Forensic Tests for Saliva: What you should know

By Maher Noureddine, Ph.D.

First in the series: Bodily Fluids and Forensics

You may encounter cases in which the prosecution claims that SBI tests establish the presence of human saliva on the victim as part of its proof that an assault occurred. This type of evidence is often seen in sexual assault cases.  In cases like this it is important to understand how the SBI or law enforcement tests for saliva and how the problem of false positives limits those tests.

The non-invasive nature of saliva sample collection has earned that bodily fluid significant medical attention. Saliva tests can reveal certain disease markers, viral infections, and the presence of therapeutic as well as illicit drugs in the body. Saliva is rich in the enzyme alpha-amylase (a.k.a. α-amylase, salivary amylase, or Ptyalin), an enzyme that breaks down complex carbohydrates into smaller sugar molecules. The biological function of this enzyme was first described by Erhard Friedrich Leuchs in 1831.

Almost 100 years later (1928), the German investigator B. Mueller conceived the use of alpha-amylase as a forensic target to validate the presence of saliva on a given surface. This idea gained popularity, and for the next 50 years several investigations endeavored to refine and enhance the methods to test for alpha-amylase. However, some important caveats continue to limit this test as a presumptive or screening test for human saliva, even to this day. Alpha-amylase is an enzyme that has changed very little throughout the process of evolution: alpha-amylases from bacteria, fungi, or chimps are very similar in structure and function to that of the human alpha-amylase. In humans, there are at least four variants (or versions) of alpha-amylase, two of which are found in saliva, and the other two are secreted in the pancreas. Those variants are almost indistinguishable at the enzymatic activity level. Because the presumptive test for saliva detects the enzymatic activity of alpha-amylase, and not alpha-amylase molecule itself, that test will yield a positive result if any alpha-amylase enzyme is present, regardless of the organism it came from.

Crime labs, including the SBI lab (See p. 13 of their protocol here) use a reagent (chemical) called Phadebas to conduct this presumptive test for alpha-amylase. This test is relatively cheap, quick, and highly sensitive to any alpha-amylase enzymatic activity. Myers, J. and Adkins W., Comparison of modern techniques for saliva screening, 53 J Forensic Sci 4 (2008).  However, it is important to keep in mind that this test alone cannot confirm the presence of human saliva because this presumptive test will give a positive result if the alpha-amylase enzyme from any organism is present.

Laboratory tests for saliva remained presumptive until the late 1980s, when a group of researchers in Japan succeeded in developing a monoclonal antibody that is specific for the alpha-amylase variant that is present in human saliva in particular. Ito, K. et al., Preparation of human salivary alpha-amaylase specific monoclonal antibody, 97 J Biochem 5 (1985). Therefore, instead of testing for enzymatic activity, now we can detect the alpha-amylase molecule itself, and specifically, the alpha-amylase from human saliva. This ushered the development of test kits that are now being used in forensic laboratories around the world to screen for human saliva (known to many as Lateral Flow Immunochromatographic Strip Test or Rapid Stain Identification (RSID) Saliva kits).  The SBI lab used a combination of the presumptive Phadebas test and the RSID test to “confirm” the presence of human saliva (See p. 14 of their old protocol here).  The SBI lab protocols have been updated recently and reflect important changes in interpretation language.  Under current protocols (see p. 2, available here), the SBI lab acknowledges that the RSID test for saliva is a presumptive test.

The RSID-Saliva kits have been tested on samples from various types of surfaces such as paper, cigarette butts, plastic and glass bottles, and metal cans. The specificity of RSID-Saliva kits has been scrutinized by researchers. Although RSID-Saliva kits were found to be sensitive and specific to human saliva, positive reactions were also noted in samples containing alpha-amylases from  mammals such as  gorillas and rats. Positive reactions were also noted in other bodily fluids such as semen, urine, and sweat. See Old J. et al., Developmental Validation Studies of RSID-Saliva Lateral Flow Immunochromatographic Strip test for the forensic detection of Saliva, Independent Forensics (2010); Pang, B. et al., Applicability of Two Commercially Available Kits for Forensic Identification of Saliva Stains, 53 J Forensic Sci 5 (2008); Casey and Price, The sensitivity and specificity of the RSID-saliva kit for the detection of human salivary amylase in the Forensic Science Laboratory, Dublin, Ireland, Forensic Sci Int (2010) 3:67–71. The RSID-Saliva test gives positive results from breast milk, likely because the presence of alpha-amylase aids the nursing infant in food digestion. High reactivity of this test is also observed in samples containing human feces. Not surprisingly, humans swallow copious amounts of saliva, which travels through the entire digestive system and, as a result, alpha-amylase from saliva (as well as alpha-amylase from the pancreas) is thought to reach the colon where it can mix with fecal material. You will note that the SBI lab’s procedure is to not use this test on rectal samples. Reactivity was also noticed in urine samples, a result that remains inconsistent between studies. Butterworth, P. et al., Human α-amylase and starch digestion: An interesting marriage, Starch 63 (2011). These limitations clearly demonstrate why the RSID-Saliva test is presumptive in nature.

It is important to consider these limitations when examining or reviewing tests for the presence of the assailant’s saliva based on swabs from sexual assault kits. Improper swabbing and other factors relating to personal hygiene, personal behavior, and indirect saliva transfer from mouth to surface can result in “false” positives. Since saliva can harbor mucosal cells from the perpetrator, the validation of the saliva sample can be coupled with forensic DNA analysis (STR/Y-STR) to rule in or rule out the connection between an individual and the forensic evidence. If DNA analysis not completed, it is worth questioning whether the positive test for saliva could be caused by personal hygiene practices or another innocent explanation for the presence of the salivary amylase.