Identification and Characterization of Blood and Bloodstains

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Identification and Characterization of Blood and
Bloodstains

• Dr. Jason Linville
• University of Alabama at Birmingham
• jglinvil_at_uab.edu

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Summary

• Introduction to Blood
• Identification of Blood as Blood
• Determining Species Origin of Blood
• Identifying Humans using Blood

Suggested Reading Material Forensic Science
Handbook, vol 1, Chapter 7
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Introduction to Blood

• What you should already know

Its that red stuff that comes out of your
fingers following a kitchen accident.
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Introduction to Blood
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Introduction to Blood
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Introduction to Blood gt Components
Red Blood Cells No DNA
White Blood Cells DNA in nucleus
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Introduction to Blood gt Components
Platelets
Plasma
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Introduction to Blood gt Components
ABO blood groups found on outside of cell
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Introduction to Blood gt Components
ABO blood groups found on outside of cell
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Introduction to Blood gt Components

• RBC no DNA WBC yes DNA

• All blood cells have blood groups on outside of
  cell.

• Red Blood Cells contain the protein hemoglobin,
  which carries oxygen

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Introduction to Blood gt Components
Hemoglobin picks up and drops off oxygen
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Forensic Analysis of Blood

• Visual examination of evidence

• Presumptive screening test (Is it blood?)

• Confirmation test (Seriously, is it blood?)

• Determine species origin (human blood?)

• Identify the blood (whose blood is it?)

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Forensic Analysis of Blood

• Visual examination of evidence

• Presumptive screening test (Is it blood?)

• Confirmation test (Seriously, is it blood?)

• Determine species origin (human blood?)

• Identify the blood (whose blood is it?)

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Visual Analysis of Blood
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Visual Analysis of Blood

• Microscopic Analysis (no reagents)

• Might see erythrocytes and leukocytes.

• Microscopic exam affected greatly by aging and
  environmental factors

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Forensic Analysis of Blood

• Visual examination of evidence

• Presumptive screening test (Is it blood?)

• Confirmation test (Seriously, is it blood?)

• Determine species origin (human blood?)

• Identify the blood (whose blood is it?)

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Presumptive Test
Negative result means the questioned stain is not
likely blood.

• Positive result means the questioned stain is
  likely blood.

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Presumptive Test

• Presumptive tests produce a color reaction or
  release of light

• Tests rely on catalytic properties of blood
  (hemoglobin presence)

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Presumptive Test
Reaction Participants

• Chromogen (color changing chemical, i.e.
  benzidine, TMB, luminol, etc.)

• Hydrogen Peroxide (H2O2) oxidant

• Catalyst (hemoglobin or peroxidase)

H2O2 oxidizes colorless chemical into colored
chemical.
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Presumptive Test
General Reaction
Donor H2O2 ltgt Oxidized Donor 2 H2O
In blood test, TMB (or other) is the donor
TMB colorless H2O2 ltgt TMB (blue) 2 H2O
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Presumptive Test

• Color Tests
• Apply chromogen (color changing chemical)
• Apply oxidizing agent (hydrogen peroxide)
• The catalyst of the reaction is hemoglobin

Rapid color change is a positive result. This
means the stain is blood.
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Presumptive Test
Color Test Method

• Sample stain with clean cotton swab
• Add drop of chromogen
• Add drop of hydrogen peroxide

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Presumptive Test
Color Test Method

• Sample stain with clean cotton swab
• Add drop of chromogen
• Add drop of hydrogen peroxide

• Alternatively, cut out part of stain add
  chromogen and hydrogen peroxide in spot plate

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Presumptive Test
False Positive A positive result given by a
substance that is not blood.
Remember, other non-blood substances might
catalyze the reaction also.

• Chemical Oxidants
• Plant materials

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Presumptive Test
False Negative A negative result given even
though the substance is blood.

• The stain may be too aged or dilute to produce a
  reaction.
• Substances may interfere with the reaction.
  (reduction compound, acid)
• DNA testing may still be possible.

