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Saliva: new interest in a nontraditional specimen.

The technology exists to perform a wide range of tests, including HIV detection, using this safe, easily obtained diagnostic medium.

SALIVA--long overlooked as a multipurpose diagnostic fluid--is gaining popularity and attention from laboratorians accustomed to gleaning data from other diverse body fluids including cerebrospinal, synovial, pericardial, pleural fluids and ascites, sweat, semen, vaginal secretions, and even tears.

Several basic questions are likely to be asked by the laboratorian about the practical aspects of saliva assessment. What makes saliva attractive for use as a diagnostic specimen by the clinical laboratory? How will patients react to a request to spend up to five minutes, for example, producing a saliva specimen? What analytes are detectable in saliva? Can currently available technologies and procedures used with serum, plasma, or urine be modified easily for use with saliva? What types of collection devices are used to obtain the specimen? What volume needs to be collected for analysis?

* Constituents. Whole saliva is a mixture of salivary gland secretion, gingival fluid, cellular debris, and microorganisms of the oral cavity. Gingival fluid, a serum transudate present in the mouth, is considered by some to be the main source of salivary immunoglobulin G (IgG), which is especially useful in HIV testing.

* Low concentrations. The volume of saliva produced daily averages between 1 and 1.5 L, over 99% of which is water. Analytes present include those produced by the salivary glands and those transferred from plasma.

Although most molecules found in blood and urine are also present in saliva, they exist in substantially lower concentrations, as little as one tenth to one thousandth the blood concentration.|1~ Therefore, detecting these substances requires extremely sensitive assays.

Enzymes found in saliva include amylases, cellulases, and proteases secreted by the body and by oral microorganisms. The pretreatment of saliva with enzyme inhibitors may be required to prevent the degradation of the target analyte and to reduce possible interference in subsequent assay procedures. Enzyme immunoassays (EIAs) are especially susceptible to interference.

* Elevated pH. An acidic fluid, saliva normally has a pH of 6.5 to 7.2, although the pH of "resting saliva" can be as low as 5.9. A variety of factors influence salivary pH. Psychological factors such as anxiety can lower it. Enhanced flow rate has the opposite effect, with salivary pH usually rising to and leveling off between 7.4 and 7.6, thought it may climb to 7.8.|2~

* Stimulation. Chemicals such as citric acid can be used to stimulate salivary flow. Mechanical stimulation of the mouth wall can trigger impulses from touch receptors. Smooth objects in the mouth enhance salivation while rough objects inhibit it. Collection devices that are smooth to the touch or that contain or are coated with citric acid will increase salivation when held in the mouth.

* Component migration. Substances enter saliva via four basic routes:|2,3~

Passive diffusion. An analyte may move from the plasma through the cells of a salivary gland and into the saliva by passive intracellular diffusion. The component's molecular weight, lipid solubility, and degree of ionization influence transfer across the lipid bilayer of the cell membrane. By affecting ionization and therefore net charge, salivary pH directly influences the transfer of material across cell membranes. The distribution of acidic and basic drugs between the saliva and blood is pH dependent.|2,4~

The saliva-to-plasma distribution ratio of electrically neutral compounds is essentially pH independent. Currently, tremendous clinical interest is focused on lipophilic, non-ionized substances that enter saliva by this intracellular route. For lipid-soluble, unconjugated steroids such as cortisol, estriol, testosterone, and progesterone, salivary concentration closely reflects plasma concentration.|5~

Active transport. Some analytes enter saliva by active transport. The outcome of this energy-consuming process depends on the overall physiologic status of the organism and the plasma concentration of the analyte.|2~

Ultrafiltration. The most common mode of extracellular transport of analytes is ultrafiltration through the tight junctions between gland cells. Lipid-insoluble materials appear to enter saliva by this mechanism. For such substances, salivary concentrations are highly dependent upon the rate of filtration and therefore do not necessarily parallel plasma concentrations.|2,3~

Leakage or rupture. Material may enter saliva when damaged cells either leak or rupture. Even in a healthy population, blood components are found in 20% to 50% of the saliva specimens that are collected. Some drugs, such as phenytoin, cause gingivitis (redness, swelling, and bleeding of the gums). This condition increases the likelihood that blood components will enter the saliva.

The mechanism by which HIV antibodies enter saliva is transudation from the serum via gingival fluid, but some additional, not yet understood transport mechanism also appears to be involved in the process.|6~

* Salivary advantages. Saliva is a natural ultrafiltrate of plasma.|4~ It is an outstanding diagnostic medium for the evaluation of free, physiologically active substances present in the circulation (drugs and hormones, for example). In addition, it is used worldwide for the detection of antibodies to HIV and hepatitis A, B, and C. Furthermore, saliva has the potential to be used for the detection of antibodies to the Epstein-Barr virus as well as the infectious agents responsible for rubella and Lyme disease.|7,8~

Saliva is easy and inexpensive to collect, ship, and store. The fact that it presents a low risk of infection to the laboratory professional handling it makes it an extremely attractive alternative to blood or urine. In addition, the specimen may be analyzed using already available serum or urine assay techniques with only minor procedural adaptations. The necessity to increase specimen volume and correct for matrix effects are examples of the changes necessary.

