxification of foreign components such as
drugs is through hydroxylation in the liver by cytochrome P-450-linked reactions. Recently, this pathway has also been demonstrated in isolated lung microsomes. These organelles from lung contain, on a weight basis, approximately 25 % of the
cytochrome P-450 activity of microsomes isolated from liver.
Sputum is a pathological secret that is formed in the case of respiratory diseases and lung and released during coughing or spitting. In healthy people, excluding smokers, singers and teachers, sputum is not released. Sputum is not homogeneous. It consists of mucus (secret of mucosa of the respiratory tract), pus, blood, serous fluid, fibrin. All these elements can be present in sputum or only some of them.
Composition and properties of sputum depend on the nature of the pathological process in the respiratory organs.
Laboratory investigation of sputum has great diagnostic value to detect inflammatory and destructive processes in the lungs and airways, as well as to identify the pathogen agent of diseases ( in case of pulmonary tuberculosis). Laboratory investigation of sputum helps to determine the degree of pathological process and its severity.
Rules of sputum collection
Very often sputum is collected for the overall clinical investigation, investigation under the microscope for its morphology. Often sputum is examined for detection of mycobacterium tuberculosis.
In the hospital sputum is collected and examined often. In ambulatory patients with chronic diseases of the respiratory tract sputum should be examined periodically, at least 1-2 times a month. Investigation on mycobacterium tuberculosis also is performed periodically. In clinical analysis should be sent fresh material, collected in a clean glass dish that prevents projects its oxidation and drying. Patients prefer to gather sputum in individual pocket spittoon graduated with dark glass, which covers twist. This dish is easy to clean and disinfect, it is convenient for both the patient and for sending material in the laboratory.
Sputum is collected in the morning before eating. In the nasopharynx and oral cavity saliva and nasal secret are mixed with sputum so that interfere with the investigation. To prevent these before collection of sputum the patient should thoroughly rinse the mouth and pharynx with boiled water. During collection of sputum, the patient should not pollute the outer edges and walls of glass dish.
Preferably as soon as possible investigate the sputum, which is collected, if there is no such possibility, sputum should keep in refrigerator or a cool place.
Often sputum is infected material, so after investigation to decontaminate it, filling the 5 % solution chloramine, or 5 % solution of carbolic acid and kept at least 4 hours. Laboratory dishes, spittoons, work place also are disinfected with the 5 % solution of chloramine. Metal spatulas and needles are disinfected over the burner flame.
Clinical sputum analysis includes the study of physical properties, microscopic and bacterioscopic investigation. Chemical investigation of is of little diagnostic value, usually limited by microchemical reaction to hemosyderin.
Physical properties of sputum
In laboratory investigated sputum is transfered in a petri dish, on black and white background and physical properties are determined.
Amount of sputum can vary from small (2-5 ml), for example in case of acute bronchitis, bronchial asthma, the catarrh of the upper respiratory tract, to a rather large (200 - 300 ml or more), which is the most typical for diseases accompanied by the formation of cavities in the respiratory organs (abscess, gangrene, bronchiectasis).
Amount of sputum is determined in a glass graduated container. If the collected sputum leave to stand, then after a while it can be distributed in layers. In the case of bronchiectasis, gangrene of lung, rot bronchitis sputum is divided into three layers: upper - foam (mucus), medium - serous (with opalescence) and bottom - crumbly, close-grained (pus). In patients with pulmonary tuberculosis lower layer of sputum is clotted, because consists of large clots covered with mucus. In the case of lung abscess sputum during standing is divided into two layers: the upper - serous and lower - purulent, yellow color.
a. Two-layers sputum: the upper – serous layer and bottom – pus.
b. Three-layers sputum: upper – colorless foam (mucus), medium - serous fluid and bottom - pus
Smell. Fresh sputum is odorless. Rot smell is typical of an abscess, gangrene of lung, as well as rot bronchitis, when rot flora is joined to sputum. Evil-smelling odor of sputum can be in patient with the decomposition of lung tissue (cancer).
