Saliva and Detergents
Written by Heather Payne, MS and Heather Miller Coyle, PhD   

IN THE REALM OF FORENSIC SCIENCE, false positive results are a serious consideration. The tests that are utilized in a forensic science laboratory should be fully validated, and the scientists should be aware of any problems that can occur (National Research Council). Several detergents and other soap products contain bacterial amylase (Niyonzima) (Hole). This study delves into false positive results that can transpire from detergents or detergent residues when testing for saliva using the Phadebas Tube Test.

Amylase is a common enzyme that is found in high concentrations in saliva and is the typical target substance for detection, as an indicator of saliva (Phaldebas, n.d.) (Whitehead) (Myers). Amylase in detergents has been shown to be completely inactivated at washing temperatures of 100ᵒ and 60ᵒC, but amylase remained 40-60% active at 40ᵒC (HERA Project). A typical cold, warm, and hot wash cycle is 30ᵒ, 40ᵒ, and 50ᵒC respectively (Textile Industries Affairs). So, it is likely that amylase is still active after a typical wash cycle, which means that it is reasonable to assume that detergents containing amylase may leave behind residues that can produce false positive results when testing for saliva.

Studies have shown that some undiluted detergents can produce fluorescence with an alternate light source (ALS) and give false positive results using the Phadebas Tube Test, thus misidentifying saliva stains. A study executed in Australia showed fluorescent stains appear under Polilight from detergents on fabrics before and after washing (Vandenberg).

Previous studies have shown that detergents produce false positive results using tests to identify saliva (Feia) (Phadebas, 2007). Detergents and other soap products used for laundry purposes and cleaning glassware may be encountered at a crime scene. Therefore, it is possible that residues left behind from these products containing amylase can affect the results of a forensic examiner when seeking to identify human saliva (Phadebas, 2007). Five other undiluted detergents provided false positive results with the Phadebas Tube Test in a different study (Feia). These brands of detergents were Tide liquid, Kirkland Signature, Oxiclean gel, Gain, and Era.

The purpose of this study was to examine comparatively some additional detergents and cleaning products with the Phadebas Tube Test to identify other brands that can result in false positives. This is a key concept when reaching back in history to casework where this form of saliva identification was used exclusively by forensic biology units. In some cases, it may be valuable to revisit the trial transcripts and the test results where the saliva identification was pivotal in prosecution for kidnappings, homicides, and child abuse cases and was identified by only the Phadebas Tube Test. Confirmation tests are now available using antibodies that recognize human amylase and could be used to re-evaluate the old, stored evidence. A form of human-specific testing was validated and published in 2009 (RSID-Saliva; Independent Forensics, Lombard, IL).

Background on Detergents

The first development of an all-purpose laundry detergent was not presented in the United States until 1946 (Ainsworth). Since then, the make-up of detergents has been enhanced by using bleach, enzymes, and other products to make detergents safer to use and more effective for cleaning.

A main component of detergents is surfactants, which are also known as surface active agents (Ainsworth) (Talmage). Surfactants serve the purpose of dispersing the surface tension of water so that items can be cleaned. There are three classifications of surfactants: anionic, nonionic, and cationic. Most detergents have either anionic surfactants, nonionic surfactants, or a combination of the two. Surfactants are typically made from chemicals that are derived from petroleum or chemicals that come from fats and oils.

Other ingredients that can be added to detergents to aid in creating the best cleaning result are bleaches, bluing, boosters, and enzymes. Bleach is used to whiten the fabric and remove various stains. However, bleach can also produce a false positive result in forensic scenarios when an alternate light source is being used to identify potential biological stains, especially in combination with a blood-detection reagent such as luminol. Bluing removes the yellow tint in old and worn fabrics. Boosters aid in removing soil and other stains. Enzymes also help remove tough stains and soil, which is why the bacterial amylase enzyme is added to some detergents.

Phadebas Tube Test

The Phadebas Tube Test is a presumptive test that is commonly used in a forensic crime lab to detect amylase activity (Virkler). A serological presumptive test allows forensic analysts to test samples and detect the activity of typical compounds found in particular body fluids. The Phadebas Tube Test utilizes Phadebas tablets that contain blue dye cross-linked starch microspheres (Phadebas, n.d.) (Myers). These tablets, along with distilled water, are mixed in with the sample (cutting or swabbing of the stain), and if amylase activity is present, then the starch will be digested. The digestion releases the water-soluble dye, which creates the blue color change in the solution and represents a positive result. If the sample does not contain amylase activity, then the starch will not be digested and no color change will occur, representing a negative result and leaving the water in the test tube clear.

