Saliva As A Forensic Evidence

Saliva is a complex biological fluid secreted by acinar cells of the major and minor salivary glands. It is an indicator of various plasma constituents.

In recent years, its role as a diagnostic and forensic tool is being increasingly researched upon. Besides maintaining the homeostasis of oral structures such as tooth integrity, it also plays a critical role in genomics, proteomics, and bioinformatics.

It is an important discriminating element in forensic biology, acting as an indicator of salivary gland conditions and toxicological and drug monitoring.

Saliva is an extracellular fluid produced and secreted by salivary glands in the mouth. In humans, saliva is 99.5% water plus electrolytes, mucus, white blood cells, epithelial cells (from which DNA can be extracted), enzymes (such as amylase and lipase), antimicrobial agents such as secretory IgA, and lysozymes.

COMPOSITION OF SALIVA

Majorly water
electrolytes : Sodium, potassium, calcium, magnesium, chloride, bicarbonate, phosphate.
mucus
Antibacterial combinations
enzymes : Basicaly three are main:

A. α-Amylase or ptyalin which initiates absorption of starch. Its ideal pH is 7.4.

B.Lingual lipase : It has a pH optimum ~4.0 ; so it is not triggered till arriving the acidic situation of the stomach.

C. Kallikrein– an enzyme that proteolytically splits high-molecular-weight kininogen to form bradykinin, which is a vasodilator.

Antimicrobial enzymes: Such as Lysozyme, lactoferrin etc.
Cells: Perhaps as numerous as 8 million human and 500 million bacterial cells per mL.
Opiorphin, a recently investigated pain-killing element established in human saliva.

FUNCTION OF SALIVA

The enzymes found in saliva are essential in beginning the process of digestion of dietary starches and fats. These enzymes also play a role in breaking down food particles entrapped within dental crevices, thus protecting teeth from bacterial decay.

Saliva also performs a lubricating function, wetting food and permitting the initiation of swallowing, and protecting the oral mucosa from drying out.

Saliva As A Forensic Evidence

Saliva is an important evidence which can provide useful information about the personal contact of victim and perpetrator.

The presence of saliva can be ascertained by Starch-iodide and Phadebas tests.Starch-iodide and Phadebas tests, however, do not confirm the presence of Human saliva.

These are merely the test for amylaze activity regardless of whether that amylase has come from human or any other source.

An advanced, monoclonal antibody based test kit is used to identify human specific salivary amylase. With Rapid Stain Identification Series (RSID) method, if a stain gives positive reaction, then it is confirmed that human saliva is present.

In addition, ABO group antigens can also be detected in saliva, if the person is secretor.

A secretor is an individual whose saliva and other body fluids contain ABO antigens. Approximately 80% individuals are known to be secretors.

Finally, since saliva may also contain buccal mucosa cells, it is possible to identify the DNA profile of the person in question using advanced DNA profiling techniques.

LOCATION OF SALIVA AS AN EVIDENCE

Saliva is found on clothes, cigarette butt, bottles, cup, Handkerchief, etc. In cases of biting during struggle, assault, kidnapping, Hanging, spitting or tobacco spitting at scene of crime, saliva provides lot of evidentiary data.

Saliva plays an important role in crime investigation because DNA profiling or grouping test can be performed by saliva due to presence of antigen in it.

Blood group can detect by saliva although saliva is contaminated by lipstick or blood.

If saliva is present as dry stain then wet with the help of normal water and make cotton swab of it and then preserve in tube or bag without contaminating.

If chewing gum, tobacco spit etc. are present at crime scene then preserve in airtight bag as such.

FORENSIC EXAMINATION OF SALIVA

Once the sample is collected and packed , it is immediately sent to the laboratory for the analysis. There are various test for the examination of the saliva.

AMYLASE

Amylase are ubiquitous enzymes found in both animals and plants. They are responsible for catalysis of the components of starch, amylose and amylopectin into smaller, less complex sugars. Amylases can be subdivided into alpha and beta categories:
Beta amylase attacks only the bonds at the ends of polyglucan chain, unlike alpha amylase which catalyzes bonds within chains.
Primates, pigs, elephants, and certain rodents have high level of amylase.

Starch- Iodine Test

Reagent Preparation:

1.0.5% soluble starch solution (50 mg soluble starch / 10 ml H2O).

2.Lugol’s iodine solution.

Procedure

1. We place 3 tubes in a rack and add the following:

a.In the first tube, place a 5 mm x 5 mm piece of sample to be tested.

b.In the second tube, place a similar sized unstained control piece.

c.In the third tube, place a 5 mm x 5 mm piece of a known saliva stain.

