Transcript Identification and Characterization of Blood and Bloodstains

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:
It’s that red stuff that comes out of your
fingers following a kitchen accident.
Introduction to Blood
Introduction to Blood
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.
Introduction to Blood
Red Blood Cells = No DNA
White Blood Cells = DNA in nucleus
Introduction to Blood
White Blood Cells = DNA in nucleus
Introduction to Blood

RBC = no DNA; WBC = yes DNA

All blood cells have blood groups on
outside of cell.
Introduction to Blood
ABO blood groups found on outside of cell
Introduction to Blood
ABO blood groups found on outside of cell
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
Introduction to Blood
Hemoglobin picks up and drops off oxygen
Forensic Analysis of Blood
1.
Visual examination of evidence
2.
Presumptive screening test (Is it blood?)
3.
Confirmation test (Seriously, is it blood?)
4.
Determine species origin (human blood?)
5.
Identify the blood (whose blood is it?)
Forensic Analysis of Blood
1.
Visual examination of evidence
2.
Presumptive screening test (Is it blood?)
3.
Confirmation test (Seriously, is it blood?)
4.
Determine species origin (human blood?)
5.
Identify the blood (whose blood is it?)
Forensic Analysis of Blood
1.
Visual examination of evidence
2.
Presumptive screening test (Is it blood?)
3.
Confirmation test (Seriously, is it blood?)
4.
Determine species origin (human blood?)
5.
Identify the blood (whose blood is it?)
Presumptive Screening Tests
Negative result means the questioned
stain is not likely blood
 Positive result means the questioned stain
is likely blood

Presumptive Screening Tests
Presumptive tests produce a color reaction
or release of light
 Tests rely on catalytic properties of blood
(hemoglobin presence)

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.
Presumptive Screening Tests
Color Test Method
 Sample stain with clean cotton swab
 Add drop of chromogen
 Add drop of hydrogen peroxide
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
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.
Presumptive Screening Tests
5 Types of Color Tests
 Benzidine
 Phenolphthalein
 O-Tolidine
 Tetramethylbenzidine (TMB)
 Leucomalachite Green (LMG)
Presumptive Screening Tests
Benzidine
 Positive result = blue color

Carcinogen (cancer causing) in 1974

No longer used by sane scientists
Presumptive Screening Tests
Phenolphthalein
 Positive result = pink

Some other substance produce colors
other than pink (not blood)

Still used today
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
Presumptive Screening Tests
Tetramethylbenzidine (TMB)
 Positive result = Blue-green

Most common test for blood
1. Rub stain with moist swab
2. Add TMB
3. Add peroxidase
Look for quick blue color
Presumptive Screening Tests
Tetramethylbenzidine (TMB)
 Positive result = Blue-green

Most common test for blood
1. Rub stain with moist swab
2. Add TMB
3. Add peroxidase
Look for quick blue color
Presumptive Screening Tests
Leucomalachite Green (LMG)
 Positive result = Green

Not as sensitive as TMB or specific as
phenolphthalein
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
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
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
Presumptive Screening Tests
Luminol
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
Presumptive Screening Tests
Fluorescein
Forensic Analysis of Blood
1.
Visual examination of evidence
2.
Presumptive screening test (Is it blood?)
3.
Confirmation test (Seriously, is it blood?)
4.
Determine species origin (human blood?)
5.
Identify the blood (whose blood is it?)
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

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
Forensic Analysis of Blood
1.
Visual examination of evidence
2.
Presumptive screening test (Is it blood?)
3.
Confirmation test (Seriously, is it blood?)
4.
Determine species origin (human blood?)
5.
Identify the blood (whose blood is it?)
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
Species Origin

Antigen = serum protein

Antibody = produced when foreign serum
protein is detected

Certain antibody will only attach to one
species’ serum protein
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
Species Origin
Ring Precipitin Test

Blood sample (dilute)
in top layer

Antiserum in heavy
bottom layer
Species Origin
Ring Precipitin Test

Blood sample (dilute)
in top layer
Precipitate means blood and
antiserum species match

Antiserum in heavy
bottom layer
Species Origin
Ring Precipitin Test
Yes line = Yes human blood
Human Antiserum
Species Origin
Ring Precipitin Test
No line = Not human blood
Human Antiserum
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
Species Origin
Crossed-Over Electrophoresis
Antiserum
Blood Stain
Gel
Holes

Antiserum and Blood move toward each other
Species Origin
Crossed-Over Electrophoresis
Antiserum
Blood Stain

Antiserum and Blood move toward each other

If line forms, antiserum and blood match
Forensic Analysis of Blood
1.
Visual examination of evidence
2.
Presumptive screening test (Is it blood?)
3.
Confirmation test (Seriously, is it blood?)
4.
Determine species origin (human blood?)
5.
Identify the blood (whose blood is it?)
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
Blood Group Markers
Many blood group markers, including:
ABO Markers
 Lewis System
 Rhesus System

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
Introduction to Blood
ABO blood groups found on outside of cell
Introduction to Blood
ABO blood groups found on outside of cell
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
Blood Group Markers
ABO Markers (antigen and antibodies)
Blood Type Antigen
(blood group)
A
B
AB
O
Antibody
% population
Blood Group Markers
ABO Markers (antigen and antibodies)
Blood Type Antigen
Antibody
% population
(blood group)
A
B
AB
O
A
Anti-B
40
Blood Group Markers
ABO Markers (antigen and antibodies)
Blood Type Antigen
Antibody
% population
(blood group)
A
A
Anti-B
40
B
B
Anti-A
10
AB
O
Blood Group Markers
ABO Markers (antigen and antibodies)
Blood Type Antigen
Antibody
% population
(blood group)
A
A
Anti-B
40
B
B
Anti-A
10
AB
A&B
none
5
O
Blood Group Markers
ABO Markers (antigen and antibodies)
Blood Type Antigen
Antibody
% population
(blood group)
A
A
Anti-B
40
B
B
Anti-A
10
AB
A&B
none
5
O
H
Anti-A &
Anti-B
45
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
Blood Group Markers
ABO Markers

Tests can get complicated with absorbing
and releasing cells

Final step is usually testing for
agglutination (blood clumping)
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
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
Protein or Enzyme Markers
+
Charge makes proteins
move through gel
 Different shapes move
at different rates
 After several minutes,
their location will tell
what type they are.

-
Ladder Type 1 Type 2 Type 3
Protein or Enzyme Markers
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
Case File
Case File
Case File
Case File
Ring Precipitin Test
Yes line = Yes human blood
Human Antiserum
Case File