The character of sputum is determined by its composition:
1) Mucus sputum - a colorless, vitreous, viscous - released in the early stages of bronchitis and bronchial asthma;
2) Purulent sputum - without admixture of mucus occurs very rarely, for example, when empyema breaks in the lumen of the bronchus or in patients with bronchiectasis;
3) Mucous-purulent or purulent-mucous sputum occurs in case of most inflammation in the lungs, bronchi and trachea, cloudy viscous mass, which closely mixed pus and mucus;
4) Bloody sputum contains blood clots, released in case of pulmonary tuberculosis, tumors (cancer lungs).
5) Serous sputum - liquid, foamy - often is observed during pulmonary edema.
The color depends on the nature of sputum. Gray or grayish-yellow is observed in mucous-purulent, yellowish-gray - in purulent-mucous sputum. Yellow is asthmatic sputum through accumulation of eosinophils in its clots. Bloody sputum is red, brown, rusty or crimson. Brown (chocolate) or brown sputum becomes due to destruction of hemosiderin. Presense of bilirubin can dye it in yellow or green color. Black color - whether caused by coal dust.
Consistence of sputum depends on its nature and can be:
1. Viscous - the presence of mucus;
2. Sticky or moderately sticky - the presence of pus;
3. Liquid - the presence of blood or serous fluid;
4. Gelatinous - the presence of fibrin and mucus.
Viscous mucus is found in the case of lobar pneumonia, viscous - in case of chronic bronchitis, bronchopneumonia, bronchiectasis, lung abscess, liquid - in case of pulmonary hemorrhage, edema of the lungs, gelatinous - in case of allergic bronchitis, bronchial asthma.
Form. There is sputum grainy, clotty and piece-like. Normally, mucus is clotty (clots of mucus) piece-like or mixed form. Clotted- piece-like form - the characteristic feature of severe pneumonia with destruction or lung cancer when mucus contained pieces of lung tissue. In case of enhanced desquamation of the epithelium of the alveoli in pneumonia, when the sputum contains a large number of casts of the alveoli, sputum has granular (grainy) form.
Pathological components of sputum that are visible with the naked eye:
Kurshman’ spirals- whitish, winding, spiral –like mucous formations, sharply differentiated from other mucous sputum, have diagnostic value in case of bronchial asthma;
Fibrinous clots - look like whitish- red balls, elastic consistence, consist of mucus and fibrin. Occur in sputum in case of fiber bronchitis, lobar pneumonia;
Rice-like bodies (Koch lenses) – not transparent tight greenish-white formation with cheesy consistency, there size like head of pins. They are formed in caverns and contain products of lipid decomposition, detritus, fiber, cholesterol crystals and a large number of mycobacteria tuberculosis. They are found in sputum in case of cavernous tuberculosis of lungs;
Ditryh Tubes - purulent clots of whitish-gray or yellow-gray color size from pinhead to a bean with sharp odor. They consists of products of cell decomposition, detritus, crystals of fatty acids and bacteria. They are found in sputum in case of bronchiectasis, lung gangrene;
Necrotic pieces of lung tissue can appear in the sputum during the destructive processes in lung tissue (gangrene of lung and lung abscess). They have black co lor, various size;
Pieces of lung tumors are found in sputum in case of tumors in the lungs and bronchi. They look like, grayish, brown or bloody formations of different size, often not larger than the pin head;
In the sputum also it is possible to find eggs and larvae of worms, hooks of ehinokok and scraps of his shell.
Foreign bodies usually are found in sputum accidently. They can include: seeds, berries, cereal ears, coins, needles, etc. Foreign bodies may be in the bronchi during years. Sometimes there is inflammation around the foreign body.
Microscopic examination: morphology of the sputum elements
For microscopic examination of sputum, first of all native preparations are used. Usefulness of investigation depends on the correct preparing and number of revised native preparations. To prepare native preparations (at least two or three) all suspicious pieces and formations, other than mucus and impurities are selected from sputum and transferred on the a glass slide, covered with a coverage lenses. Preparation has to be thin and not go beyond covering lenses.
