Presumptive Tests For Blood

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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
2
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

3
The Mission Never Miss Anything

• The Tools
• The Evidence Cascade
• Your Brain
• Experience

Logical Critical Thinking
The Underlying Science
Understanding the Technology
4
The Evidence Analysis Cascade
Gross Visual Examination
Stereomicroscopy
Trace Evidence
Impression Evidence
Biological Evidence
Scrapping
Tape lift
Pattern Analysis
Blood, Semen, Saliva
Hairs
Soil/Paint/Glass
Presumptive Testing
Pattern Analysis
Fibers
Tactile Analysis
Chemical Testing
Enzyme Testing
ALS
Immuno. Chromat.
Confirmatory Testing
Confirmatory Testing
Microscopy
Instrumental Analysis
Immuno Chromat.
Species Testing
Microcrystal Analysis
Lectins
Genetic Marker Testing
Pattern Analysis
DNA
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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

7
Locating Biological Evidence

• Your Eye
• Touch
• Hi-Intensity Alternate Light Sources
• Chemical Tests
• Enzymatic Tests
• Immunological Tests

8
Blood
9
Blood
Cells
Liquid
Plasma
Serum
Red Blood Cells White Blood Cells
10
Forensically Speaking What is Blood?
Blood Complex Connective Tissue
Plasma
Cells
Antigens
Hormones
White Cells
Red Cells
Salts
Drugs
Antibodies
Genetic Markers
Blood Group Substances
Enzymes
Blood Group Antigens
HLA Antigens
Individual Specific Antibodies
DNA
Isoenzymes
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Forensically Critical Information from Blood
Drugs of Abuse Prescriptions
Genetic Markers
Identification
Psychological Behavior
Disease Susceptibility
Ancestry
Sexing
Individual Identity
Racial Identity
DNA Profiling
Antibody Profiling
12
The Scientific Faces of Forensic Biology

• Chemistry
• Immunology/Chemistry
• Serology/Biochemistry
• Molecular Biology
• Population Genetics

13
The Investigators JobFind Those Stains!
Biological Evidence
Blood, Semen, Saliva
Presumptive Testing Tools
Tactile/Visual Analysis
Chemical Testing
Enzyme Testing
ALS
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Using Alternate Light SourcesTo Find Biological
Evidence
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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
492-577
577-597
597-622
622-700
Violet
Blue
Green
Yellow
Orange
Red
Infrared Region
gt700
IR
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ALS WavelengthsApplications to Finding
Biological Evidence
Evidence Type MiniScope 400
settings Goggle Camera Filter
Bone 455/CSS/515 Orange Orange Teeth Fing
ernails
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
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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
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Detecting Blood _at_ the Scene
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Historical Overview

• Classification

1862
Chemistry
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Chemical Testing
Catalytic Tests
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Blood Presumptive tests
Based on the peroxidase properties of hemoglobin
globin
heme
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Blood Presumptive tests
heme
iron
porphyrin
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Catalytic Tests Presumptive Testing for Blood
Van Deens or Days Test 1862
Kastle Meyer 1901
Benzidine 1904
Tetramethylbenzidine 1976
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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

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Presumptive TestingPractical Variations

• One step
• All reagents added together
• Considered to be most sensitive
• Doesnt allow for identifying false positives
• Two Step
• Reagent added to the stain
• Peroxide added last
• Three Step
• Alcohol added first
• Reagent second
• Peroxide last

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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

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Kastle-Meyer TestHow 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

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Blood Presumptive testsGeneral Considerations

• heme has peroxidase activity

Heme Fe
Heme Fe
2H2O2
2O 2H2O
Oxygen free radicals cleaved from peroxide group
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Blood Presumptive testsGeneral Considerations

• Free radicals interact
• with organic chemicals (dyes)

O
chemical oxidized
Chemical reduced
Presumptive test detects oxidized organic dyes
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Kastle-Meyer Test
false negatives

• Rare
• Some substances inhibit reaction
• Blood can mask the color change
• Acidic solutions can mask the reaction

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BlueStarTM
N2
3-aminophthalate
light
Luminol
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Using IR to Search for Blood
Camera Viewfinder
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Semen
34
Forensically Speaking What is Semen?
Semen Complex Connective Tissue
Plasma
Cells
Hormones
White Cells
Sperm Cells
Pre-Sperm Cells
Genetic Markers
Salts
Drugs
Antigens
Genetic Markers
Enzymes
Antibodies
Blood Group Antigens
HLA Antigens
Blood Group Substances
DNA
Isoenzymes
Isoenzymes
Individual Specific Antibodies
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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
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Identifying Semen

