Saliva As A Diagnostic Tool In Forensic Odontology

Over the last few years investigators and researchers have been repeatedly magnetized by the use of saliva in the forensics. Popularity saliva has gained amongst other body fluids owed to its multipurpose qualities that include easy and painless collection, hazard free handling, close relationship with plasma and resourceful composition. This review emphasizes on the comprehensive use of saliva in forensics including crime detection, personal identification, alcohol and drug abuse, hormone identification and serological testing.

Mandel in 1990 quoted that “saliva lacks the drama of blood, the sincerity of sweat and the emotional appeal of tears1.’’ But now there has been a growing interest in saliva and it is emerging as a diagnostics tool as well as an alternative to blood and urine. Saliva is an accessible, inexpensive, non-invasive and easy-to-use diagnostic aid for drug monitoring, Serological and cellular analysis, detecting heavy metal poisoning by reflecting ionic imbalance and excretion of various poisons by this route. In clinically difficult situations, where blood sampling could be a difficult act to perform such as in children, handicapped and anxious patients saliva can be used.

In past forensic investigations have revealed saliva which is deposited in prints, bite marks, cigarette butts, marks and prints left on stamps and other objects helps in individual identification to a greater extent because saliva consists of exfoliated oral epithelial cells, microorganisms, DNA and RNA proteins. When saliva enters the oral cavity it makes its way through the salivary ducts and coalesces with blood cells, micro-organisms of the oral cavity, food and upper airway secretions. So this increases the chances of saliva containing vital information about the quality of life, health and general wellbeing of a person.

The dearth of the role of saliva in forensic odontology in literature evokes an extensive search of the existing literature to outline the vast topic into a comprehensive update. Hence, the current review article emphasize on the use of saliva in forensics.

Saliva in personal identification

In many homicides, assault and other criminal cases saliva is deposited in bite marks5. Since saliva remains invisible this makes saliva stains difficult to collect from skin, clothing, paper or other inanimate objects. Moreover saliva deposited on substrate (mainly skin) cannot be directly submitted to extraction procedures.

Methods for saliva recovery from skin:

• Single wet cotton technique or wet filter paper technique (classical method).
• Double swab technique by sweet et al (1997).
• Phenol- chloroform method for DNA extraction.

Methods to detect dried saliva stains:

• Chemicals: Dried saliva stains can be detected from stamps and envelopes by using alkaline phosphatase, starch and amylase which gives a red insoluble precipitate. Nitrate and thiocyanate salts have also been used for the detection.
• Lasers and ultraviolet light, quartz arch tube and argonion laser.
• Fluorescent spectroscopy: Tryptophan is an aromatic amino acid present in α salivary amylase. It possess a good sensitivity in detecting dried saliva stains on the skin thus giving characteristic emission spectrum on fluorescent spectroscopy.
• Immunological methods: ELISA is not widely used method. Alpha -amylase activity in saliva stains is detected using a horseradish peroxidase conjugate which is combined with monoclonal antibodies.

Analysis of drugs of abuse in saliva

Urine is the most frequent and reliable biological specimen for drug monitoring. Direct observed collection of urine is advisable to prevent adulteration but this hinders the privacy of person. Due to improved analytical techniques large number of drugs can be detected in small amount of oral fluid.

Drugs enter the saliva by simple passive diffusion which is characterized by the transfer of drug molecules down a concentration gradient with no expenditure of energy7. Many drugs like amphetamines, barbiturates, benzodiazepines, phencyclidine, cocaine and opioids can be identified in saliva. A number of researchers have used radioimmunoassay (RIA) to detect marijuana, phenobarbital, amphetamine and morphine in saliva and salivary strains.

Recently, Securetec (Ottobrunn, Germany) introduced Drug wipe, which is used for the detection of drugs of abuse on surfaces. It is a pen sized, immunochemical based test strip. Separate tests are available for opiates, cocaine, cannabinoids and the amphetamine group. The wiping part enables the user to sample drug particles from any kind of surface.