Elements of sputum microscopy are divided into four groups: cellular, fibrous, crystalline and flora.
Leukocytes - round cells, the size of 10-15 microns, grainy - neutrophils. They are constantly detected in sputum, in mucous - single in the field of view, in purulent or mucous-purulent - completely cover the field of view.
Eosinophils - round cells, darker colored than neutrophils, have dense cytoplasm, with granules. They are constantly detected in sputum in case of bronchial asthma and other allergic diseases.
Red blood cells - round cells, greenish-yellow color, there diameter is smaller than leukocytes, grain free. Isolated red blood cells are constantly detected in sputum. The presence of a large number of RBC is characteristic of bloody sputum. Under the influence of rot processes red blood cells are destroyed.
Flat epithelium looks like flat colorless cells, 10 times greater than the leukocytes; has a round or polygonal shape and a small round nucleus located in the centre of the cells. This is desquamated epithelium of the mucous membranes of the oral cavity and nasopharynx. Some squamous cells as always detected in the sputum, in large numbers - if sputum is mixed with saliva, so there is no diagnostic value.
Cylindrical ciliary epithelium looks like elongated cells expanded at one end and narrow at the other (form of wineglass). At the expanded end of the cells are placed cilia, and in the narrower end - oval nucleus. These cells are placed in groups. In the fresh material can be seen active movement of cilia. Very often cylindrical epithelium modifies: losing cilia, changing the shape of cells (becoming triangular or round).
The detection in sputum ciliary cylindrical epithelium, which lining the mucosa of the trachea and bronchi, shows damage of these organs (asthma, bronchitis, tracheitis, etc.).
Alveolar macrophages - cells of reticulo-histiotsytic origin, freely moving to the place of inflammation and are capable of phagocytosis. They have oval or round shape, size, twice or three times larger than white blood cells, the nucleus is located eccentrically bean-like or round form, in the cytoplasm are inclusions (dark brown particles). Macrophages absorb dust, leukocytes, erythrocytes, bacteria, etc. In the case of chronic inflammatory processes macrophages often undergo fatty degeneration. Cytoplasm of such cells filled with fat droplets (granular balls).
Alveolar macrophages in sputum occur as clusters in patients with pneumonia, bronchitis, professional lung diseases and in smokers. Macrophages also are detected in sputum of patients with cancer, actinomycosis, pulmonary tuberculosis.
Syderophages - are alveolar macrophages containing hemosiderin in the form of golden-yellow color inclusions. The old name of these cells - "cells of heart defects," as they appear in the sputum in case of stasis in the lungs, which is typical for heart disease, myocardial infarction. To differentiate syderophages from alveolar macrophages containing dust particles and nicotine, should make the reaction to Prussian blue, which is positive in syderophages (hemosiderin contained in syderophages is painted in blue or blue-green color).
Tumor atypical cells are found in sputum in case of tumors in the lungs and bronchi. Features of malignancy cells are their size polymorphism, violation of nuclear-cytoplasmic ratio in the direction of increasing the nucleus, the presence of hyperchromatic nuclei, changing shape of the nucleus, the presence in it nucleoli of irregular shape, mitosis of the cells. These cells are arranged separately or as clusters (complexes) with no clear boundaries. Polymorphism of the cells size and nuclei shape, randomly placing the cells in the complex - is characteristic feature of malignancy.
Elastic fibers are elements of the connective tissue, they are detected in sputum in case of pathological processes such as destroying of lung tissue (lung abscess, tuberculosis, cancer, etc.). Identification of elastic fibers is important for differentiating lung abscess from lung gangrene. As with cavernous tuberculosis, and for lung abscess in sputum elastic fibers are present; in the case of gangrene elastic fibers are absent due to rapid destruction.
Elastic fibers in the native preparation look like shiny, double, fibrous strands that sometimes are packed in bunches, often they follow the structure of the alveoli. In the case of pulmonary tuberculosis in clusters and fragments of elastic fibers Mycobacterium tuberculosis is detected. In the case of lung abscess elastic fibers look like clusters among the pus together with hematoidyn crystals.
