It consists of preliminary and confirmatory methods of saliva analysis the biological fluid gets at the crime scene.

1. SALIVA ANALYSIS
Dr. Archana Mahakalkar
Department of Forensic Biology
Government Institute of Forensic Science, Nagpur

2. INTRODUCTION
• On average, a human being secretes 1.0 to 1.5 liters of saliva every day.
• It is a fluid largely composed of water with little amounts of electrolytes and
enzymes.
• It is secreted by parotid and submandibular salivary glands in the mouth.
Forensically, it is often seen in sexual assault cases.
• Saliva samples can be analyzed from various types of surfaces such as body parts,
paper, envelopes, cigarette butts, plastic and glass bottles, and metal cans, etc.
• Saliva is an important evidence which can provide useful information about the
personal contact of victim and perpetrator.

3. • 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 amylase 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 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.

4. Saliva
Analysis
Methods
Starch –Iodine
test
DNA
Identification
ABO group
identification
Phadebas
Test
RSID test

5. HUMAN SALIVARY GLANDS
• About 70% of saliva is produced
from the submandibular salivary
glands
• 25% from the parotids
• 5% from the sublingual salivary
glands
• The digestion of carbohydrates
in the diet begins in the oral
cavity, where amylase in the
saliva breaks down
carbohydrates such as starch.
• Thus, detecting amylase
indicates the presence of saliva.

6. AMYLASES
• Amylases are enzymes that cleave polysaccharides such as starches that are
composed of D-glucose units connected by α1→4 linkages.
• Starches contain two types of glucose polymers: amylose and amylopectin
• Amylose consists of long, unbranched chains of glucose residues connected by
α1→4 linkages.
• Amylopectin is highly branched and consists of linear chains of glucose residues
connected by α1→4 linkages with the branch points connected by α1→6 linkages.

7. Two types of amylases can be
characterized.
1. β-amylases found in plant and
bacterial sources cleave only at the
terminal reducing end of a
polysaccharide chain. The end of a
chain with a free anomeric carbon
(not involved in a glycosidic bond)
is called the reducing end.
2. Human α-amylases cleave at
α1→4 linkages randomly along the
polysaccharide chain.

8. • Human α−amylases have two major isoenzymes (multiple forms that differ in
their amino acid sequences).
• Human salivary a-amylase (HSA) is encoded by the Amy1 locus, synthesized
at the salivary glands and secreted into the oral cavity.
• Human pancreatic a-amylase (HPA), encoded by the Amy2 locus, is
synthesized by the pancreas and secreted into the duodenum through the
pancreatic duct.
• The amino acid sequences of the HSA and HPA are highly homologous.
However, HSA is inactivated by acids in the stomach, while most HPA is
inactivated in the lower portions of the intestine, and some amylase activity
remains in the feces.

9. IT COMPRISES THE FOLLOWING COMPONENTS:
1. Water;
2. Electrolytes: sodium (lesser than blood plasma), potassium (greater than plasma), calcium (similar to plasma),
magnesium, chloride (lesser than plasma), bicarbonate (greater than plasma), phosphate;
3. Iodine (mmol/L commonly greater than plasma, but reliant on variable according to nutritive iodine consumption);
4. Mucus- Mucus in saliva chiefly contains of mucopolysaccharides and glycoproteins;
5. Antibacterial combinations (thiocyanate, hydrogen peroxide, and secretory immunoglobulin A);
6. Epidermal growth factor or EGF;
7. Several enzymes- There are 3 main enzymes present in saliva.
8. α-Amylase, or ptyalin, produced by the acinar cells of the parotid and submandibular glands, initates the absorption
of starch. It has an ideal pH of 7.4.
9. Lingual lipase produced by the acinar cells of the sublingual gland, have a pH optimum ~4.0 so it is not triggered till
arriving the acidic situation of the stomach.
10. Kallikrein- an enzyme that proteolytically splits high-molecular-weight kininogen to form bradykinin, which is a
vasodilator. It is released by the acinar cells of all three major salivary glands.
11. Antimicrobial enzymes: Lysozyme, Salivary lactoperoxidase, Lactoferrin, Immunoglobulin A.
12. Proline-rich proteins (role in enamel creation, Ca2+-binding, microbe death and lubrication).

