Identification and characterization of blood and bloodstains
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Identification and Characterization of Blood and Bloodstains. Summary. Introduction to Blood Identification of Blood as Blood Determining Species Origin of Blood Identifying Humans using Blood. Introduction to Blood. What you should already know:.

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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:

It’s that red stuff that comes out of your fingers following a kitchen accident.




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Introduction to Blood

What you should already know:

It’s 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

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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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

  • Human antiserum will only attach to human blood sample

Antibody is in antiserum

Antigen(serum protein) is in 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

Human blood

Ouchterlony Double Diffusion

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

  • ABO Markers

  • Lewis System

  • Rhesus System

Many blood group markers, including:


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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 doesn’t 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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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

Ring Precipitin Test

Yes line = Yes human blood

Human Antiserum



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