Forensic Bio

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Forensic Bio
2010-04-28 00:13:18
Saliva Forensic Bio BU

Saliva Lecture
Show Answers:

  1. What are the components of saliva?
    • 98% water
    • 2% (Electrolytes, Mucus, Antibacterial compounds, Enzymes, Cells)
  2. 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
  3. What is the daily production of Saliva in humans?
    0.75-1.5 liters
  4. 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
  5. Identification of saliva is based on the detection of:
    • inorganic anions (thiocynate, nitrites)
    • Enzymes (alkaline phosphatase, Amylase)
    • antigens (HSA)
  6. Thiocyanate
    • -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
  7. Nitrites
    • -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
  8. 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
  9. b amylase (beta)
    • -found in plants; breaks starch into sugar in ripening fruit
    • -yeast, mold and bacteria
  10. 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
  11. 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
  12. 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.
  13. Feces can sometimes exhibit amylase activity higer than saliva. (T/F)
  14. 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
  15. 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
  16. 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)
  17. Individualizing AMY1 and AMY2
    • -isoenzymes (multiple molecular forms) will separate with polycrylamide or agar gel
    • -electrophoresis:
    • -AMY1 has 7 phenotypes
    • -AMY2 has 3 phenotypes
  18. What types of forensic samples could saliva be recovered from?
    • -bitemarks (objects or skin)
    • -cigarette butts
    • -drinking vessels
    • -masks
    • -condoms
    • -vaginal/penile swabs
    • -stamps/envelopes
    • -expirated blood
  19. 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)
    • -mapping
  20. Which body fluids flouresce?
    • -Semen
    • -saliva
    • -urine
    • -breast milk
  21. 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
  22. 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
  23. Oral swab standards are replacing drawn blood samples for DNA samples. (T/F)
    True. because of ease of collection
  24. 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
  25. What are presumptive tests for saliva?
    Starch gel, Phadebas
  26. What are the confirmatory tests for saliva?
    RSID, RNA-based

    - also detection of high levels of salivary amylase and observation of glycogen free nucleated epithelial cells = saliva confirmation
  27. 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
  28. What are the advantages and disadvantages of the
    starch-iodine radial diffusion test?
    • Advantages:
    • -Cheap
    • -Fairly easy
    • -can run multiple samples on same gel
    • -identifies amylase
    • disadvantages:
    • -difficult to use as locater
    • -not specific to saliva
  29. 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
  30. 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
    • activity
  31. 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
  32. 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
  33. 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
  34. Gel electrophoresis/diffusion cannot differentiate between AMY 1 and AMY 2. (T/F)
  35. 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
  36. 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
  37. Monoclonal antihuman salivary a-amylase antibodies from what animal is used in the RSID test?
  38. 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
  39. 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
  40. 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
  41. A DNA profile can be generated from as few as 100NSEC but may not contain detectable levels of amylase. (T/F)
  42. Saliva reporting guidelines for positive result
    Analysis of ___ gave chemical indications for the presence of amylase, a component of saliva.
  43. Saliva reporting guidelines for negative result
    Amylase, a constituent of saliva, was not detected on ___.