Identification and Characterization of Blood and Bloodstains

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Identification and Characterization of Blood and
Bloodstains
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Summary

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

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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

• What you should already know

Its that red stuff that comes out of your
fingers following a kitchen accident.
Lets look at blood.
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Introduction to Blood
Red Blood Cells No DNA
White Blood Cells DNA in nucleus
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Introduction to Blood
White Blood Cells DNA in nucleus
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Introduction to Blood

• RBC no DNA WBC yes DNA

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

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Introduction to Blood
ABO blood groups found on outside of cell
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Introduction to Blood
ABO blood groups found on outside of cell
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Introduction to Blood

• 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
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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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 Screening Tests

• Negative result means the questioned stain is not
  likely blood
• Positive result means the questioned stain is
  likely blood

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Presumptive Screening Tests

• Presumptive tests produce a color reaction or
  release of light

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

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Presumptive Screening Tests

• 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 Screening Tests

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

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Presumptive Screening Tests

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

• Alternatively, collect stain on thread add
  chromogen and hydrogen peroxide in spot plate

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Presumptive Screening Tests

• Color Tests

Remember, other non-blood substances might
catalyze the reaction also.

• Chemical Oxidants
• Plant materials

False Positive A positive result given by a
substance that is not blood.
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Presumptive Screening Tests

• 5 Types of Color Tests
• Benzidine
• Phenolphthalein
• O-Tolidine
• Tetramethylbenzidine (TMB)
• Leucomalachite Green (LMG)

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Presumptive Screening Tests

• Benzidine
• Positive result blue color

• Carcinogen (cancer causing) in 1974

• No longer used by sane scientists

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Presumptive Screening Tests

• Phenolphthalein
• Positive result pink

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

• Still used today

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Presumptive Screening Tests

• O-Tolidine
• Positive result blue

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

• No longer used gradually replaced by TMB

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Presumptive Screening Tests

• Tetramethylbenzidine (TMB)
• Positive result Blue-green

• Most common test for blood

• Rub stain with moist swab
• Add TMB
• Add peroxidase

Look for quick blue color
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Presumptive Screening Tests

• Tetramethylbenzidine (TMB)
• Positive result Blue-green

• Most common test for blood

• Rub stain with moist swab
• Add TMB
• Add peroxidase

Look for quick blue color
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Presumptive Screening Tests

• Leucomalachite Green (LMG)
• Positive result Green

• Not as sensitive as TMB or specific as
  phenolphthalein

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Presumptive Screening Tests

• Chemiluminescence and 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 Screening Tests

• Chemiluminescence and 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 Screening Tests

• 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 Screening Tests

• Luminol

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Presumptive Screening Tests

• 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 this makes it
  more effective on vertical surfaces

• Study showed no interference with DNA analysis

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Presumptive Screening Tests

• 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

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Confirmatory Tests

• 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

• Ring Precipitin Test

Yes line Yes human blood
Human Antiserum
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Species Origin

• Ring Precipitin Test

No line Not human blood
Human Antiserum
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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

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Species Origin

• Crossed-Over Electrophoresis

Antiserum
Blood Stain
Gel
Holes

• Antiserum and Blood move toward each other

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Species Origin

• Crossed-Over Electrophoresis

Antiserum
Blood Stain

• Antiserum and Blood move toward each other

• If line forms, antiserum and blood match

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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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Genetic Markers in Blood

• If a stain is blood, and it is human blood, then
  whose is it?

• Blood Group Markers

• Protein/Enzyme Markers

• DNA

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Blood Group Markers
Many blood group markers, including

• ABO Markers
• Lewis System
• Rhesus System

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Blood Group Markers

• ABO Markers

• Look at antigens on Red Blood Cells

• In this case, antigens are glycoproteins and are
  attached to the outside of the cell

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Introduction to Blood
ABO blood groups found on outside of cell
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Introduction to Blood
ABO blood groups found on outside of cell
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Blood Group Markers

• ABO Markers (antigen and antibodies)

• A person will have antibodies (A or B) to
  whatever blood group he/she doesnt have

• Otherwise, a persons blood would clump up and
  cause death

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Blood Group Markers

• ABO Markers (antigen and antibodies)

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Blood Group Markers

• ABO Markers (antigen and antibodies)

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Blood Group Markers

• ABO Markers (antigen and antibodies)

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Blood Group Markers

• ABO Markers (antigen and antibodies)

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Blood Group Markers

• ABO Markers (antigen and antibodies)

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Blood Group Markers

• ABO Markers

• Testing is similar to species test

• An anti-A, anti-B, or anti-AB antiserum
  (containing antibodies) is reacted with the blood
  stain to detect blood cells

• A, B, or AB blood cells are reacted with a blood
  stain to detect antibodies

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Blood Group Markers

• ABO Markers

• Tests can get complicated with absorbing and
  releasing cells

• Final step is usually testing for agglutination
  (blood clumping)

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Blood Group Markers

• ABO Markers

• A cells clump with anti-A
• B cells clump with anti-B
• AB cells clump with both
• O cells do not clump

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Other Markers

• Protein or Enzyme Markers

• Some proteins or enzymes can be in different
  forms (different shapes)

• These differences can be detected by separating
  the proteins in a gel by electrophoresis

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Protein or Enzyme Markers

Ladder
Type 1
Type 2
Type 3

• Charge makes proteins move through gel
• Different shapes move at different rates
• After several minutes, their location will tell
  what type they are.

-
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Protein or Enzyme Markers
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Genetic Markers in Blood

• ABO blood typing and protein analysis may help
  eliminate a suspect.

• Since there are only a small number of types (ABO
  4 types), a match does not mean the stain
  definitely came from a certain person

• DNA testing can identify a person, and is
  becoming just as easy as the above tests

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Case File
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Case File
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Case File
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Case File

• Ring Precipitin Test

Yes line Yes human blood
Human Antiserum
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Case File