Coral-like fibers in sputum are present in case of chronic lung diseases such as cavernous tuberculosis. In the cavernous cavity elastic fibers are covered with fatty acids and alkalis and became coarser, like sea coral.
Calx fiber - this elastic fibers are impregnated with calx salts. They lose their elasticity, become fragile, brittle and take the form of dotted lines, that composed of separate, gray rods that refract light. They are found in sputum in the amorphous mass of calx salt and drops of fat, which indicates the presence in the lungs calx caseous decomposition that is characteristic for pulmonary tuberculosis.
Ehrlich Tetrada – these are elements, which get into a sputum from calx primary tuberculous center. The composition of Ehrlich tetrady includes four elements:
1) Calx elastic fibers
2) Calx detritus
4) Mycobacterium tuberculosis.
Kurshman’ spirals- whitish, winding, spiral –like mucous formations, sharply differentiated from other mucous sputum, have diagnostic value in case of bronchial asthma
Charcot-Leyden crystals have the form of colorless, transparent, elongated rhombus of various sizes. Typically, they are detected in sputum containing eosinophils. The formation of Charcot-Leyden crystals is associated with the break down of eosinophils, that’s why very often fresh sputum does not contain these crystals, they formed it in 24-48 hours. They are detected in sputum in patients with bronchial asthma, eosinophilic bronchitis, worms invasion of lungs.
Cholesterol crystals have the form of colorless rectangular with cut corners; situated separately or superimposed on each other. They are form due to the collapse of fatty-rebirth cells, staing sputum in the cavities and are placed on the detritus. They are detected in sputum in patients with purulent processes in the lung (abscess), tuberculosis, tumors.
Micropreparation of sputum. Flat epithelium, cylindrical epithelium, WBC.
Micropreparation of sputum. Alveolar macrophages, flat epithelium, cylindrical epithelium WBC
Micropreparation of sputum. Alveolar macrophages, which contain in the cytoplasm inclusions of hemosiderin dark-blue color
Micropreparation of sputum. Alveolar macrophage
Microflora. In stained preparations of sputum it is possible to find various microorganisms, which in small quantities always are present in the airways of healthy humans. In particularly adverse conditions (cooling, decreased resistance, this flora becomes pathogenic and causes disease. The most important diagnostic significance has detection Mycobacterium tuberculosis in sputum, which is agent of pulmonary tuberculosis. Mycobacterium tuberculosis - acid and alcohol resistant microorganisms. Due to Tsile-Nielsen Mycobacterium tuberculosis colored in red color (thin, slightly curved rods of different lengths, placed together or separately), and all other organisms have blue color.
In the sputum it is possible to detect these microorganisms: streptococci, staphylococci, pneumococci, Klebsiella in Gram stained preparations. Bacteria that are colored by Gram stain, are called gram-positive (blue) and those that are not painted - Gram-negative (red). Streptokoky, staphylococci, diphtheritic rod and so on are gram-positive. Klebsiella, typhoid rods, catarrhal micrococcuses et al. - gram-negative.
DIAGNOSTIC SIGNIFICANCE OF SPUTUM INVESTIGATION
Acute bronchitis. At the onset of the disease is excreted small amount of mucous, viscous sputum. Further increasing the amount of sputum. It becomes mucous-purulent. During microscopic studies is detected a lot of cylindrical epithelium, leukocytes, sometimes - red blood cells.
Chronic bronchitis. As a rule, is excreted a lot of mucous-purulent sputum, often with streaks of blood. Microscopically is detected a large number of leukocytes, erythrocytes, cylindrical epithelium, alveolar macrophages, many different microorganisms.
Micropreparation of sputum. Inflammation. Alveolar macrophages, leukocytes.
Bronchial asthma. It is excreted a small amount of mucous, viscous, glassy - like sputum. Kurshman’ spirals can be detected macroscopically. Microscopic studies is particularly characterized by detection of eosinophils and cylindrical epithelium. Charcot-Leyden crystals also are present.
