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Forensic Biology by Richard Li

Chapter 9: Identification of Saliva and Other Biological Fluids. Forensic Biology by Richard Li. Saliva: Biological Characteristics. Human salivary glands produce 1.0-1.5 liters per day Mostly water Amylase- digests starch in the oral cavity and small intestine Two types of amylase

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Forensic Biology by Richard Li

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

  2. Saliva: Biological Characteristics • Human salivary glands produce 1.0-1.5 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

  5. 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

  6. 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

  7. 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)

  8. 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

  9. 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

  10. 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

  11. Misc. Biological Fluids

  12. 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

  13. 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

  14. 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

  15. 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

  16. Muscle cells Fat cells Epithelial cells Onion cells

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