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Presumptive Test
5 Types of Color Tests

• Benzidine
• Phenolphthalein
• O-Tolidine
• Tetramethylbenzidine (TMB)
• Leucomalachite Green (LMG)

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Presumptive Test gt Color

• Benzidine

• Positive result blue color

• Carcinogen (cancer causing) in 1974

• No longer used by sane scientists

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Presumptive Test gt Color

• Phenolphthalein

• Positive result pink

• Some other substance produce colors other than
  pink (not blood)

• More specific not affected by vegetable
  peroxidases

• Still used today

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Presumptive Test gt Color

• O-Tolidine

• Positive result blue

• Similar to benzidine still carcinogenic as it
  can be metabolized to benzidine

• No longer used gradually replaced by TMB

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Presumptive Test gt Color

• Tetramethylbenzidine (TMB)

• Positive result Green to blue-green

• Component of Hemastix, kit for field test

Plastic strip with filter paper on tip containing
TMB and peroxide.
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Presumptive Test gt Color

• Tetramethylbenzidine (TMB)

• Cotton swab is moistened with water

• Blood stain is sampled

• Blood smeared swab is rubbed on hemastix

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Presumptive Test gt Color
Leucomalachite Green (LMG)

• Positive result Green

• Not as sensitive as TMB or specific as
  phenolphthalein

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Presumptive Test

• Chemiluminescence Fluorescence

Chemiluminescence light is emitted as a product
of the chemical reation
Fluorescence light is emitted when a substance
is exposed to a shorter wavelength of light
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Presumptive Test
Chemiluminescence Fluorescence

• More sensitive than color tests

• May damage blood stain (no blood/DNA typing)

• Used to locate and define areas of blood
• old blood stains
• cleaned floor

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Presumptive Test
Luminol

• Method similar to color test

• Luminol is combined with oxidant and sprayed over
  area thought to contain blood

• Emits a blue-white to yellow green glow

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Presumptive Test

• Luminol

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Presumptive Test
Fluorescein

• Fluorescein is combined with oxidant and sprayed
  over area thought to contain blood

• Fluoresces when treated with a UV light

• Fluorescein includes a thickener it is more
  effective on vertical surfaces

• Study showed no interference with DNA analysis

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Presumptive Test

• Fluorescein

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Forensic Analysis of Blood

• Visual examination of evidence

• Presumptive screening test (Is it blood?)

• Confirmation test (Seriously, is it blood?)

• Determine species origin (human blood?)

• Identify the blood (whose blood is it?)

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Confirmatory Tests

• Due to the possibility of false positives with
  the presumptive tests, confirmatory tests are
  necessary
• Confirmatory tests involve making crystals that
  detect the presence of hemoglobin
• Species testing and DNA testing may also be
  considered confirmatory tests.

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Confirmatory Test

• Teichmann Test and Takayama Test

• Small amount of blood added to microscope slide

• Chemical solution is added

• Slide is heated to form crystals

• Crystals viewed under microscope

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Forensic Analysis of Blood

• Visual examination of evidence

• Presumptive screening test (Is it blood?)

• Confirmation test (Seriously, is it blood?)

• Determine species origin (human blood?)

• Identify the blood (whose blood is it?)

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Species Origin

• Most methods test for serum proteins

• Serum proteins are found in all animals, but are
  slightly different

• Species ID methods based on antigen/antibody
  interactions

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Species Origin

• Antigen serum protein

Antibody produced when foreign serum protein is
detected
Certain antibody will only attach to one species
serum protein
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Species Origin
Antibody is in antiserum
Antigen (serum protein) is in blood sample

• Human antiserum will only attach to human blood
  sample

• Rabbit antiserum will only attach to rabbit blood
  sample

• Dog antiserum will only attach to dog blood sample

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Species Origin

• Ring Precipitin Test

Blood sample (dilute) in top layer
Antiserum in heavy bottom layer
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Species Origin

• Ring Precipitin Test

Blood sample (dilute) in top layer
Precipitate means blood and antiserum species
match
Antiserum in heavy bottom layer
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Species Origin

• Ouchterlony Double Diffusion

Human blood
Human antiserum
Not human blood

• Antiserum placed in center

• Several bloodstains tested at one time

• White line means antiserum and blood match