The Biological Crime Scene

"Every Contact Leaves a Trace".

Dr. Edmond Locard

Blood at the Scene is the most visible example of the Locard Exchange Principle

Solving Forensic Problems

Steps to problem solving Understand the Problem

The parameters The issues

Knowledge Key to any successful analysis

Understand the science Know the technology

The Mission: Never Miss Anything

The ToolsThe Evidence Cascade

Your Brain

Experience

The Underlying Science

Understanding the Technology

Logical & Critical Thinking

Pattern Analysis

The Evidence Analysis Cascade

Trace Evidence

Presumptive Testing

Scrapping Tape lift

Gross Visual Examination

Tactile Analysis

Stereomicroscopy

Genetic Marker Testing

ImmunoChromat.

HairsFibers

Soil/Paint/Glass

Pattern Analysis

Impression Evidence

BiologicalEvidence

ChemicalTesting

EnzymeTesting

Confirmatory Testing

Species Testing

MicrocrystalAnalysis

ALS

Blood, Semen, Saliva

Lectins

DNA

Confirmatory Testing

MicroscopyInstrumentalAnalysis

Immuno.Chromat.

Pattern Analysis

Forensically Important Biological Substances

What are the they? Blood Semen Saliva Urine Feces Vomit Fingerprint residue Sloughed off cells

What else? Bacteria Plant material Pollen Viruses

Locating Biological Evidence

Your Eye Touch

Hi-Intensity & Alternate Light SourcesChemical Tests

Enzymatic Tests Immunological Tests

Blood

Blood

Cells Liquid

Red Blood CellsWhite Blood Cells

Plasma Serum

Plasma Cells

BloodComplex

Connective Tissue

Salts

Blood GroupAntigens

Antigens

Drugs

Hormones

Antibodies

DNA

HLA Antigens

Blood Group Substances

White Cells Red Cells

Enzymes

Isoenzymes

Genetic Markers

Individual Specific

Antibodies

Forensically Speaking: What is Blood?

Genetic Markers

Drugs of AbusePrescriptions

IdentificationPsychological Behavior

Disease Susceptibility

Individual Identity

Ancestry Sexing

Racial Identity

Forensically Critical Information from Blood

DNA Profiling

Antibody Profiling

The Scientific Faces of Forensic Biology

Chemistry

Immunology/Chemistry

Serology/Biochemistry

Molecular Biology

Population Genetics

The Investigators JobFind Those Stains!

Presumptive Testing Tools

Tactile/Visual Analysis

BiologicalEvidence

ChemicalTesting

EnzymeTesting

ALS

Blood, Semen, Saliva

Using Alternate Light SourcesTo Find Biological Evidence

The Electromagnetic Spectrum Using Light to Find Biological Evidence

190-290 290-400

Ultraviolet Region

Shortwave

Longwave

Visible Region

400-455 455-492

>700

492-577 577-597 597-622 622-700

Infrared Region

Violet Blue Green Yellow Orange Red

IR

ALS WavelengthsApplications to Finding Biological Evidence

Bone 455/CSS/515 Orange OrangeTeethFingernails

Body Fluids CSS Orange 1-2 Orange Dk Surfaces UV Clear/Yellow None “ w/crust White/oblique Clear None

Evidence Type MiniScope 400 settings Goggle Camera Filter

Hair untreated Blk White/oblique Clear None treated-red/bld 415/CSS Yellow/Orange Yellow/Orange

Blood 415, 455 Clear/Yellow None

Choosing a Goggle Color

Color Range ALS Setting (nm) GoggleLong wave UV 300-400 ClearViolet 515-445 YellowBlue/green 455-515 OrangeGreen-red 536 Red - - - - - - CSS Orange

Detecting Blood @ the Scene

Historical Overview

Classification

1862

Chemistry

Chemical Testing

Catalytic Tests

Blood – Presumptive testsBased on the peroxidase properties of hemoglobin

globin

heme

Blood – Presumptive tests

heme

porphyrin

iron

Catalytic Tests: Presumptive Testing for Blood

Van Deen’s or Day’s Test 1862

Kastle Meyer 1901

Benzidine 1904

Tetramethylbenzidine 1976

Common Presumptive Tests

phenolphthalin (Kastle-Meyer)

leucomalachite green (LMG)

