The Biological Crime Scene
L- 5
Dr. Varun Vyas
"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
Forensic serology is a crucial branch of forensic science that
deals with the detection, identification, and characterization of
biological fluids like blood, semen, saliva, and other bodily fluids
found at crime scenes.
⚫ 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 Tools
The Evidence Cascade
Your Brain
Experience
Logical & Critical
Thinking
The Underlying Science
Understanding the Technology
The Evidence Analysis Cascade
Gross Visual Examination
Stereomicroscopy
Biological Impression Trace
Evidence Evidence Evidence
Pattern Scrapping Tape lift
Blood, Semen, Saliva
Analysis Soil/Paint/Glass Hairs
Presumptive Testing Pattern Analysis
Fibers
Tactile Chemical Enzyme Immuno.
ALS
Analysis Testing Testing Chromat. Confirmatory Testing
Confirmatory Testing Instrumental Microscopy
Analysis
Species Immuno Microcrystal
Lectins
Testing Chromat. Analysis
Genetic Marker Testing
Pattern
DNA
Analysis
The Evidence Analysis Cascade
Gross Visual Examination
Stereomicroscopy
Biological Impression Trace
Evidence Evidence Evidence
Pattern Scrapping Tape lift
Presumptive Testing Pattern Analysis Analysis Soil/Paint/Glass Hairs
Fibers
Tactile Chemical Enzyme Immuno .
ALS
Analysis Testing Testing Chromat . Confirmatory Testing
Confirmatory Testing Instrumental Microscopy
Analysis
Species Immuno Microcrystal
Lectins
Testing Chromat . Analysis
Pattern
Genetic Marker Testing Analysis
DNA
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 Sources
Chemical Tests
Enzymatic Tests
Immunological Tests
Blood
Blood
Cells Liquid
Red Blood Cells Plasma Serum
White Blood Cells
Forensically Speaking:
What is Blood?
Blood
Complex
Connective Tissue
Plasma Cells
Hormones Antigens White Cells Red Cells
Salts Drugs Antibodies Genetic Markers
Blood Group
Enzymes Substances
HLA Blood Group
Antigens Antigens
Individual
Specific DNA Isoenzymes
Antibodies
Forensically Critical
Information from Blood
Drugs of Abuse
Prescriptions
Genetic Markers
Psychological Behavior Identification
Disease Susceptibility Ancestry Sexing
Racial Identity Individual Identity
DNA Antibody
Profiling 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 Chemical Enzyme
ALS
Analysis Testing 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 Long
wave wave
Visible Region
400-455 455-492 492-577 577-597 597-622 622-700
Violet Blue Green Yellow Orange Red
Infrared Region
>700
IR
ALS Wavelengths
Applications to Finding Biological Evidence
Evidence Type MiniScope 400
settings Goggle Camera Filter
Bone 455/CSS/515 Orange Orange
Teeth
Fingernails
Body Fluids CSS Orange 1-2 Orange
Dk Surfaces UV Clear/Yellow None
“ w/crust White/oblique Clear None
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) Goggle
Long wave UV 300-400 Clear
Violet 515-445 Yellow
Blue/green 455-515 Orange
Green-red 536 Red
------ CSS Orange
Detecting Blood @ the Scene
Historical Overview
⚫ Classification
1862
Chemistry
Chemical Testing
Catalytic Tests
Blood – Presumptive tests
Based on the peroxidase
properties of hemoglobin
globin
heme
Blood – Presumptive tests
heme
iron
porphyrin
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 Testing
Practical 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
Kastle-Meyer reagent
⚫ phenolphthalin
⚫ Organic molecule that becomes an indicator
⚫ potassium hydroxide
⚫ makes solution basic
⚫ Ethanol/water
⚫ used to dissolve stain
⚫ zinc dust
⚫ Used to reduce Phenolphthalein to phenolphthalin
⚫ Continued presence in final solution slows
spontaneous oxidation of phenolphthalein
Kastle-Meyer Test
How run the Test
⚫ Dissolve the stain in water/ethanol
⚫ KM reagent added to stain
⚫ color change at this point: false positive
⚫ Add 3% H2O2
⚫ Pink: KM positive
Blood – Presumptive tests
General Considerations
heme has peroxidase activity
Heme Heme
Fe+++ Fe++
2H2O2 2O· + 2H2O
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
BlueStarTM
+N2
3-aminophthalate +
Luminol
light
Using IR to Search for Blood
Camera Viewfinder
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 AMY1 v AMY2
Plant Extracts - Lectins
Monoclonal Antibodies agains AMY 1 & 2 (Salivary
Amylase)
Immunological Chromatography
ABA Card Saliva Test – Amy 1
RSID – Amy A
Two Amylases
in the Human Body
AMY 1 AMY 2
Blood
Blood Pancreas
Saliva Vag. Sec.
Detection of Saliva Using Light Sources
Article Color Fiber W Light UV Laser Old ALS
pants blue Cot/poly --------- ------ -------- ---------
- -
-
-
Bed sheet white Cotton 1-2 1-8 1-16 1-16
panties white Nylon --------- ------ neat ---------
-
-
-
shirt Cream/r acetate --------- ------ 1-2 ---------
d -
-
-
shirt Y/brown polyester --------- ------ -------- ---------
- -
-
-
sweater Gray/blk Poly/ct/ray --------- ------ -------- ---------
- -
-
Urine
Forensically Speaking:
What is Urine?
Urine
Liquid Cells
Epithelial
Hormones Cells
Salts Drugs Genetic Markers
DNA
Locating Urine Stains
⚫ Based on the detection of inorganic anions & organic compounds
typically found in urine
⚫ Inorganic anions
⚫ Phosphate
⚫ Sulfate
⚫ Organic compounds
⚫ Creatine
⚫ Creatinine
⚫ Steroid derivatives
⚫ Urinary indican
⚫ Urochrome
⚫ Free purine & pyrimidine
⚫ Urea
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 1400- 16-35 0-18.1 72 12-57
3500
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: can help locate
color varies
ALS: fluoresces weakly under different
wavelengths
Odor
Gentle heating
Kirk (1953) said most specific test for urine
Identifying Urine Stains
Urea – Xanthydrol Crystal Test
Alcoholic xanthydrol & Acetic Acid + fibers
Crystals form w/in 30 minutes
Kirk (1953): Didn’t put much stock in identifying urea to ID Urine
1914
Policard: Suggested using xanthydrol for urea crystals
Test carried out on few threads of stained material
1915
Maiocchi Found false positives with serum, saliva, tears
1922
Balthazard Negative with blood, egg white, semen, milk, feces
1947
Ishler False positives with xanthydrol crystal test
Identifying Urine Stains
Urea – Enzymatic Tests: Urease
Urease catalyzes decomposition of Urea
urea + water CO2 + 2 NH3
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 –Rapid Stain Identification of
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 1:1,000,000 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