Analyte detection and quantitation are possible using various widely available methods (immunoassay, TLC, HPLC, and GC/MS, for example) that are sensitive enough to detect the low concentrations of analytes in saliva. Both conventional and rapid EIA techniques have been used for HIV screening on saliva specimens.

* The HIV question. Infectious HIV is seldom found in saliva.|9~ In fact, salivary mucins may play a role in inhibiting the infectivity of HIV-1.|10~ The route by which the virus enters saliva is not entirely clear; however, transmission of HIV from one person to another by way of that medium is believed to be extremely unlikely.|11~

Saliva is particularly useful as a specimen in HIV surveillance studies and field research. With it you can avoid the problems associated with collecting blood among mobile street-based populations and geographically remote communities. In fact, the convenience of saliva testing could make it the preferred method of insurance screening for HIV.

* Convenience. Specimen collection can take place at home or in the workplace at almost any time. Both patient privacy and confidentiality are easily maintained.

Multiple specimens can be readily collected over any desired time interval. Such temporal flexibility is essential to chronobiologic, pharmacokinetic, and endocrine function studies, as well as to compliance monitoring in forensic and therapeutic situations.

Specimen adulteration is difficult, and disposal presents no problem since, unless the saliva is contaminated with blood, it is not an infectious waste.

Certain patients, such as some athletes and astronauts, are often anxious or fearful about diminished performance as a possible consequence of needlesticks or sharp injury. The use of saliva eliminates this fear and offers a painless alternative for any patient on whom phlebotomy is difficult or inadvisable (pediatric, geriatric, and obese patients, for example).

* Diagnostic capabilities. Saliva-based diagnostic testing currently encompasses detection and monitoring of viral antigens and antibodies; evaluation of hormone levels; monitoring of therapeutic drugs; testing for drugs commonly abused; monitoring compliance; and screening for certain cancers. A cursory literature search yields a fascinating diversity of reports on salivary diagnostics (Figure 1).

With so many advantages offered by the use of saliva as a diagnostic medium, why are we not further along in its clinical utilization? Two explanations are often offered:

Inertia. The medical community is accustomed to reference ranges established for assays of blood and urine. Some physicians resist implementing routine analysis of a body fluid with substantially different analyte reference ranges.

Memories. Equally plausible is the suggestion that a prejudice against the use of saliva exists among laboratory workers due to unpleasant memories of old collection and processing methods, which required patients to spit into containers, among other equally distasteful techniques.

To encourage salivation, patients were often asked to chew on rubber bands, small blocks of paraffin, or other substances. A solution of citric acid placed on the tongue also served to stimulate salivary flow. Following collection, the saliva was frozen, thawed, and centrifuged to remove mucus and particles and leave a clear supernatant.

Opportunities for error abounded. Sources of error included pipetting inaccuracies due to the specimen's high viscosity, frothing of the saliva as it was forcefully expelled from the mouth, nonspecific binding of the analyte to the mechanical stimulant, and adherence of the analyte to salivary sediment following centrifugation. The specimens were often contaminated with blood proteins that readily bind to drugs and make an accurate determination of their concentration in saliva difficult.|12~

* Sampling procedures. Stability without refrigeration plus the convenience of multiple sampling makes saliva an ideal specimen.|13~ New collection devices are available that provide a sterile, high-quality specimen while working quickly, safely, and in a manner that is inoffensive to both patient and technologist. These patented, noninvasive instruments enhance the clinical usefulness of saliva for analyte detection.

No matter what device is used, patients should avoid food and fluid (except water) for at least one hour before collection to minimize contamination and discoloration. Enzymes, inhibitors, and binding proteins in foods can interfere with immunoassays used for the detection of analytes.

Following are descriptions of four patented saliva sampling systems in varying stages of FDA approval and availability.

* Oral diffusion. Instrument A is an oral diffusion sink (ODS) sample that permits collection of an ultrafiltrate of saliva directly in the mouth.|12~

The device consists of a semipermeable membrane (either cellulose or a synthetic copolymer) surrounding an osmotically active substance such as granular sucrose. The ODS is essentially a soft, smooth, pliable plastic pouch about the size of a quarter. Placed in the mouth and gently agitated with the tongue, the disc collects about 1 ml of liquid in five minutes. Upon removal from the pouch by syringe, the clear, non-viscous ultrafiltrate is ready for use.