Materials

All liquid detergent samples were purchased from a commercial store that had the product in stock or an online shopping site. Major detergent manufacturing companies in the United States include: Colgate-Palmolive, Procter & Gamble, Ecolab, and Zep (U.S. Dept of Commerce).

The majority of detergents selected for this study were manufactured by Procter & Gamble because their website contains a readily available list of ingredients for every product. Palmolive Oxy Power Degreaser was the only detergent not manufactured by Procter & Gamble; it was manufactured by Colgate-Palmolive. All microcentrifuge tubes, sample vials, and test tubes were purchased online from Fisher-Scientific. Phadebas tablets were purchased from Magle-Life Sciences. Two yards of white cotton fabric was purchased from a Jo-Ann Fabric and Craft Store (West Haven, CT).

Methods

Collection of Saliva—A single subject chewed a stick of sugar-free gum for five minutes to stimulate saliva production (Dabbs). After five minutes, the subject discarded the gum and began to expel saliva into a 15 mL sterile Fisherbrand vial. Once the vial was full, it was sealed and stored at room temperature in a secured, locked drawer in the laboratory for up to 30 days (Quinque). Samples collected for research purposes were labeled with a numerical coding system. When more saliva samples were needed, the same subject repeated the previously described procedure. University of New Haven Institutional Review Board guidelines for human subjects were followed. Any remaining saliva samples were disposed of properly at the conclusion of the study.

Alternate Light Source Examination and Phadebas Tube Test Prior to Washing—The alternate light source (ALS) used was the Mini-CrimeScope MCS-400. Each dried fabric sample was labeled and placed under the CrimeScope light. The researcher wore orange goggles and then viewed the sample under the ultraviolet and visible wavelengths of light (SPEX Forensics) (Schulz). The light in the room was turned off to try to make the room as dark as possible for the best results. The light intensity on the Mini-CrimeScope MCS-400 was used at the highest intensity. Any products that produced a fluorescent stain on the fabric were then tested using the Phadebas Tube Test. The tests were repeated in triplicate for each product. Positive, negative, and substrate controls were performed for each Phadebas assay for quality control purposes. The positive control was a known sample of human saliva, the negative control was a known sample of distilled water, and the substrate control was a sterile cotton swab.

Post Wash Cycle Examination with Phadebas Tube Test—The same products that were tested with the Phadebas Tube Test were used in a cold wash cycle. White cotton 3 x 3-in. fabric swatches were washed separately in each of these products on a cold wash setting. There were three fabric swatches per detergent and three blank fabric swatches that did not contain any detergent for a substrate control, respectively. Each fabric swatch was coated separately in 30 mL of each detergent product, and then placed in the washing machine, one per load. Between each new cold wash cycle, there was an empty hot wash cycle to rinse out any residual detergent from the previous wash. The washed materials were then dried separately in a standard dryer on the “Quick Fluff” ten-minute setting. A small 1 cm2 cutting was taken from the middle of each swatch and tested for amylase activity using the Phadebas Tube Test.

The Phadebas Tube Test Method

Undiluted Detergent Examination—Undiluted detergent (450 uL) was added to the tip of separate sterile cotton swabs. Each swab tip was transferred to a sterile tube. Sterile distilled water (0.5 mL) was added to each tube and the swab was allowed to soak for one minute (Whitehead). The tubes were then vortexed for 30 seconds. Approximately 0.5 mL of each supernatant was pipetted into different tubes and one half of a Phadebas tablet was added to each tube. Distilled water was added to the tubes until the total volume reached 1 mL and the tubes were then vortexed for five seconds. The tubes were incubated at 37°C and centrifuged at 900 rpm for 30 minutes, removed, and then vortexed again for five seconds. The tubes were then centrifuged at 10,000 g for one minute. After centrifugation, results were read and recorded. A positive amylase reaction produced a blue colored solution. A clear solution represented a negative amylase result.

Post Wash Cycle Examination—A 1 cm2 cutting was taken from the middle of each fabric swatch and tested using the Phadebas Tube Test. Each cutting was placed in a separate test tube and 2 mL of distilled water was added to each tube. The tubes were vortexed for 30 seconds each and then 1.5 mL of the sample was pipetted into separate microcentrifuge tubes that each contained one quarter of a Phadebas tablet. Then each microcentrifuge tube was vortexed for five seconds and incubated at 37°C and centrifuged at 900 rpm for 30 minutes. The tubes were removed from the incubator, vortexed for five seconds and centrifuged at 10,000 g for one minute. After centrifugation, results were read and recorded.

