The Biological Crime Scene Every Contact Leaves a

The Biological Crime Scene "Every Contact Leaves a Trace". Blood at the Scene is the most visible example of the Locard Exchange Principle Dr. Edmond Locard

Solving Forensic Problems l Steps to problem solving l Understand the Problem l l l The parameters The issues Knowledge l Key to any successful analysis § § Understand the science Know the technology

The Mission: Never Miss Anything The Tools The Evidence Cascade Your Brain Experience Logical & Critical Thinking The Underlying Science Understanding the Technology

The Evidence Analysis Cascade Gross Visual Examination Stereomicroscopy Impression Trace Evidence Pattern Scrapping Tape lift Analysis Soil/Paint/Glass Hairs Presumptive Testing Pattern Analysis Fibers Tactile Chemical Enzyme Immuno. ALS Analysis Testing Chromat. Confirmatory Testing Biological Evidence Blood, Semen, Saliva Confirmatory Testing Species Immuno Microcrystal Testing Chromat. Analysis Instrumental Analysis Lectins Genetic Marker Testing Pattern Analysis DNA Microscopy

Forensically Important Biological Substances l l What are they? l Blood l Semen l Saliva l Urine l Feces l Vomit l Fingerprint residue l Sloughed off cells What else? l Bacteria l Plant material l Pollen l Viruses

Locating Biological Evidence Your Eye Touch Hi-Intensity & Alternate Light Sources Chemical Tests Enzymatic Tests Immunological Tests

Blood

Blood Cells Liquid Red Blood Cells White Blood Cells Plasma Serum

Forensically Speaking: What is Blood? Blood Complex Connective Tissue Cells Plasma Salts Hormones Antigens Drugs Antibodies Enzymes Blood Group Substances Individual Specific Antibodies White Cells Red Cells Genetic Markers HLA Antigens Blood Group Antigens DNA Isoenzymes

Forensically Critical Information from Blood Drugs of Abuse Prescriptions Genetic Markers Psychological Behavior Disease Susceptibility Identification Ancestry Racial Identity Sexing Individual Identity DNA Profiling Antibody Profiling

The Scientific Faces of Forensic Biology Chemistry Immunology/Chemistry Serology/Biochemistry Molecular Biology Population Genetics

The Investigators Job Find Those Stains! Biological Evidence Blood, Semen, Saliva Presumptive Testing Tools Tactile/Visual Analysis Chemical Enzyme ALS Testing

Using Alternate Light Sources To Find Biological Evidence

The Electromagnetic Spectrum Using Light to Find Biological Evidence Ultraviolet Region 190 -290 290 -400 Short wave Long wave Visible Region 400 -455 455 -492 Violet Blue 492 -577 Green 577 -597 597 -622 Yellow Orange 622 -700 Red Infrared Region >700 IR

ALS Wavelengths Applications to Finding Biological Evidence Type Bone Teeth Fingernails Body Fluids Dk Surfaces “ w/crust Mini. Scope 400 settings Goggle 455/CSS/515 Orange CSS UV White/oblique Camera Filter Orange Clear/Yellow Clear 1 -2 Orange None Hair untreated Blk White/oblique treated-red/bld 415/CSS Clear Yellow/Orange None Yellow/Orange Blood Clear/Yellow None 415, 455

Choosing a Goggle Color Range ALS Setting (nm) Goggle Long wave UV Violet Blue/green Green-red ------ 300 -400 515 -445 455 -515 536 CSS Clear Yellow Orange Red Orange

Detecting Blood @ the Scene

Historical Overview l Classification 1862 Chemistry

Chemical Testing Catalytic Tests

Blood – Presumptive tests Based on the peroxidase properties of hemoglobin heme

Blood – Presumptive tests heme iron porphyrin

Catalytic Tests: Presumptive Testing for Blood Van Deen’s or Day’s Test Kastle Meyer 1862 1901 Benzidine Tetramethylbenzidine 1904 1976

Common Presumptive Tests phenolphthalin (Kastle-Meyer) leucomalachite green (LMG) Luminol (Blue. Star) 3, 3’, 5, 5’-Tetramethylbenzidine (TMB) leucocrystal violet (LCV) o-tolidine Benzidine: Carcinogenic o-toluidine hydrogen peroxide: Bubbles