No centrifugation is required. Because substances with a molecular weight greater than 12,000 daltons cannot cross the membrane, the ODS excludes particulate matter and most proteins. Blood contamination of specimens therefore has no effect on the contents of the device. Drugs or hormones that are associated with proteins cannot diffuse into it.

Only the free form of the analyte can accumulate within the collector. The disc can be sterilized by irradiation prior to its use and, since microorganisms cannot cross the membrane, the specimen collected is a sterile one that will not deteriorate in storage or during shipment, even at room temperature. Ongoing research in ODS technology suggests that these devices are suitable for sampling saliva for corticosteroids, progesterone, estradiol, and testosterone.|14~

* Oral antibody sampling. A relatively new toothbrush-like oral antibody collection device consists of a fibrous pad, impregnated with salts and preservatives in a buffering system, attached to a stick.

After vigorous stroking of the inside of the cheek, the device is placed between the lower cheek and gums, where it draws mucosal transudate from the tissues. This fluid is highly likely to contain anti-HIV IgG antibodies in seropositive individuals.|8~

The pad is then placed in a vial containing a liquid preservative that is absorbed by the pad. The stick is removed from the pad. The vial is then transferred to a centrifuge tube for centrifugation in order to elute the specimen from the pad. A standard EIA is carried out on the eluate.

* Collection system. Figure 4 shows a system that includes a collection device, a solution for stabilizing the specimen, and a transport tube for delivering the sample to the laboratory.

The paddle-shaped sterile cotton pad is placed under the tongue for two minutes. Specimen adequacy is indicated by a color change in the stem of the collector when the pad is saturated. The collector is sealed in the transport tube, which contains a stabilizing fluid composed of a detergent, a preservative, a proteolytic enzyme inhibitor, and an antimicrobial agent.

Once in the laboratory, vigorous agitation or vortexing dislodges the wick pad from its stem. Expression of the saliva solution from the pad with a filter separator yields a specimen of approximately 1.2 ml for testing. Conventional ELISA procedures may be used to detect anti-HIV antibodies in the filtered specimen.

Both conventional and rapid EIA techniques can be used on saliva collected with this device to determine if HIV-1 and HIV-2 antibodies are present. When saliva obtained with the sampling pad was used with a conventional rapid HIV rDNA test kit, a highly accurate, rapid, on-site AIDS test was achieved, the results of which matched both serum and saliva results obtained with the rDNA EIA. All positive results from the saliva test were confirmed by Western blot analysis.|15~

* Quantitative alcohol. After collecting specimens with the above devices, they are assayed using many existing protocols and procedures.

A new system has received final FDA clearance specifically as a quantitative saliva alcohol test. In clinical trials, alcohol levels measured with the instrument shown in Figure 5 displayed excellent correlation with whole blood alcohol levels measured by gas chromatography.|16~

The test is an enzyme-based assay in which the alcohol dehydrogenase reaction is combined with a proprietary colorimetric chemical reaction system to generate a "color bar." When saturated after about one minute of swabbing around the cheeks and gums, the swab is pressed into the entry port of a thermometer-like test instrument; saliva fills the central reaction capillary and reacts with the enzyme strip.

The height of the color bar that develops after two minutes is read on a scale as ethanol concentration measured in mg/dl. The test is available in two detection ranges: 0 to 150 mg/dl (0 to 0.15%) and 0 to 350 mg/dl (0 to 0.35%). The test version with the lower alcohol detection range is designed for use in the workplace or in counseling settings with people suspected of alcohol intoxication or those being monitored for compliance.

The higher range test is intended for patients in emergency situations and for addiction unit populations who have chronically high alcohol levels. The usefulness of noninvasive testing for alcohol, particularly in the transportation industry and the military, is readily apparent.

* Limitations. Unfamiliarity with the reference ranges of analytes in saliva is a temporary inconvenience, easily corrected with education. A more substantive issue is our still limited knowledge of the correlation of analyte concentrations in saliva, blood, and urine. For example, more study is required to determine the concentrations of drugs and their metabolites in saliva as a function of dose and time after intake.|4~

* Invaluable tool. The benefits associated with the use of saliva in clinical diagnosis--namely, the ease of collecting, handling, shipping, and storing samples--clearly outweigh the temporary limitations. The urgent need for a method of HIV surveillance that minimizes the exposure of health care workers and others to infectious materials is reason enough to accept saliva as an invaluable diagnostic specimen.

Sharon M. Miller is associate dean of the College of Professional Studies, Northern Illinois University, DeKalb, Ill.