10. 13. Minor enzymes comprise salivary acid phosphatases A+B, N-acetylmuramoyl-
L-alanine amidase, NAD(P)H dehydrogenase (quinone), superoxide dismutase,
glutathione transferase, class 3 aldehyde dehydrogenase, glucose-6-phosphate
isomerase, and tissue kallikrein (function unidentified).
14. Cells: Perhaps as numerous as 8 million human and 500 million bacterial cells
per mL. The existence of bacterial products (small organic acids, amines, and
causes saliva to occasionally display foul odor.
15. Opiorphin, a recently investigated pain-killing element established in human
saliva.
16. Haptocorrin, a protein which fixes to Vitamin B12 to safeguard it besides
deprivation in the stomach, previously it bind to Intrinsic Factor.

11. PRESUMPTIVE ASSAYS
• 1. Visual Examination The lighting
techniques used to search for semen stains
can be employed in searching for saliva
stains.
• For example, a 470- nm excitation
wavelength can be used with colored
goggles or interference filters of higher
wavelengths (555 nm) to allow visualization
of fluorescence.
• However, the fluorescence of a saliva stain is
usually less intense than that of a seminal
stain.
Examining saliva stains using an ALS technique. (© Richard C.
Li.)

12. 2. MICROSCOPIC EXAMINATION
• Microscopic examination with proper histological staining can also be
performed to identify the buccal epithelial cells that indicate a saliva
stain.

13. 3. DETERMINATION OF AMYLASE ACTIVITY
• Starch–Iodine Assay — Iodine is used to test for the presence of starch.
• The amylose in starch reacts strongly with iodine to form a dark blue complex,
• Amylopectin develops reddish-purple colors. In the presence of amylases, starch is broken down
to mono or disaccharides.
• An agar gel containing starch is prepared.
• A sample well is created by punching a hole in the gel and an extract of the questioned sample is
placed into the well.
• If amylase is present in the sample, it diffuses from the sample well and hydrolyzes the starch in
the gel.
• The gel is then stained using an iodine solution. A clear area in the gel indicates amylase activity
and the
size of the clear area is proportional to the amount of amylase in the sample.
• A linear standard curve (in log scale) can be prepared using a series of standard amylases with
known concentrations.
• The amount of amylase can be quantified by comparing the results with the standard curve.

14. 3.1 STARCH–IODINE ASSAY

15. 3.2 COLORIMETRIC ASSAYS
Amylase mapping for saliva stains.
(a) Amylase substrate (amylose and amylopectin which
are components of starch) is sprayed on a sheet of
filter paper.
(b) Substrate-containing paper is placed over the area to
be tested. The orientation of the filter paper is marked
to aid in locating the stain.
(c) The filter paper is dampened by spraying it with
water, and plastic wrap is placed on top to prevent
the paper from drying.
(d) Blue color indicates a positive reaction. (© Richard C.
Li.)
(e) These assays are based on the principle that amylase
is water soluble and can be transferred from evidence
to filter paper and then analyzed via colorimetric
assay. This procedure is also referred to as a press
test. The sensitivity of the method is similar to that of

16. CONFIRMATORY ASSAYS
1. IMMUNOCHROMATOGRAPHIC TEST

17. 2. ELISA

18. 3. RNA BASED TEST
• Mouth contains cells that are specific to it.
• These cells contain specific protein coding genes that render them to be exclusively identified.
• Two such genes are HTN3 and STATH.
• HTN3 gene encodes histan-3 protein which is involved in defense mechanism in oral cavity.
• STATH encodes statherin which is prevents the precipitation of calcium phosphate salts in salivary glands.

19. REFERENCES
• Li, Richard- Forensic Biology ( Second Edition
)

20. Thank You