Presumptive Testing Practical Variations l One step l l Two Step l l l All reagents added together l Considered to be most sensitive l Doesn’t allow for identifying false positives Reagent added to the stain Peroxide added last Three Step l l l Alcohol added first Reagent second Peroxide last

Blood – Presumptive tests General Considerations heme has peroxidase activity Heme Fe+++ 2 H 2 O 2 Heme Fe++ 2 O· + 2 H 2 O Oxygen free radicals cleaved from peroxide group

Blood – Presumptive tests General Considerations Free radicals interact with organic chemicals (dyes) O· + Chemical reduced chemical oxidized Presumptive test detects oxidized organic dyes

Kastle-Meyer Test false negatives Rare Some substances inhibit reaction Blood can mask the color change Acidic solutions can mask the reaction

Blue. Star. TM +N 2 3 -aminophthalate Luminol + light

Using IR to Search for Blood Camera Viewfinder

Semen

Forensically Speaking: What is Semen? Semen Complex Connective Tissue Plasma Salts Hormones Genetic Markers Drugs Antigens Enzymes Antibodies Blood Group Substances Isoenzymes Individual Specific Antibodies Cells White Cells Sperm Cells Pre-Sperm Cells Genetic Markers HLA Antigens Blood Group Antigens DNA Isoenzymes

Semen Origins Contributing gland Percentage of ejaculate Testicles and epididymus 5 per cent Seminal vesicles 46 to 80 per cent Prostate gland 13 to 33 per cent Bulbourethral and urethral glands 2 to 5 per cent

Identifying Semen Test Type Specificity Microscopic Tests Spermatozoa ……………… 19 th Century……. . . Specific Visual Tests Crusty stains ………………………. Color of stain ………………………. UV Light Phosphorescence …… 1950’s, 1970’s……. . Alternate Light Sources ………… 1980’s……………. . . Nonspecific Chemical / Crystals Tests Florence Test ………… 19 th Century. . ………………… Barberio Test ………… 19 th Century. . ………………… Fructose/Zn …………. . 1950’s……………. . Nonspecific Enzyme/Protein Tests Acid Phosphatase ……………… 1950’s………………. Lactic Dehydrogenase-X ……… 1980’s………………. Sperm/seminal Esterases ……… 1980’s……………… Gamma seminoprotein …………… 1980’s……………. Nonspecific Specific for sperm Nonspecific Specific for seminal plasma Immunological Tests Prostate Specific Antigen (p 30). . . 1980’s. . . ABACard p 30 Test …………………. 1990’s…………. Specific for seminal plasma

Locating Dried Semen Visual Examination First l Stain Appearance l l l Crusty stains Yellow stains on aging Mixed with blood l Light red (diluted appearing) or streaks with blood § § § l Tactile l l Menstrual blood Blood from trauma Blood of assailant Feel the “crust” of the stain Stereomicroscopic Examination l Characteristic “look” of dried biological material l Extremely small stains

Semen – Presumptive tests l visual exam l l touch UV light (λ ~ 495 nm or CSS) l l enzymatic test l l Long wave UV w clear goggles acid phosphatase (AP) reagent crystal test l l Florence test - Choline Barberio test - Spermine

Semen – Presumptive tests Acid phosphatase test l Sodium a-naphthyl phosphate l Fast Blue l l l o-dianisidine combines with naphthyl group produces scarlet red color Acid Phosphatase l a-naphthyl phosphate o-dianisidine l broken down by AP frees naphthyl group Scarlet Color

Detection of Semen Using Light Sources Article Color Fiber W Light pants blue Cot/poly --------- Bed sht white Cotton 1 -4 1 -16 1 -8 panties white Nylon 1 -4 1 -2 shirt Cream/rd acetate 1 -2 1 -16 shirt Y/brown polyester --------- 1 -2 ----- sweater Gray/blk Poly/ct/ray --------- 1 -4 ----- sweater navy Nylon/acry neat 1 -2 1 -2 sock gray Poly/ct/ray ----- 1 -4 1 -4 sock white Poly/ct/ray 1 -2 1 -4 1 -8 Auvdel, M: UV Laser Comparison of Laser & UV Techniques Used in Detection of Body Secretions JFS: 32(2) 1987, 326 -345. Old ALS

