4.1. Oral Fluid Specimens
The alternative specimen receiving the most recent interest appears to be saliva or, more appropriately, oral fluid (11-15). Although saliva has been the commonly used term to describe fluid specimens from the oral cavity, this fluid, as collected by current simple swabbing or absorbent pad devices, is really a complex mixture of several different oral fluids, including saliva. Accordingly, the broader term oral fluid is preferred. Oral fluid represents a mixture of not only the saliva from the three oral salivary glands (parotid, sub-mandibular, and submaxillary), but other oral fluids as well (e.g., gingival crevicular fluid).
The first experiments to measure biological analytes in saliva were performed in the mid-19th century. Further experiments in the 1930s demonstrated the role of lipid solubility and ionizability in the partitioning of solutes into saliva. Oral fluid has been used for a wide variety of analytes, including steroids, hormones, enzymes, antibodies, DNA typing, therapeutic drugs, and drugs of abuse. From the earliest days of immunoassay development for drug testing in the early 1970s, saliva has been considered a suitable specimen. In fact, one of the first papers published on the use of homogeneous immuno-assays for the detection of drugs (a spin immunoassay developed by Syva Company, called free radical assay technique [FRAT], a forerunner of the now well-established enzyme-multiplied immunoassay technique [EMIT] assay), specified in the title the use of both urine and saliva for morphine testing (16).
The key advantage of oral fluid for drugs-of-abuse testing is the ease of specimen collection, without invoking privacy or gender concerns. Oral fluid for drug testing offers great promise for roadside driving-under-the-influence scenarios, which prove prohibitive for the collection of a urine specimen (see Chapters 8 and 17). Furthermore, there is the potential for immediate test results with on-site, noninstrumented immunoassays already developed. In addition, there has been the promise that saliva drug levels may correlate better with blood levels than urine, thereby allowing for better assessment of the level of likely impairment. Unfortunately, a close review of the literature indicates that although oral-fluid levels may correlate better with blood levels than urine drug levels, the correlation is not so strong that a clear relationship with impairment exists. Finally, the possibility of specimen adulteration appears to be minimal. Some limitations are the very low specimen volume and the low analyte levels. Although oral fluid as a specimen for drugs-of-abuse testing is receiving active research interest, oral fluid has been widely studied for use in therapeutic drug monitoring (17).
Ethanol was apparently first reported in saliva in 1875. Saliva ethanol levels have been shown to demonstrate excellent correlation with blood alcohol levels, with a saliva/blood ratio close to 1; this is why saliva as a specimen for initial alcohol testing is authorized under the Department of Transportation (DOT) program as well as under several state driving statutes (18-20). The DOT regulations detail saliva collection and testing procedures. In conjunction with the DOT rulemaking, the National Highway Traffic and Safety Administration (NHTSA) included performance evaluations of nonevidential alcohol-screening devices for saliva for use in the DOT testing program. Those devices fulfilling NHTSA's criteria are listed in their Conforming Products List (CPL), periodically published in the Federal Register (20). In addition, there is ongoing review by SAMHSA's Division of Workplace Testing of the use of saliva for federally regulated workplace testing for other drugs of abuse as well (4). There is also a program in Europe called Roadside Testing Assessment (ROSITA), which examines a variety of specimens and technologies for their suitability in roadside testing, with much attention paid to saliva (21).
Saliva is effectively an ultrafiltrate of blood. All of the organic compounds present in plasma have been detected in saliva, albeit in trace amounts for some analytes. Saliva is about 98% water, with a specific gravity of 1.00-1.02. Saliva contains both electrolytes (primarily Na, K, Cl, and HCO3) and proteins, and its osmolality is less than or equal to that of plasma. The electrolyte concentrations and pH are markedly dependent upon saliva flow rate. Accordingly, stimulating saliva flow for speed of specimen collection can alter the partitioning of drugs between blood and oral fluid and thus affect the saliva:blood ratio. The protein concentration in saliva is less than 1% of that in plasma. However, saliva has proven a suitable specimen for forensic DNA analysis as well as antibody testing for human immunodeficiency virus (HIV).
Typ ical daily saliva secretion is 500-1500 mL—average 0.6 mL/min (range 0.1-1.8; during sleep, 0.05 mL/min). Production rates for stimulated saliva have been reported to average about 2 mL/min but have reached as high as 7 mL/min. Saliva pH is typically 6.7 (5.6-7.9, flow rate dependent), vs 7.4 for plasma.
The mechanism by which drugs are found in saliva is passive diffusion, although there are examples of active secretion (e.g., lithium). The major factors affecting drug entry into saliva are lipid solubility and degree of ionization at saliva pH.