Difficulty in analysis arises when traditional identification methods fail because of the effects of heat; trauma and autolytic processes then the techniques involving DNA analysis are of great dependency. DNA profiling is unique for every individual. Although soil bacteria which are rich in nucleases have an effect on stability of human DNA but they didn’t pose a great threat as the probes used in DNA profiling are specific to humans’ only.

As saliva contains leukocytes and exfoliated epithelial cells from the host it acts as a very useful DNA source. Blood can also be a source of DNA but the difference lies in the fact that saliva is easy to access, collect and handle for diagnostic procedure since it doesn’t clot. Also saliva collection is a non-invasive and safer than venepuncture which leads to lesser chance of cross infection.

A major drawback in using saliva is that a very small amount is available for molecular diagnostics. But with help of Polymerase chain reaction (PCR) replication of thousands of copies of a specific DNA sequence in vitro can be obtained. The basis of all inherited polymorphisms lies within the DNA molecule and they are unique as well as do not change over the lifetime of an individual. DNA samples are amplified by polymerase chain reaction for DNA typing using short tandem repeats (STRs). Short Tandem Repeat (STR) are DNA markers, also referred to as microsatellites or simple sequence repeats (SSRs), consisting of repeated DNA sequences with a core repeat of 1–5 base pairs (bp). The polymorphic repair of STR mainly in small fragments also makes it possible to evaluate DNA from samples with a significant grade of degradation.

Microsatellite loci are endowed with several properties that render them desirable markers for maternity/paternity determination and forensic analysis, because, in conjunction with the amplification process, they require as little as 0.5ng of DNA template.

A mitochondrial DNA (mtDNA) cell is present in saliva. The main advantage of mtDNA is that there is a high copy number in each cell caused by the high number of mitochondria present in most cells. Chromosomal DNA is inherited from both the mother and father whereas mtDNA is strictly maternally inherited. Thus when identifying a set of human remains, if close relatives are not available, distant maternal relatives can be used as a reference source to support identification. Therefore, mtDNA testing may be successful when nuclear DNA testing fails or when genomic DNA cannot be analyzed, possibly because it is too degraded.

Sex determination from saliva in bitemarks

Saliva is a very useful DNA source because it contains leukocytes and exfoliated epithelial cells from the host; this increases the possibility of sex determination of the suspect. Two parameters can be used to identify the sex that is detection of sex chromatin. Barr bodies in case of females and F bodies in case of males can be observed and the levels of sex hormones; testosterone and 17β-estradiol can be determined by radioimmunoassay.

Salivary biomarkers

Saliva contains DNA, RNA and proteins which carry vital information about person and these are considered to be of great value to the forensic odontologist. Saliva biomarkers have been used in diagnosis of various diseases but they have wider role in forensics. Although saliva helps in cases involving bite marks but is also useful in identification of people in situations of mass disasters. Salivary biomarkers for any particular disease can be matched and tracking that disease to hospital visits positive identification can be arrived. So in this way saliva biomarkers can be used to identify people whose DNA are not on any database.

Determination of hormone levels

Recently saliva is used in detecting steroid hormones such as cortisol, cortisone and testosterone as it is an accepted thing that salivary level of these steroids reflects the free, unbound circulating fraction. These hormones help in determining the mental state of person. For example low morning basal salivary cortisol levels coupled with traumatic brain injuries and recent abuse, is associated with dangerous violent criminal behavior and high testosterone in saliva shows that the person is violent and less compliant and is associated with personal harm.

Conclusion

When most people think of saliva, which they rarely do it as a convenience, handy for licking stamps and sealing envelopes. Practicing dentists find it’s a nuisance, to be sponged, evacuated or dammed. But saliva is much more than water. Saliva is a most valuable oral fluid. Over the years in forensic laboratories the need and importance of saliva as an investigative body fluid is increasing rapidly due to its wide range of use in forensics. Automated settings for saliva should be present for saliva as are routinely done for blood and urine. More research dedicated towards this particular innocuous body fluid should be aimed at for gaining detailed information in forensic sciences.