Luminol (BlueStar)

3,3’,5,5’-Tetramethylbenzidine (TMB)leucocrystal violet (LCV)

o-tolidine

Benzidine: Carcinogenic

o-toluidine

hydrogen peroxide: Bubbles

Presumptive TestingPractical Variations

One step All reagents added together

Considered to be most sensitive

Doesn’t allow for identifying false positives

Two Step Reagent added to the stain Peroxide added last

Three Step Alcohol added first Reagent second Peroxide last

Blood – Presumptive testsGeneral Considerations

heme has peroxidase activity

2H2O2

2O· + 2H2O

Heme Fe+++

Oxygen free radicals

cleaved from peroxide group

Heme Fe++

Blood – Presumptive testsGeneral Considerations

Free radicals interact

with organic chemicals (dyes)

O· chemical oxidized

Presumptive test detects oxidized organic dyes

+ Chemical reduced

Kastle-Meyer Test

Rare

Some substances inhibit reaction

Blood can mask the color change

Acidic solutions can mask the reaction

false negatives

BlueStarTM

LuminolLuminol3-3-aminophthalateaminophthalate

+N+N22

+ + lightlight

Using IR to Search for Blood

Camera Viewfinder

Semen

Plasma Cells

SemenComplex

Connective Tissue

Salts

Blood GroupAntigens

AntigensDrugs

Hormones

Antibodies

DNA

HLA AntigensBlood Group

Substances

White Cells

Sperm Cells

Enzymes

Isoenzymes

Genetic Markers

Individual Specific

Antibodies

Pre-Sperm Cells

Genetic Markers

Isoenzymes

Forensically Speaking: What is Semen?

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

Semen Origins

Identifying SemenTest Type Specificity

Microscopic TestsSpermatozoa ………………19th Century…….............. Specific

Visual TestsCrusty stains ……………………………………………. NonspecificColor of stain ……………………………………………. NonspecificUV Light Phosphorescence ……1950’s, 1970’s…….. Nonspecific Alternate Light Sources …………1980’s……………... Nonspecific

Chemical / Crystals Tests Florence Test …………19th Century..………………… NonspecificBarberio Test …………19th Century..………………… NonspecificFructose/Zn …………..1950’s……………………….. Nonspecific

Enzyme/Protein TestsAcid Phosphatase ………………1950’s………………. NonspecificLactic Dehydrogenase-X ……… 1980’s………………. Specific for spermSperm/seminal Esterases ………1980’s……………… NonspecificGamma seminoprotein ……………1980’s……………. Specific for seminal plasma

Immunological TestsProstate Specific Antigen (p30) ......1980’s.............. Specific for seminal plasmaABACard p30 Test ………………….1990’s…………. Specific for seminal plasma

Locating Dried SemenVisual Examination First

Stain Appearance Crusty stains Yellow stains on aging Mixed with blood

Light red (diluted appearing) or streaks with blood Menstrual blood Blood from trauma Blood of assailant

Tactile Feel the “crust” of the stain

Stereomicroscopic Examination Characteristic “look” of dried biological material

Extremely small stains

Semen – Presumptive tests visual exam

touch UV light (λ ~ 495nm or CSS)

Long wave UV w clear goggles

enzymatic test acid phosphatase (AP) reagent

crystal test Florence test - Choline Barberio test - Spermine

Sodium a-naphthyl phosphate broken down by AP frees naphthyl group

Fast Blue o-dianisidine combines with naphthyl

group

produces scarlet red color

Acid phosphatase testSemen – Presumptive tests

a-naphthyl phosphate

Acid P

hosphatase

o-dianisidine

Scarlet Color

Article Color Fiber W Light UV Laser Old ALS

pants blue Cot/poly --------- --------- --------- ---------

Bed sht white Cotton 1-4 1-4 1-16 1-8

panties white Nylon 1-4 1-2 1-4 1-2

shirt Cream/rd acetate 1-2 1-16 1-16 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 1-2

sock gray Poly/ct/ray --------- 1-4 1-4 1-4

sock white Poly/ct/ray 1-2 1-4 1-8 1-8

Detection of Semen Using Light Sources

Auvdel, M: Comparison of Laser & UV Techniques Used in Detection of Body SecretionsJFS: 32(2) 1987, 326-345.