Unfortunately, oral fluid has not proven very sensitive for detection of cannabis use, as it appears that cannabinoids are not secreted from the blood into oral fluid. Rather, it is only from contamination of the oral cavity after smoking or oral ingestion of cannabis that cannabinoids may be detected in oral fluid. Accordingly, detection of cannabis use is likely only for several hours after use (22,23). However, this may prove beneficial in testing programs where the goal is to demonstrate a likelihood of impairment. If an appropriate threshold cut-off is established, then a positive result in oral fluid would clearly represent use within the past few hours, with demonstrated cognitive and psychomotor deficits.
Analysis of oral fluid for drugs is relatively straightforward. However, there are limitations in repeat and multiple confirmation tests as a result of low specimen volumes. Both on-site and laboratory-based methods have been developed (24,25).
Specimens may be collected through a variety of techniques, although simple expectoration (spitting) into plastic (polypropylene) tubes (either stimulated or unstimulated) or absorption of oral fluid with an absorbent material (foam pad, cotton fiber wad) are the most common. Spitting causes a saliva secretion rate of approx 0.5 mL/min. The flow of saliva can be stimulated through a variety of techniques, such as chewing paraffin, or through the use of chemical stimulants, such as citric acid or sour candy drops. Of course, the use of any foreign material to stimulate saliva must be carefully considered so that the specimen is not altered or contaminated in a way that might limit subsequent analyses or interpretations. Chewing paraffin will cause secretion rates of 1-3 mL/min, but paraffin may absorb highly lipophilic compounds, causing a reduction in measured saliva levels. Citric acid candies are potent stimulators, leading to secretion rates of 5-10 mL/min. Stimulated saliva appears to have a fairly narrow pH range (approx 7.4) relative to the broader range for unstimulated saliva. Again, the variability in pH may be important for the saliva/plasma ratios of weakly basic or weakly acidic compounds. The pH of saliva increases from approx 6.2 to 7.4 as the secretion rate increases. It is generally approx pH 7 for stimulated saliva, whereas unstimulated saliva shows a greater pH variation. This variation in saliva pH resulting from variations in secretion rates can have a significant impact on the saliva/plasma ratio for certain drugs, depending on their pKa.
Generally, a specimen is collected with an absorbent pad placed in the mouth for a few minutes. After the pad is saturated with oral fluid or a specific amount has been absorbed, the pad is placed in a tube of buffer for shipment to the laboratory. On-site methods may similarly collect the specimen with an absorbent pad from which the specimen is applied to a noninstrumented or instrumented immunoassay device. There is even a device that aspirates a specimen directly into a bench-top analyzer. However, drug levels in oral fluid are generally much lower than those found in urine specimens, except when there is direct contamination of the oral cavity.
Specimen handling is relatively straightforward. Saliva has been shown to be source of infectious microorganisms, and appropriate precautions should be taken in the handling of oral fluid (5). Court cases have addressed the relative infectivity of saliva when one subject has been bitten by another.
One promise of oral fluid testing is a supposed better correlation with blood levels and, accordingly, impairment. Unfortunately, a detailed review of the literature indicates that although oral fluid levels generally correlate better with blood levels than, for example, urine, the correlation is not so close as to allow a strong prediction of blood levels. This is especially so shortly after drug use by oral ingestion, smoking, or nasal insufflations, when contamination of the oral cavity by drug can lead to dramatically elevated drug levels, much greater than corresponding blood levels, at least for several hours. Another issue is that oral fluid testing is relatively insensitive for the detection of cannabis use, as it appears that cannabinoids are not secreted from the blood into oral fluid. Rather, detection of cannabis use is possible only as long as there is contamination of the oral cavity with cannabinoids. This period of detection is much shorter than the several days for detection of cannabinoids in urine. However, by choosing an appropriate cut-off, one can insure that a positive cannabis result in oral fluid can occur only within a few hours of use, and thus provides a clear indication of likely impairment.
It has been demonstrated that a very wide variety of ingested drugs and/or their metabolites may be found in hair specimens. Hair specimens from ancient mummies have been demonstrated to contain cocaine. Several famous deceased persons have also had their hair analyzed for drug exposure (Napoleon Bonaparte, Ludwig van Beethoven, William Butler Yeats) (26,27).
Hair testing has gained interest because of its ability to provide a history of drug use, dependent on the length of hair tested (see Chapter 11). Unlike other conventional biological specimens used for drug testing with detection times measured in days, drugs have been demonstrated to remain in hair for extended periods of time: years, decades, and even longer. Current hair-testing protocols examine segments of hair representing about 3 mo of growth (head hair typically grows approx 1 cm/mo). That drug residues may be detected in hair over extended periods of time has been amply demonstrated in a large number of published studies. Hair specimens examined include not only head hair, but also beard hair, axillary hair, body hair, and even pubic hair. Furthermore, even neonatal hair has been analyzed to demonstrate possible prenatal drug exposure (28).