• Test Type Specificity
• Microscopic Tests
• Spermatozoa 19th Century..............
  Specific
• Visual Tests
• Crusty stains . Nonspecific
• Color of stain . Nonspecific
• UV Light Phosphorescence 1950s,
  1970s.. Nonspecific
• Alternate Light Sources 1980s... Nonspec
  ific
• Chemical / Crystals Tests
• Florence Test 19th Century.. Nonspeci
  fic
• Barberio Test 19th Century.. Nonspeci
  fic
• Fructose/Zn ..1950s.. Nonspecifi
  c
• Enzyme/Protein Tests
• Acid Phosphatase 1950s. Nonspecific
• Lactic Dehydrogenase-X 1980s. Specific
  for sperm
• Sperm/seminal Esterases 1980s Nonspecif
  ic

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Identifying SemenHistorical Perspectives

• Medico legal identification of semen in sexual
  assault cases
• Early part of 19th century
• First systematic efforts used chemical tests
• 1826
• Ollivier, Dangers Barruel
• Stain extd w/water ETOH
• Stain extract in ETOH had Spermatic odor
• Suspect had claimed stain was fat from uncooked
  animal meat.

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Identifying SemenHistorical Perspectives

• 1827
• Orphilia Case of 13 year old girl
• Testified
• 1. as defense expert after a physician said that
  hed isolated semen from vagina after 9
  days
• 2. No chemical tests available to ID semen
• 3. Girl had mucous discharge
• Designed tests
• Based on appearance of the stains
• Changes in color consistency on heating,
  immersion in water, odor emitted by moistened
  stain behavior of aqueous extract toward
• Compared with vaginal discharges, nasal mucus
  saliva stains
• Found sperm in fresh seminal samples in 18 year
  old dried semen
• (van Leeuwenhoek who credited medical student Ham
  with the discovery in 1677)

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Identifying SemenHistorical Perspectives

• 1837
• Rattier
• Published paper on ID of sperm in semen stains
• Suggested the procedure for medico-legal
  proceedings
• 1839
• Devergie
• Identified sperm in 10 month old semen stains
• Suggested that sperm ID was a more certain
  criteria for identifying semen than chemical
  tests
• Orphilia disagreed
• Bayard
• Published extensive paper on the use of the
  microscope in examining semen stains for sperm
• Procedures became widely accepted used from
  this point.
• Early chemical methods slowly abandoned.

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Identifying SemenHistorical Perspectives

• 1858
• Lassaigne
• Series of reagents to use with semen stains
• 1896
• Crystal tests
• First non-morphological test for semen that
  persisted
• Still used in very few laboratories

41
Identifying SemenHistorical Perspectives

• Crystal tests
• Florence Test
• 1896
• 1.65g KI, 2.54g I2 in 30mL water
• Reagent allowed to diffuse under cover slip on
  microscope slide
• Florence considered it a useful presumptive test
• Characteristic crystals not found with nasal,
  vaginal mucus, urine, sweat, saliva, tears, milk,
  cerebral fluid or leucorrheal discharge
• Seminal component called virispermine
• 1897
• Richter
• Positive crystals from vaginal uterine mucus
  from dead bodies
• Found that lecithin gave the test postulated
  that it was choline in semen that gave the
  crystals
• Whitney
• 2 ½ year old semen stains
• Positive test with morphnine, strychnine other
  alkaloids
• Thought it was the choline
• 1902
• Bocarius established that it is choline in semen
  that gives the test

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Identifying SemenHistorical Perspectives

• Crystal tests
• Florence Test
• 1937
• Kahane Levy
• Conducted biochemical studies tissue
  distribution of choline
• 11.2-14.4 mg choline/100 mL semen
• Any tissue with this concentration will give the
  Florence test

43
Identifying SemenHistorical Perspectives

• Crystal tests
• Barberio Test
• 1905
• Microscope slide test
• Saturated picric acid (water or abs ETOH)
  refractive yellow crystals
• Positive reaction with semen, semen stains,
  putrefied seminal material semen heated to 150
  deg C
• 1906
• Cevidalli
• Proposed test carried out in glycerin w/picric in
  ETOH
• Neg rxn with dog, horse, pig semen
• 1907
• Lecha-Marzo
• Regarded the test more specific than the Florence
  Test
• 1913
• Baecchi
• Suggested the crystals were spermine picrate
• 1924
• Rosenheim
• Confirmed crystals as spermine picrate

44
Identifying SemenHistorical Perspectives

• Crystal tests
• Barberio Test
• 1932
• Harrison confirmed that crystals were spermine
  picrate
• Proposed modified test using 2.5 TCA to extract
  stain centrifuge protein

CH3

N
H
C
H
CH3
OH-
O
H
C
2
2
CH3
Spermine 20-250mg/100 mL semen
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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

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Semen Presumptive tests

• visual exam
• touch
• UV light (? 495nm or CSS)
• Long wave UV w clear goggles
• enzymatic test
• acid phosphatase (AP) reagent
• crystal test
• Florence test - Choline
• Barberio test - Spermine

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Semen Presumptive tests
Acid phosphatase test

