Card Set Information
Saliva Forensic Bio BU
What are the components of saliva?
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:
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)
-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)
-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
-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
: Salivary amylase (HSA)- saliva, breast milk, perspiration, tears
: 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)
How would you begin to detect saliva stains?
-use an ALS
-look at obvious locations where saliva would be (i.e. the end of a cigarette)
Which body fluids flouresce?
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)
- 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?
: bitemarks (on skin, lightly rub with moistened swab); Cans, cups, bottles (swab inside and outside of rim)
: 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?
-can run multiple samples on same gel
-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?
-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
-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 ___.