Using the ALS To Locate

Semen Stains

1968 Case Analyzed in 2005CSS 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

Prostate Specific Antigen (p30)

Cross-over electrophoresisRocket electrophoresisElisaImmunological Chromatography

ABAcard p30 Test CardRSID (semenogelin )

Saliva

Oral Fluid Cells

SalivaComplex

Connective Tissue

Salts

Blood GroupAntigens

AntigensDrugs

Hormones

AntibodiesDNABlood Group

Substances

Epithelial Cells

Enzymes

Isoenzymes

Genetic Markers

Individual Specific

Antibodies

Genetic Markers

Forensically Speaking: What is Saliva?

Using the ALS To Locate Saliva Stains

1968 Case Analyzed in 2005CSS setting – Orange Goggles

Total AMY Levels in Semen & Saliva

Saliva ………………………………………………… 94x10Semen (vasectomized) …………………………….. 10Semen (aspermatic) ………………………………... 18

3

Body Fluid Avg. [AMY]

Two Amylases in the Human Body

AMY 1

Blood Saliva

AMY 2

Blood PancreasVag. Sec.

Identifying Saliva

Presumptive Tests for AmylaseIdentification of High Levels of Amylase

Phaedebas – Scene versions of the testDiffusion into Starch Agarose – Scene adaptable

Differentiation of AMY1 v AMY2Plant Extracts - LectinsMonoclonal Antibodies agains AMY 1 & 2

mmunologyical ChromatograplhyABA Card Saliva Test – Amy 1RSID – Amy A

Article Color Fiber W Light UV Laser Old ALS

pants blue Cot/poly --------- ---------

---------

---------

Bed sht white Cotton 1-2 1-8 1-16 1-16

panties white Nylon --------- ---------

neat ---------

shirt Cream/rd

acetate --------- ---------

1-2 ---------

shirt Y/brown polyester --------- ---------

---------

---------

sweater Gray/blk Poly/ct/ray --------- ---------

---------

---------

sweater navy Nylon/acry neat neat neat neat

sock gray Poly/ct/ray --------- ---------

---------

---------

sock white Poly/ct/ray --------- ---------

---------

---------

Detection of Saliva Using Light Sources

Urine

Liquid Cells

Urine

Salts Drugs

Hormones

DNA

Epithelial Cells

Genetic Markers

Forensically Speaking: What is Urine?

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 1400-3500

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

Concentrations of Components of Urine -v- Other Fluids

Locating Urine StainsMicroscopic, UV & Odor

FluorescenceUV Light: can help locate

color variesALS: fluoresces weakly under different

wavelengths

OdorGentle heating Kirk (1953) said most specific test for urine

FecesOdorColor

Visual appearanceDissolution in water followed by heating

Cellular Material (vegetable – cells w/DNA)

Confirming Human OriginImmunology in a Card Format

Immunological Chromatography

BloodSemen

Saliva - ID

Human BloodCross-reaction with Ferret BloodAnti-human Hemoglobin

Human BloodCross-reaction with Ferret BloodAnti-human Hemoglobin

Human Blood – RSIDGlycophorin-A

Saliva – RSID CardAmy A

Human Blood – OBTIHuman-Ferret Cross-reaction

Old StainsCold Cases

Never Trust a Negative Presumptive Test

Typical Problems1.Degradation2.Oxidation3.Contamination4.Solubility5.Stupidity

Quality Assurance

Reagent:Test Date 1:10,000 1:100,000 1:1,000,000 Comments

Kastle Meyer:4/15/09 + + Wk Cut off @ 100,000

Kastle Meyer:9/15/09 + + Wk Cut off @ 1,000,000

Kastle Meyer: 1/15/10 + wk - Does not meet standard

Kastle Meyer: 1/15/10 + + = Fresh reagent – Meets Standard