References

1. Malamud D. Saliva as a diagnostic fluid. Br Med J. 1992; 305: 207-208.

2. Haeckel R. Saliva, an alternative specimen in clinical chemistry. J Int Fed Clin Chem. 1990; 2: 208-217.

3. Vining RF, McGinley RA, Symons RG. Hormones in saliva: Mode of entry and consequent implications for clinical interpretation. Clin Chem. 1983; 29(10): 1752-1756.

4. Schramm W, Smith RH, Craig PA. Drugs of abuse in saliva: A review. J Anal Toxicol. 1992; 16: 1-9.

5. Tunn S, Mollmann H, Barth J, et al. Simultaneous measurement of cortisol in serum and saliva after different forms of cortisol administration. Clin Chem. 1992; 38(8): 1491-1494.

6. Holstrom P, Syrjanen S, Laine P, et al. HIV antigens detected in gingival fluid. AIDS. 1992; 6(7): 738-739.

7. Major CJ, Read SE, Coates RA, et al. Comparison of saliva and blood for human immunodeficiency virus prevalence testing. J Infect Dis. 1991; 163: 699-702.

8. Thieme T, Yoshihara P, Piacenpini S, et al. Clinical evaluation of oral fluid samples for diagnosis of viral hepatitis. J Clin Microbiol. 1992; 30(5): 1076-1079.

9. Barr CE, Miller LK, Lopez MR, et al. Recovery of infectious HIV-1 from whole saliva. J Am Dent Assoc. 1992; 123(2): 36-48.

10. Archibald DW, Cole GA. In vitro inhibition of HIV-1 infectivity by human salivas. AIDS Res Hum Retroviruses. 1990; 6(12): 1425-1432.

11. Lifson AR. Do alternate modes for transmission of human immunodeficiency virus exist? JAMA. 1988; 295: 1353-1356.

12. Schramm W, Smith RH. An ultrafiltrate of saliva collected in situ as a biological sample for diagnostic evaluation. Clin Chem. 1991; 37 (1): 114-115.

13. Chen YM, Cintron NM, Whitson PA. Long-term storage of salivary cortisol samples at room temperature. Clin Chem. 1992; 38(2): 304.

14. Wade SE. Less-invasive measurement of tissue availability of hormones and drugs: Diffusion-sink sampling. Clin Chem. 1992; 38(9): 1639-1644.

15. Seymour EH, Barriga GA, Stramer SL. Use of a rapid test for the determination of salivary HIV 1/HIV 2 antibodies. Presented at the VIII International Conference on AIDS. Amsterdam, The Netherlands, July 19-24, 1992.

16. Stolz R. A quantitative, rapid test for saliva alcohol determinations. Presented at the 25th annual American Society of Addictions Medicine Conference. Boston, Mass., April 18-21, 1991.

Figure 1

Applications of saliva testing from recent research

Abused substances

* Saliva has proven to be extremely useful in screening drivers for impairment of judgment and performance associated with alcohol or drug abuse.

* Saliva is a better indicator of current or recent marijuana intoxication than a urine specimen.

* Because saliva can reveal high levels of amphetamines, it has been suggested as the specimen for diagnostic evaluation.

* The feasibility of using saliva for detection of cocaine and benzoylecgnonine with analysis by immunoassay is under study as a means of screening for recent cocaine use.

HIV

* In HIV epidemiologic studies conducted around the world, accurate prevalence rates were obtained using only saliva, as long as specimens of adequate were obtained.

* The anonymous saliva testing of prison inmate volunteers to determine HIV prevalence has been useful for institutional policy development and resource and service planning.

* Saliva is an alternative to neonatal heelstick specimens for use in monitoring the status of children born to HIV-infected mothers.

Endocrinology

* Salivary cortisol is a potential valid measure of serum cortisol changes in response to acute exercise.

* In the field of work physiology, results indicate that saliva cortisol is a useful strain indicator of short-term stress situations.

* The relationships between salivary cortisol levels and several psychological variables are being studied in cases of postpartum depression.

* Cortisol levels are determined on saliva collected during space flight to study changes in adrenal function during the body's adaptation to weightlessness.

* The circadian variation in melatonin levels among astronauts before and after space flights has been studied using saliva. The hormone, which sets the body's internal clock, acts on the parts of the brain that generate sleep and alertness rhythms.

* Salivary progesterone has been used successfully to monitor ovarian function in cases of female infertility.

* Saliva CA125 levels have been suggested as a new way to screen for malignant ovarian tumors.

* The assessment of salivary testosterone is an effective means of evaluating the success of medical or surgical orchiectomy in patients with prostatic carcinoma.

* Salivary testosterone measurements appear to be useful in research on sexual and aggressive behaviors.
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Title Annotation:saliva as a multipurpose diagnostic fluid
Author:Miller, Sharon M.
Publication:Medical Laboratory Observer
Date:Apr 1, 1993
Words:3287
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