Forensic Biology by Richard Li

1. Chapter 5: Serology Techniques Section 5.1 only Forensic Biologyby Richard Li

2. Serological Techniques • Forensic Serology = Detection and identification of bodily fluids • Enzymatic assays • Blood: peroxidase in heme group of hemoglobin • Semen: acid phosphatase • Saliva: amylase • Antigen-antibody assays • Primary binding assays • Secondary binding assays Forensic Biology by Richard Li

3. Enzymatic Assays: Blood • Enzymatic assays • Kastle-Meyer test for blood • Tests for presence of peroxidase activity • Peroxidases break down hydrogen peroxide to water and oxygen free radicals (O-) • Oxygen free radicals are strong oxidants and strip hydrogens off the K-M reagent (oxidize it) • The reduced form of K-M is colorless but the oxidized form is bright pink • Not the same dark red color of blood

5. Method • Moisten Q-tip swab in distilled water • Lightly touch suspected blood stain with tip of Q-tip • Add one drop K-M reagent • Add one drop hydrogen peroxide • Look for fast color change to bright pink

6. Limitations • False positive reactions • Any strong oxidizing agent (e.g. bleach) • Will oxidize K-M reagent even in the absence of peroxidase • Any substance with peroxidase activity • Bacteria • Plants • Not species-specific • Reacts with blood from any animal • Sensitivity (too high?) • Will detect blood in urine, saliva, and other body fluids if trace amounts are present

7. α-naphthyl acid phosphate monosodium salt sodium phosphate + naphthol Acid phosphatase napthol + Brentamine Purple azo dye Coupling reaction Enzymatic Assays: Semen • Enzymatic assays • AP test for semen • Tests for presence of acid phophatase activity • AP liberates naphthol from alpha-naphthol and the naphthol then reacts with brentamine to form a purple-colored dye

8. Method • Spray a Whatman paper circle with distilled water • Lay the paper down over the suspected semen stain • Leave in contact with stain 30-60 seconds • Remove paper circle from stain and spray with AP spot solution • Look for a rapid color change to purple

9. Limitations • False positive reactions • Any substance with acid phophatase activity • Bacteria • Plants • Vaginal fluid • Not species-specific • Reacts with semen from any animal • Not usually a big problem in forensic casework • Animal sperm are morphologically distinct from primate sperm under the microscope

10. Enzymatic Assays: Saliva • Enzymatic assays • Amylase test for saliva • Tests for presence of amylase enzyme • Amylase is present in saliva and small intestine • Salivary amylase = ptyalin • Pancreatic amylase = amylopsin • Breaks down starch to simple sugars • Two types of starch: amylose and amylopectin • Amylose changes color from clear to deep blue-black in the presence of iodine

11. amylose amylopectin Forensic Biology by Richard Li

12. Method • Spray a Whatman paper circle with solution of soluble starch • Lay the paper down over the suspected saliva stain • Leave in contact with stain for 20 minutes • Incubate in a 37 deg moisture chamber for 1 hour, then dry • Spray with iodine and look for a lack of color change to deep blue-black

13. Limitations • False positive reactions • Any substance with amylase activity • Bacteria • Plants • Vomit • Not species-specific • Reacts with saliva from any animal that produces it • Not usually a big problem in forensic casework • Cats and dogs do not produce amylase

14. Primary Binding Assay • Enzyme-Linked Immunosorbent Assays (ELISA) is most common type used • Very sensitive • Colorimetric or fluorometric signal is proportional to the amount of bound antigen • Often performed in wells • Detected by color change • Two methods: • Well • Cassette

16. Immunochromatographic ELISAs • Rapid and simple test • Used as screening test in the field for seminal and saliva stains and for species identification • High-dose effect • Highly sensitive (too sensitive?) • Specificity still under debate • We will perform these in lab

17. T C T Positive test

18. T C Negative test