BIOCHEMISTRY OF SALIVA. REGULATION AND PATHOLOGY OF SALIVA
SECRETION.
LABORATORY DIAGNOSTICS OF THE
DISEASES OF ORAL CAVITY AND
PERIODONT
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.
http://www.youtube.com/watch?v=HUhdVaH2xMM&feature=related
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.
http://www.youtube.com/watch?v=HrFtnFudzYw&feature=related
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:
Mandibulartori
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 Composition
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).
Salivary Dysfunction
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.
Tooth
Erosion
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.
Taste
Disorders
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.
Candidiasis
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.
SALIVA
COLLECTION
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.
SEROLOGIC
EVALUATION
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
Hereditary diseases
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.
Malignancy
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.
Infectious diseases
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).
(2b) HIV
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.
Drug Monitoring
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.
Surfactant
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
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.
Cellular elements
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.
Fiber formations
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
3)
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
Flora
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
morning to
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.
Tuberculosis
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
detritus, 3)
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.
Sputum Microscopy
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.