• high concentration in semen
• not unique to semen
• vaginal secretions
• blood of males with prostate cancer
• Enzymatic activity
• drops after three months
• keep in mind during exam!
• Negative results must be questioned

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Semen Presumptive tests
Acid phosphatase test

• Sodium a-naphthyl phosphate
• broken down by AP
• frees naphthyl group
• Fast Blue
• o-dianisidine
• combines with naphthyl group
• produces scarlet red color

a-naphthyl phosphate
Acid Phosphatase
o-dianisidine
Scarlet Color
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Semen Presumptive tests
Crystal tests

• Choline (Florence) test
• detects choline
• iodine solution
• microscopic choline-iodine crystals
• Barberio test
• detects spermine
• picric acid
• microscopic spermine picrate crystals

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Detection of Semen Using Light Sources
Article Color Fiber W Light W Light UV Laser Old ALS
pants blue Cot/poly --------- --------- --------- --------- ---------
Bed sht white Cotton 1-4 1-4 1-4 1-16 1-8
panties white Nylon 1-4 1-2 1-2 1-4 1-2
shirt Cream/rd acetate 1-2 1-16 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 1-2
sock gray Poly/ct/ray --------- 1-4 1-4 1-4 1-4
sock white Poly/ct/ray 1-2 1-4 1-4 1-8 1-8
Auvdel, M Comparison of Laser UV Techniques
Used in Detection of Body Secretions JFS 32(2)
1987, 326-345.
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Using the ALS To Locate Semen Stains
1968 Case Analyzed in 2005 CSS setting Orange
Goggles
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Vaginal Secretions
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Electrophoretic Separation of Vaginal Seminal
Acid Phosphatases
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SAP/VAP Electrophoresis
SAP
VAP
Bacterial
Laboratory Technique for Separating Seminal from
Vaginal Acid Phosphatase
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Immunological Tests for Semen

• Prostate Specific Antigen (p30)
• Cross-over electrophoresis
• Rocket electrophoresis
• Elisa
• Immunological Chromatography
• ABAcard p30 Test Card
• RSID (semenogelin )

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Saliva
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Forensically Speaking What is Saliva?
Saliva Complex Connective Tissue
Oral Fluid
Cells
Hormones
Epithelial Cells
Genetic Markers
Salts
Drugs
Antigens
Genetic Markers
Enzymes
Antibodies
Blood Group Antigens
DNA
Blood Group Substances
Isoenzymes
Individual Specific Antibodies
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Using the ALS To Locate Saliva Stains
1968 Case Analyzed in 2005 CSS setting Orange
Goggles
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An Historical Overview
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Total AMY Levels in Semen Saliva
Body Fluid
Avg. AMY
3
Saliva 94x10 Semen
(vasectomized) .. 10 Semen
(aspermatic) ... 18
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Two Amylases in the Human Body
AMY 2
AMY 1
Blood Pancreas Vag. Sec.
Blood Saliva
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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
• mmunologyical Chromatograplhy
• ABA Card Saliva Test Amy 1
• RSID Amy A

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Detection of Saliva Using Light Sources
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 --------- --------- --------- ---------
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Urine
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Forensically Speaking What is Urine?
Urine
Liquid
Cells
Epithelial Cells
Hormones
Salts
Drugs
Genetic Markers
DNA
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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

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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 1400-3500 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
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Locating Urine StainsMicroscopic, 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
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Identifying Urine StainsUrea Xanthydrol
Crystal Test
Alcoholic xanthydrol Acetic Acid
fibers Crystals form w/in 30 minutes
Kirk (1953) Didnt 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 Balthaza
rd Negative with blood, egg white, semen, milk,
feces 1947 Ishler False positives with
xanthydrol crystal test
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Identifying Urine StainsUrea Enzymatic Tests
Urease
Urease catalyzes decomposition of Urea urea
water CO2 2 NH3
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Feces
Odor Color Visual appearance Dissolution in water
followed by heating Cellular Material (vegetable
cells w/DNA)
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Confirming Human OriginImmunology in a Card
FormatImmunological Chromatography
Blood Semen Saliva - ID
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Human Blood Cross-reaction with Ferret
Blood Anti-human Hemoglobin
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Human Blood Cross-reaction with Ferret
Blood Anti-human Hemoglobin
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Human Blood RSIDGlycophorin-A
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Saliva RSID CardAmy A
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Human Blood OBTIHuman-Ferret Cross-reaction
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Old StainsCold Cases

• Typical Problems
• Degradation
• Oxidation
• Contamination
• Solubility
• Stupidity

Never Trust a Negative Presumptive Test
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Quality Assurance
ReagentTest Date 110,000 1100,000 11,000,000 Comments
Kastle Meyer4/15/09 Wk Cut off _at_ 100,000
Kastle Meyer9/15/09 Wk Cut off _at_ 1,000,000
Kastle Meyer 1/15/10 wk - Does not meet standard
Kastle Meyer 1/15/10 Fresh reagent Meets Standard