There have been numerous national and international scientific meetings specifically addressing hair testing, with the establishment of a few professional societies dedicated to hair testing. In 1990, there was a small conference addressing this new technology convened in Washington, DC, by the Society of Forensic Toxicology, the National Institute on Drug Abuse, and the National Institute of Justice. Although most attendees were critical of hair testing, subsequent research has demonstrated its utility. The first international meeting addressing hair testing was held in Genoa in 1992 (29); an international workshop was held in 1994 in Strasbourg (30); a joint The International Association of Forensic Toxicologists (TIAFT)/Society of Forensic Tox icologists (SOFT) meeting was held in 1994 in Tampa with a special session dedicated to hair testing; another international meeting held in 1995
in Abu Dhabi; and the first European meeting held in Genoa in 1996 (31). The proceedings of several of these meetings have been published as full issues of the journal Forensic Science International. Since then, hair-testing science and technology has been extensively addressed at numerous forensic science and toxicology meetings. A large body of experimental and epidemiological scientific data has been published (32-36).
The mechanism of drug incorporation in hair has been found to be not as simple as originally proposed. It was thought that drugs within the blood capillaries bathing the follicle were transferred into the growing hair shaft and effectively locked in place. However, it has been shown that such a simple mechanism does not account for all the experimental observations. It has been demonstrated that drugs can also enter the hair shaft via sweat and sebum. Also, environmental contamination of the hair has been demonstrated. One question is whether hair analysis can differentiate between drugs in hair from actual drug use as opposed to environmental exposure (see Chapter 11).
Hair analysis is performed by cutting a segment of hair from close to the scalp, generally representing about three months' growth. Hair typically grows approx 1 cm/mo, although there are inter-individual differences in hair-growth rates. The cut hair specimen is washed to remove potential external contamination and then digested. The digest solution is tested by immunoassay and GC-MS. In addition, some laboratories also test the initial wash solutions for an assessment of the possibility of environmental contamination and its likely contribution to the subsequent test results.
Unlike the multitude of laboratories offering urine drug-testing services, there are only a few laboratories offering hair-testing services. There are currently no formal hair-testing laboratory regulations or guidelines, although there are a few professional societies as well as some proficiency-testing programs (37). However, hair testing is on the list of alternative specimens proposed for federally regulated workplace testing, with some laboratory and testing standards established, at least in draft form (4).
The main issues facing hair testing are (1) distinguishing environmental exposure/contamination of the hair from drug incorporation in the hair shaft from use and (2) addressing the possibility of hair-color bias. Both of these issues have been reasonably well investigated but still appear to remain subjects of controversy (38).
Hair-testing laboratories claim that they can distinguish between actual drug use and contaminating environmental exposure by a comparison of the levels of drugs that might be found in the preliminary wash solutions and the level of drugs found in the actual hair digest. If there are high levels of drugs in the wash solutions relative to those found in the digest, external contamination is considered likely. However, there appears to remain some controversy surrounding these claims.
It has been well demonstrated that drugs bind to hair differentially, dependent upon the physicochemical properties of the drug in question and those of the hair. It is known that many drugs bind preferentially to dark-pigmented hair over fair-colored hair, leading some to make claims of a hair-color bias in hair testing, unfairly identifying those with heavily pigmented hair over those with fair hair. Some have even called this a racial bias (39-42).
Another issue is the possibility of specimen adulteration (43-45). It has been demonstrated that hair color plays a significant role in binding of drugs to hair and that bleaching or other treatments can dramatically reduce the amount of drug found in hair. In addition, there are shampoos being sold on the Internet claiming that they can rid the hair of drugs (43). It seems clear that the opportunity to thwart hair testing through such chemical treatments exists. Of course, drug users could also shave their heads and even other body hair to prevent testers from obtaining an incriminating specimen.
Some claim that by segmental analysis of the hair shaft, a time profile of drug use may be obtained, although others challenge the scientific validity of such segmental analysis (46-48). Some experimental studies have challenged the simple view that drugs are neatly deposited along the hair shaft from the blood capillaries bathing the hair follicle, and remain in place as the hair shaft grows to provide a timeline of drug use. It has been demonstrated that this model of drug deposition and incorporation into hair is too simplistic and that drugs may be incorporated into and onto the hair shaft by a variety of mechanisms, including sweat and sebum excretion.
There have been numerous court challenges to the admissibility, probative value, and interpretation of hair drug tests. On balance, it now appears that hair testing has been generally accepted by the courts (49).
Another issue to consider when using hair testing in the workplace setting is that by examining prior drug use where there may not be current drug use, any sanctions may run afoul of the Americans with Disabilities Act. This act precludes employers from discriminating against otherwise qualified applicants or employees based on prior drug use, as long as they are not currently using drugs. Whether a positive hair test that looks back 3 mo in time represents current use appears not to have been conclusively decided in the courts.
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