CLINICAL INVESTIGATION OF
SPUTUM.
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
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.
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
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
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.
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
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 =
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.