2. Add 3 drops of soluble starch solution to each tube.

3. Mix, cork and incubate the tubes for 1 hour at 37⁰C.

4. Add 2 drops of Lugol’s iodine and note the colour formed.

5. A dark blue starch-iodine complex should be observed in the second and fourth tubes. The absence of the dark blue colour indicates that the starch has been hydrolyzed and is no longer available for complexing. Therefore, the lack of blue colour is a positive result for amylase activity, indicative of the presence of saliva. This is not a confirmatory test for saliva.

Note : To dissolve the starch, heat the solution to a boil. Allow solution to cool to room temperature. Always use soluble starch. Do not use hydrolyzed starch.

Starch solution must be freshly prepare. Never use starch solution older than 24 hours.

Phadebas Test

This test is based on the fact that amylase digests starch. Phadebas reagent consists of a dye cross-linked with starch. The presence of saliva digests the starch and releases the dye from the complex. The solution thus becomes blue in color. This indicates the presence of saliva.

The test can be used as a quantitative test by measuring the intensity of the developed color at 620nm wavelength. A standard concentration curve of known concentration of colored dye may be prepared and used for quantitative data.

The degree of coloration is proportional to the amount of amylase in the sample.

Phadebas Press Test

Phadebas paper is made by dissolving six Phadebas tablets in 30 mililiters of disilled water. Whatmann grade filter paper sheets measuring 46 by 57 cm are used. The solution is sprayed evenly onto one side of the paper using atomizer and allowed to dry.

The item to be tested must be flat and ensure good contact between it and the paper.

A piece of phadebas is placed over the entire stain area to be tested. The paper is dampened by spraying the distilled water. A piece of plastic wrap is placed on the paper to prevent drying during the test.

The test is observed for 40 minutes, with notes made at the time of initial appearance and its development over 40 minute period.

The reaction appears as a light blue smooth area in contrast to grainy appearance of test paper.

Immunochromatographic Test

Commercially developed immunochromatographic kits are available for testing saliva samples. The method is based on antigen-antibody interaction principle. One such kit is known as RSID saliva kit. The presence of saliva can be ascertained by the appearance of pink line in the RSIDsaliva kit. This technique can detect saliva as low as 1 µL.

Radial Diffusion Test for Amylase

Reagent Preparation:

1.0.1M Phosphate buffer, pH 6.9 prepared as follows:

NaH2PO4 anhydrous – 62.7 g

Na2HPO4 anhydrous – 3.9 g

NaCl- 0.2 g

Distilled water- 500 ml

2.Gel test plates (2% agarose, 0.1% soluble starch)

Buffer, pH 6.9 – 10.0 ml

Agarose – 0.2 g

Soluble starch – 1.01g

Heat to boiling and continue stirring constantly until all the agarose is dissolved. Divide gel solution and pour into 302” disposable plastic petri dishes. Allow to polymerize completely. Store gels inverted (to retard dehydration) at 4⁰C.

3.Iodine development solution

KI-1.65 g

I2- 2.54 g

Distilled water- 30 ml

Dissolve by stirring for 5 minutes at 65⁰C. Store saturated I2 solution in dark stoppered bottle.

Working solution in 1/50 dilution with distilled water.

Sample preparation:

Extract approximately 3 mm² piece of stained material with 50 ul of distilled water. Prepare an extract of unstained material (when available) in the same manner.

Procedure:

1.Punch holes in gel plate with a vacuum pipette, leaving 1.5cm between the sample wells.

2.Place samples to be tested in the sample wells using a precision pipettor. Each well holds approximately 4 μl of liquid.

3.Cover the petri dish and place in an incubator at 30⁰C for 6 hours or overnight.

4.Stain the plate by pouring a 1:50 dilution of saturated iodine solution onto the surface. Rinse with H2O.

5.Clear circles around the wells indicate areas of amylase activity. The diameter of the clear circle is proportional to the square root of the concentration of amylase.

1.Positive Controls:

a)Known dilutions of fresh liquid saliva (1/200, 1/500 in H2O)

b)Known dried saliva stain extracted with H2O (3 mm2 extracted in 50 ul H2O)

2.Negative Control: distilled H2O.

3.Unstained Control: 3 mm2 unstained material extracted in 50 μl of H2O).

Forensic Importance of Saliva As An Evidence

Saliva has recently attracted much attention among researchers, particularly in the branch of forensic sciences. Serological testing and analysis of salivary cellular component are being increasingly applied in crime detection, hormone identification, drug and alcohol abuse, cases of animal bites, and poisoning.

Safe handling, along with the easy and noninvasive procedures of saliva collection, has increased its popularity in drug abuse tests. Sex determination and identification of the suspects in scenes of crime, with the help of cytological analysis and DNA profiling of saliva, are finding widespread applications in forensic investigations.

Pros of Saliva as an evidence