Results and Discussion

The alternate light source (ALS) results are shown in Figure 1. All ten detergents were deposited on white cotton fabric swatches and allowed to dry for 24 hours. A Mini-CrimeScope MCS-400 was used as the alternate light source to view the detergent stains with ultra-violet light. An orange filter was placed between the light and the detergent stains, and all ten detergents fluoresced. The blank fabric swatch was used as a substrate/negative control and it did not fluoresce.


 Figure 1. Detergent stains viewed with ultraviolet light. The first three fabric swatch panels had dried detergent stains applied, and the final fabric swatch panel with the right was blank to use as a substrate/negative control. The top row shows detergents (from left to right): Tide, Cheer, Gain with Oxi Boost, Gain Original, and Non-Ultra Joy. The bottom row shows detergents (from left to right): ERA 2x, Ivory Snow, Gain with Bleach Alternative, Ivory Classic Scent, and Palmolive.

The typical results for the Phadebas Tube Test are shown in Figure 2. All ten undiluted detergents were tested three times each with the Phadebas Tube Test. Distilled water was used as a negative control, known saliva was used as a positive control, and a cotton swab was used as a substrate control. In all three replicates, each detergent that contains amylase (Cheer, Gain Original, Gain with Bleach Alternative, Gain with Oxi Boost, and Tide) produced a positive result and each detergent that does not contain amylase (Era 2x, Palmolive, Non Ultra Joy, Ivory Classic, and Ivory Snow) mostly produced a negative result. There was one exception in the first replicate where Era 2x produced an inconclusive result due to a weak positive result in one replicate and negative results in the other two replicates.


Figure 2. Phadebas Tube Testing with undiluted detergents. Samples from left to right: Era 2x, Cheer, Palmolive, Gain Original, Gain with Bleach Alternative, Gain with Oxi Boost, Non-Ultra Joy, Ivory Classic, Ivory Snow, Tide, Positive Control, Negative Control, Substrate Control.

Since all five detergents that contain amylase provided a positive result with the Phadebas Tube Test with each replicate, it is possible that these detergents can create false positives in a crime lab setting where clothing and evidence would be presumptively screened for stains with alternate light sources, then tested for the presence of amylase, an indicator of saliva, with the Phadebas Tube Test. Situations where this may occur could be clothing that is in the wash cycle and clothing where detergent is spilled but clothes are not laundered. Some laboratory reports note that perspiration and bacteria can give false Phadebas Tube Test results; in addition, laundry detergents containing bacterial amylase should be included in the list of substances that can yield a false positive result. Our post-washing study of fabrics did not yield sufficient trace detergent to be detected by FTIR, nor was a false positive result achieved with the Phadebas Tube Test.

The purpose of this study was to examine comparatively detergents and cleaning products with the Phadebas Tube Test to identify brands currently on the market that can result in false positives. This is important for forensic biology units, associated law enforcement, and attorneys when the saliva identification was the basis for prosecution and only the Phadebas Tube Test results were presented for plea agreement or at trial.

One original concern was that detergent bacterial amylase would be still active after a typical wash cycle, leaving behind residue that could produce false positive results when testing for saliva with the traditional method. On clean laundered clothing, this false positive issue was found not to be valid; however, with situations of bacteria, bacteria in body fluids, perspiration, and detergent stains containing bacterial amylase, a false positive is likely with the traditional Phadebas forensic test method. The impact on the court system would likely be a review required for any worn clothing tested solely with the Phadebas Tube Test and also being the crucial biological evidence to the case.


About the Authors

Heather Payne is a 2017 graduate of the Master of Science Forensic Science program at University of New Haven, a small selective private University in West Haven, Connecticut. Payne has a strong interest in biological applications to forensic science. Her main thesis research is presented in part, here, and also included some methods to attempt to classify unknown detergents to manufacturer and brand.

Heather Miller Coyle is an Associate Professor at University of New Haven in the Forensic Science program since 2005. She obtained her doctorate in 1994 in Plant Biology from the University of New Hampshire. Her research interests and scientific publications include touch DNA, methods for human identification by DNA, and forensic biology.


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This article appeared in the Summer 2018 issue of Evidence Technology Magazine.

 
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