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What are the components of saliva?
- 98% water
- 2% (Electrolytes, Mucus, Antibacterial compounds, Enzymes, Cells)
Where is saliva produced?
- Parotid gland (near the ears)
- Submandibular and sublingual glands (under the tongue)
- Also produced in two types of epithelial cells:
- mucous cells
- serous cells
What is the daily production of Saliva in humans?
Characteristics of saliva
- ~stains are invisible to faint white
- ~stains faintly fluoresce at 450nm
- ~several million human cells per milliliter
- ~several hundred million bacterial cells per milliliter
Identification of saliva is based on the detection of:
- inorganic anions (thiocynate, nitrites)
- Enzymes (alkaline phosphatase, Amylase)
- antigens (HSA)
- -inorganic anion in saliva
- -detected by adding ferric chloride (forms a red/pink product)
- - 2.5 times higher in smokers
- - concentration decreaes with time
- - found in other body fluids in lower concentrations
- - may be present in undetectable amounts- negative result does not exclude saliva
- -inorganic anions found in saliva
- -NO2 is detected by the Griess test (used in GSR)
- -undetectable after a few weeks- so positive result meanse the stain is relatively recent
- -source= ingested nitrates in diet are broken down by bacteria
- -also form when nitrogen-containing substances decompose
Detection of Amylase
- -most common method of detecting saliva
- -one of the oldest know enzymes (1831)
- -found in both plants and amylase
- -a and b amylase
b amylase (beta)
- -found in plants; breaks starch into sugar in ripening fruit
- -yeast, mold and bacteria
What is a-amylase (alpha)?
- produced by humans and some animals that have a diet of complex carbohydrates
- -a-amylase is responsible for catalyzing the breakdown of starch, amylose and amylopectin into smaller less complex sugars
- -approx. 50 times higher in saliva than most other body fluids (values can vary within an individual and between individuals)
- -relatively stable
- -originally names ptyalin
- -type of amylase typically encountered in forensic science
- -saliva has one of the highest amylase values
a-Amylase loses inactivity when:
- -loses activity upon drying (once dry, the rate at which activity loss
- occurs decreases)
- -will become inactive at high temperatures (100C),
- in the presence of strong acids or stong bases
Vaginal secretions have lower amylase activity than saliva. (T/F)
True, usually. However occasionally the amylase activity in vaginal secretions can enter the range of saliva.
Feces can sometimes exhibit amylase activity higer than saliva. (T/F)
Functions of salivary amylase
- -maintain oral health
- -lubricate the passage of food
- -breakdown of carbohydrates
other uses: breaks down starches in simple sugars in bread making; and added to detergents to dissolve starches from fabric
types of a-amylase
- AMY 1: Salivary amylase (HSA)- saliva, breast milk, perspiration, tears
- AMY 2: Pancreatic amylase (HPA)- pancreas, vaginal secretion, seminal plasma, fecal material
Distinguishing AMY 1 and AMY2
- -lectins used to preferentially inhibit one or the other
- -monoclonal antibodies used to react with one type or the other (ELISA)
Individualizing AMY1 and AMY2
- -isoenzymes (multiple molecular forms) will separate with polycrylamide or agar gel
- -AMY1 has 7 phenotypes
- -AMY2 has 3 phenotypes
What types of forensic samples could saliva be recovered from?
- -bitemarks (objects or skin)
- -cigarette butts
- -drinking vessels
- -vaginal/penile swabs
- -expirated blood
How would you begin to detect saliva stains?
- -Stiff area
- -whitish stain
- -use an ALS
- -look at obvious locations where saliva would be (i.e. the end of a cigarette)
Which body fluids flouresce?
- -breast milk
What types/conditions of ALS can be used in detection of saliva?
- -UV light (200-400nm) can damaging to DNA
- -polilight, omnichrome, ultralight- 450nm
- -needs an orange filter
- -not damaging to DNA
- -Longwave UV (365-415nm)
- -UV A
- - good for body fluids such as semen and salive- exhibit a greenish flouresence
- -blood appears black
- -no barrier filter needed, but goggles used for eye protection
What are the collection techniques for saliva stains?