Micropreparation of sputum of patient with Bronchial asthma. Kurshman’ spirals, eosinophils, Charcot-Leyden crystals.
Bronchiectasis. It is excreted a lot of purulent sputum (in the
Lobar pneumonia. At the onset of disease small amount of viscous rusty sputum is excreted. As the disease progresses sputum is secreted more, it becomes mucous-purulent. Rusty sputum frequently contains clots of fibrin and altered blood, which gives it a brown color. Microscopically red blood cells, grains of hemosyderin, crystals of hematoydin, a small amount of white blood cells, many pneumococci are identified et the beginning of disease. At the end of the disease amount of leukocytes increases and red blood cells decreases, a lot of alveolar macrophages are identified.
Lung abscess. At the time of breakthrough abscess in bronchus is excreted a lot of sputum (600 ml). After settling the sputum becomes two-layer. Microscopically it is possible to find many white blood cells, elastic fibers, scraps of lung tissue, crystals of fatty acids and cholesterol, different flora.
of the lungs. The
amount of sputum depends on the stage of the disease. In the presence of
cavities in the lungs amount of sputum can be significant. Sputum is
mucous-purulent, often mixed with blood. Macroscopically in sputum can be
detected rice-like bodies (Koch lenses),
consisting of the elements of decomposed lung tissue. Under the microscope
elastic fibers, crystals of fatty acids, hematoydin are identified. Ehrlich Tetrada – these are elements,
which get into a sputum from calx primary tuberculous center. The composition
of Ehrlich tetrady includes four elements: 1) Calx elastic fibers, 2) Calx
Lung cancer. The amount of sputum may be different. In case of breakup of tumors - is significant. It is mucous-purulent-bloody. During macroscopic study can be found fragments of lung tissue. Microscopically atypical cells and their complexes are detected.
Micropreparation of sputum of patient with lung cancer. Tumor cells.
Micropreparation of sputum of patient with lung cancer. Tumor cells.
The arrow shows the TB cavity in the upper part of the right lung.
People can have TB even if the sputum culture results come back as negative.
Active tuberculosis is an infection with Mycobacterium tuberculosis which can be transmitted to other people. Usually this is an infection of the lungs (pulmonary tuberculosis), it can be spread through droplets while coughing and sneezing.
The diagnosic tests for TB, as for all diseases, have to follow two important criteria of quality: sensitivity and specificity.
Sensitivity means that a test needs to detect all people with a disease. For example, if 100 people are being tested, and 50 of them have TB, then a good test should find all 50 people. In this case, health workers would speak of a test with a 'high sensitivity'.
Specificity means that a test only detects people with a disease, and does not wrongly detect a disease in somebody healthy. If of the 100 people tested for TB, 50 people are healthy, then a test with a good specificity would correctly identify the 50 healthy people and show 50 negative results.
No diagnostic test is ever completely foolproof – if thousands and thousands of people are tested, mistakes happen. A good diagnostic test is one with a high sensitivity (detects people with TB) and also a high specificity (correctly identifies healthy people without TB). Sensitivity and specificity are expressed in percentages. Good tests should have a sensitivity and a specificity at least above 90%. For example a TB diagnostic with a sensitivity of 95% and a specificity of 99% would correctly identify 95 out of 100 people who have TB, but it would also incorrectly identify one of out 100 people who do not have TB.
The first step to detecting TB infection, after taking a medical history and doing a physical exam, is to do a chest x-ray. This allows the health worker to examine the lungs of the person with suspected TB. On a chest x-ray from someone with TB you can often see the cavitation that the TB bacteria form in the lung tissue. The picture here shows a chest x-ray, and the arrow points to a TB cavity in the right upper part of the lung (in an x-ray, the right and left side are reversed).
TB cavities are often in these upper parts of the lung, also known as the apex (apex is latin for 'the highest point'). Chest x-rays have poor specificity. If a health worker sees something on the lung, it can also be a lot of other things, and is not necessarily TB. Therefore chest x-rays are usually an indicator of whether or not a person might have TB, but they cannot confirm the diagnosis. They are used to confirm a suspicion, and will always be followed by tests that aim at finding the TB bacterium. The diagnosis of TB cannot be made by a chest x-ray alone. It can also not be excluded by a chest x-ray.