Using the ALS To Locate Semen Stains 1968 Case Analyzed in 2005 CSS setting – Orange Goggles

Vaginal Secretions

Electrophoretic Separation of Vaginal & Seminal Acid Phosphatases

SAP/VAP Electrophoresis SAP VAP Bacterial Laboratory Technique for Separating Seminal from Vaginal Acid Phosphatase

Immunological Tests for Semen l Prostate Specific Antigen (p 30) Cross-over electrophoresis Rocket electrophoresis Elisa Immunological Chromatography ABAcard p 30 Test Card RSID (semenogelin )

Saliva

Forensically Speaking: What is Saliva? Saliva Complex Connective Tissue Oral Fluid Salts Hormones Genetic Markers Drugs Antigens Enzymes Antibodies Blood Group Substances Isoenzymes Individual Specific Antibodies Cells Epithelial Cells Genetic Markers DNA Blood Group Antigens

Using the ALS To Locate Saliva Stains 1968 Case Analyzed in 2005 CSS setting – Orange Goggles

Total AMY Levels in Semen & Saliva Body Fluid Avg. [AMY] 3 Saliva ………………………… 94 x 10 Semen (vasectomized) ………………. . 10 Semen (aspermatic) ………………. . . 18

Two Amylases in the Human Body AMY 1 AMY 2 Blood Saliva Blood Pancreas Vag. Sec.

Identifying Saliva Presumptive Tests for Amylase Identification of High Levels of Amylase Phaedebas – Scene versions of the test Diffusion into Starch Agarose – Scene adaptable Differentiation of AMY 1 v AMY 2 Plant Extracts - Lectins Monoclonal Antibodies agains AMY 1 & 2 mmunologyical Chromatograplhy ABA Card Saliva Test – Amy 1 RSID – Amy A

Detection of Saliva Using Light Sources Article Color Fiber W Light UV Laser pants blue Cot/poly ----- Bed sht white Cotton 1 -2 1 -8 1 -16 panties white Nylon ------ neat ----- shirt Cream/r d acetate ------ 1 -2 ----- shirt Y/brown polyester --------- --------- sweater Gray/blk Poly/ct/ray -------- Old ALS -----

Urine

Forensically Speaking: What is Urine? Urine Salts Liquid Cells Hormones Epithelial Cells Drugs Genetic Markers DNA

Concentrations of Components of Urine -v- Other Fluids Substance Urine Serum Saliva Semen Sweat phosphate 70 -105 2. 4 -3. 76 7. 4 -21. 1 11 . 009 -. 043 sulfate 14. 5 -122. 5 . 45 - - 0. 7 -7. 4 creatinine 105 -210 0. 6 0. 275 -0. 455 - 0. 1 -1. 3 creatine 0 -14 2. 7 - 20 - urea 14003500 16 -35 0 -18. 1 72 12 -57 uric acid 5. 6 -21 1. 6 -3. 9 0. 5 -8. 7 6 0. 07 -0. 25

Locating Urine Stains Microscopic, UV & Odor Fluorescence UV Light: ALS: can help locate color varies fluoresces weakly under different wavelengths Odor Gentle heating Kirk (1953) said most specific test for urine

Feces Odor Color Visual appearance Dissolution in water followed by heating Cellular Material (vegetable – cells w/DNA)

Confirming Human Origin Immunology in a Card Format Immunological Chromatography Blood Semen Saliva - ID

Human Blood Cross-reaction with Ferret Blood Anti-human Hemoglobin

Human Blood Cross-reaction with Ferret Blood Anti-human Hemoglobin

Human Blood – RSID Glycophorin-A

Saliva – RSID Card Amy A

Human Blood – OBTI Human-Ferret Cross-reaction

Old Stains Cold Cases Typical Problems 1. Degradation 2. Oxidation 3. Contamination 4. Solubility 5. Stupidity Never Trust a Negative Presumptive Test

Quality Assurance Reagent: Test Date 1: 10, 000 1: 100, 000 1: 1, 000 Comments Kastle Meyer: 4/15/09 + + Wk Cut off @ 100, 000 Kastle Meyer: 9/15/09 + + Wk Cut off @ 1, 000 Kastle Meyer: 1/15/10 + wk - Does not meet standard Kastle Meyer: 1/15/10 + + = Fresh reagent – Meets Standard