1. Forensic Biology by Richard Li
Chapter 9: Identification of Saliva and Other Biological Fluids
Forensic Biology by Richard Li

2. Saliva: Biological Characteristics
Human salivary glands produce liters per day
Mostly water
Amylase- digests starch in the oral cavity and small intestine
Two types of amylase
β-amylase: Plant and bacterial sources
α-amylase: HSA (human saliva), HPA (human pancreas)

3. Saliva: Presumptive Assays
Visual Examination
ALS (470nm), Microscopic
Starch-iodine assays
Iodine used to test for starch
Not specific
Colorimetric assays
Dye-labeled amylase substrates are employed
Phadebas test

4. Saliva: Presumptive Assays
Starch-iodine assays
Amylase overlay assay already discussed
We’ll perform this assay in lab
Amylase radial diffusion assay
Pour agar gel containing starch into culture plate
Create wells in solidified agar with vacuum punch
Add extract of evidence sample to well; Add standards to other wells (amylase samples at known concentrations)
Incubate overnight at 37 degrees C
Stain with iodine solution

6. Saliva: Presumptive Assays
Phadebas tablets consist of insoluble starch polymers to which a blue dye has been covalently bound
Extract sample and add tablet
When amylase is present:
Polymers are degraded
Dye is liberated and becomes soluble
Concentration of soluble dye is measured by spectrophotometry at 620 nm

7. Saliva: Confirmatory Assays
Immunochromatographic Assays
RSID-Saliva
Similar to PSA and hemoglobin tests
ELISA
Monoclonal HSA antibody
RNA-based assays
Certain genes are specifically expressed in certain types of cells
Amylase mRNA

8. Misc. Biological Fluids
Vaginal secretions, Menstrual blood, Urine, Vomitus, and Feces
Less frequently encountered
Can be probative in certain cases
Vaginal secretions
Gel mobility assays
Compare SAP and VAP mobilities in protein gel
RNA-based assays
β-defensin 1: antimicrobial peptide
Mucin 4 (membrane protein of endocervix)

9. Misc. Biological Fluids
Menstrual Blood
Color assays
Detect glycogenated nucleated squameous epithelial cells in vaginal secretions
Can also be used to test for vaginal secretions
RNA assay
Detects matrix metalloprotease, found only in menstrual blood
Involved in tissue remodeling during menstruation

10. Misc. Biological Fluids
Urine:
Presumptive assay tests for urea
All animals must get rid of excess nitrogen from breakdown of proteins and amino acids
Aquatic animals get rid of nitrogen as ammonia
Birds and terrestrial reptiles use uric acid
Mammals use urea and expel in urine
Urea

11. Misc. Biological Fluids
Assay for urea:
Prepare Whatman filter paper circle with one drop of bromothymol blue, a pH indicator dye
Yellow-green at pH 6
Aqua blue at ph greater than 7.6
Allow bromothymol blue to dry
Add drop of extract from evidence sample on top of dye, then add drop of urease
If urea present, urease will degrade to ammonia
Ammonia will raise pH and dye will turn aqua blue

12. Misc. Biological Fluids

13. Misc. Biological Fluids
Confirmatory tests for human urine
Immunological assays
ELISA or immunochromatographic test for presence of Tamm Horsefall glycoprotein
Abundant in urine
Human form can be targeted
Ouchterlony assay
Less sensitive
Based on precipitation reaction between antibody and antigen (human Tamm Horsefal proteinl) in diffusion gels

14. Misc. Biological Fluids
Vomitus
Microscopy for food particles
Enzymatic test for pepsin
Proteolytic enzyme secreted from gastric gland
Sweat
Immunological assays
Human sweat-specific antigen

15. Misc. Biological Fluids
Feces:
During heme catabolism, waste product called bilirubin is produced
Bilirubin excreted in feces: gives it its brown color
Gut bacteria break down some of bilirubin to urobilinogen, which is also excreted in feces
Urobilinogen

16. Misc. Biological Fluids
Presumptive Assay for Feces:
Prepare extract of feces stain
Oxidize to urobilinogen to urobilin
Add zinc chloride
If urobilin present, extract will fluoresce under UV light
Confirmatory Assay for Feces
Microscopy to look for animal and plant cells present due to digestion

17. Muscle cells
Fat cells
Epithelial cells
Onion cells