Instead,the diagnosis of active tuberculosis means finding the bacterium in a sample of bodily fluid from the patient. Where the bacterium is found depends on where the infection takes place. In most cases, Mycobacterium tuberculosis infects the lungs (see 'Pulmonary Tuberculosis'). If that is the case, the bacterium can be found in sputum. Sputum is a very thick bodily fluid (also called mucus), which comes from the lower airways. It is thicker than saliva and is usually coughed up.
A sputum sample. Source NHS.
If a person has difficulty coughing up sputum, it can be induced by inhaling saline air through a mask., called a nebulizer. This is often done to help children cough up sputum.
There are two ways to test sputum for Mycobacterium tuberculosis: with a sputum smear and with a sputum culture. In both tests, the aim is to find Mycobacterium tuberculosis through colouring it in the sputum.
Usually bacteria are identified by adding colouring agents to the laboratory surface on which they grow. This process is called ‘staining’. Different bacteria have different ways of responding to staining agents, and this allows scientists to differentiate between them. Because of its thick cell wall consisting of mycolic acids, Mycobacterium tuberculosis does not respond very well to most staining agents. In fact, it is very difficult to stain any mycobacterium.
For sputum smears, the TB bacterium is stained with an agent that binds to the acids on the cell wall. This is typically done using the Ziehl-Neelson method, which is named after the two scientists who described it in the late 1800s. It is also called an acid-fast stain. However, sputum smears often do not detect TB especially in people who have advanced HIV disease. Consequently sputum smear microscopy has a poor sensitivity; it misses many cases of people with active TB.
In one large study, sensitivity was only 53%. In other words TB was not detected in about half of patients who really had TB. This is very poor. In addition, this method requires much skill. But sputum microscopy is widely used, because it is one of the cheapest and quickest ways to diagnose TB. Newer flourescent microscopes have improved the sensitivity of sputum microscopy but not nearly enough.
Sputum Culture: the slow, expensive and not very good gold standard in TB diagnostics
For sputum cultures, the sputum is added to a special surface in a laboratory, under circumstances that will encourage the TB bacterium to grow. Lab technicians then check whether or not the bacterium grows. Because TB bacteria grow very slowly, this often takes 3 or 4 weeks, sometimes even more. If there are TB bacteria in the sputum, then they will start growing in round clusters, and the culture is deemed positive. A positive culture is proof for an infection with Mycobacterium tuberculosis, so it is proof that somebody has active TB.
Compared to sputum smears, sputum cultures have a much higher sensitivity, but it is still not high enough (only 82% in a large study). This means that unfortunately, sputum cultures often do not pick up every person with TB, and people can have TB even if the sputum culture results come back negative.
If a patient with presumed pulmonary tuberculosis cannot cough up sputum, there are other ways to get body fluids that contain Mycobacterium tuberculosis. Health workers can do gastric washings, a so-called laryngeal swap or get a sample through a bronchoscopy. For a gastric washing, the person with presumed TB has to swallow a tube through which the health workers sucks up a little bit of what is in the person's stomach. This sample is then examined either as a smear, or put into a culture. Gastric washings usually have to be done at least twice to gain enough for a sample. In South Africa, they are done twice on different days. Children often need to have gastric washings done to diagnose TB.
Recently, a new test has been developed to diagnose active TB. It is called the GeneXpert, and also uses sputum samples. If the sample contains TB bacterium, it multiplies its DNA (the “genes” of the bacterium, DNA is short for desoxyrubinucleic acid) through a method called PCR (polymerase chain reaction). This allows it to detect TB bacterium very reliably, and also very fast – it only takes about 2 hours for the test to come to a result. So far, it has had good sensitivity and specificity, and it can also test if the TB bacterium is resistant to one of the TB drugs, rifampicin. However, the GeneXpert machine is not widely available yet.