- -swabs: bitemarks (on skin, lightly rub with moistened swab); Cans, cups, bottles (swab inside and outside of rim)
- -Cuttings: masks, cigarette filter, vaginal swabs, envelopes
Oral swab standards are replacing drawn blood samples for DNA samples. (T/F)
True. because of ease of collection
Saliva contains ABH subtances in secretors. (T/F)
True. saliva samples were sometimes taken as reference materials for determining secretor status and stains were types using absorbtion-elution or absorption-inhibition
What are presumptive tests for saliva?
Starch gel, Phadebas
What are the confirmatory tests for saliva?
- also detection of high levels of salivary amylase and observation of glycogen free nucleated epithelial cells = saliva confirmation
Starch-Iodine Radial diffusion test
- -agarose gel with starch added
- -iodine added, dyes starch dark blue
- -absence of blue color, indicates no starch because amylase digests starch
- -indicates hydrolysis reaction
What are the advantages and disadvantages of the
starch-iodine radial diffusion test?
- -Fairly easy
- -can run multiple samples on same gel
- -identifies amylase
- -difficult to use as locater
- -not specific to saliva
What is the phadebas test?
- -1974 began to be used forensically
- -Insoluble blue dye attached to starch polymer
- -Upon hydrolysis by amylase, blue dye released
How does the phadebas test work?
- Active ingredient: starch polymer chains are interlinked to form spheres of uniform and pre-defined size- called bio-degradable starch microspheres (DSMS)
- -DSMS are insoluble in water- a water soluble
- blue dye is chemically attached and as long as the dye is bound to the DSM, it
- remains insoluble in water
- -In the presence of amylase, the DMSs are
- degraded by the enzyme, at a speed increasing with the solution’s amylase
What is the difference between the Phadebas
forensic tube test and the Phadebas forensic press test?
- Tube test:
- -useful when amylase is suspected in mixed body fluid stains
- -test quantifies amylase activity in a positive stain
- -spectrophotometer can measure optical density of the sample
- Press test:
- -used for detection of hidden saliva stains (mapping)
- - paper can be tested for semen when amylase test is complete
How can you interpret a positive result of the phadebas test?
- - Quantitative assays: free water soluble fraction of dye is measured with spectrophotometer (Concentration of dye is proportional to amylase)
- - Qualitative assays: (press test) diffusion of liberated blue dye molecules indicates the presence of amylase
- -Manufacture claims no other body fluid (except
- some fecal stains) will yield a positive result in 10 minutes
What is the mechanism behind the SALIgAE test?
- The mechanism is unknown due to proprietary nature
- - Salivary amylase reacts with colorless tests to
- produce a yellow color change in 1-10 minutes
Gel electrophoresis/diffusion cannot differentiate between AMY 1 and AMY 2. (T/F)
RSID saliva is considered confirmatory for human saliva. (T/F)
True (according to the manufacturer) however there is a low level cross reaction with human breast milk
What is the RSID saliva test?
- -immunochromatographic strip test that detects human salivary a-amylase
- - uses antibodies to detect presence of amylase rather than activity of amylase
- - only a fraction of extract is used, remainder is available for DNA analysis
Monoclonal antihuman salivary a-amylase antibodies from what animal is used in the RSID test?
Mechanism of detection of a-amylase
- -Ag-ab complex is formed
- - Ag-Ab migrates to the test area of the strip
- - Immobilized antibodies in test area “capture” the Ag-Ab complex forming an Ab-Ag-Ab sandwich
- -Colloidal gold accumulates at test area becomes visible as a pink band in the test region
No high dose hook effect when tested with 50ul saliva
Detection of a-amylase- RNA based assays
- - Use PCR to detect the expression of genes exclusive to the oral cavity
- - Relatively new application
- - Specific but can degrade easily
The failure to detect a-amylase means the absence of saliva. (T/F)
False- amylase degrades over time and starting values may be low in some samples
A DNA profile can be generated from as few as 100NSEC but may not contain detectable levels of amylase. (T/F)
Saliva reporting guidelines for positive result
Analysis of ___ gave chemical indications for the presence of amylase, a component of saliva.
Saliva reporting guidelines for negative result
Amylase, a constituent of saliva, was not detected on ___.