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Forensic Serology:
Blood
By
Mohamed Elgamal, MD, PhD
Lecturer and consultant of Forensic Medicine and Clinical
Toxicology
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Identification and
Characterization of
Blood and
Bloodstains
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Forensic Serology

-Serology – term used to describe a broad range of

laboratory tests using reactions of blood serum and
body fluid
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Forensic Serology

-The serology section of a forensic laboratory may deal with any or all of
the following:
• -blood typing
• -characterization of unknown blood
• -stain patterns for crime reconstruction
• -paternity testing
• -semen identification in rape cases
• -DNA techniques used for identification
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Summary

 Identification
of Blood as Blood
 Determining Species Origin of Blood
 Introduction to Blood
 Identifying Humans using Blood
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Forensic Characterization of
Bloodstains

Three questions that must be answered by the forensic investigator:

1) Is it blood?
-Benzidine Test
-Kastle-Meyer Test
-Hemastix (o-tolidine, TMB)
-Luminol Test
2) Is it human blood?
-Precipitin Test

3) Can it be associated with an individual?

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• Blood is the most common, well-known, and

perhaps most important evidence in the world of
criminal justice today.
• Wet blood has more value than dried blood
because more tests can be run.
–For example, alcohol and drug content can be determined
from wet blood only.
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• Blood begins to dry after 3-5 minutes of

exposure to air. As it dries, it changes color
towards brown and black.
• There's no substitute for it, whether for medical or
forensic purposes.
• Its presence always links suspect and victim to
one another and the scene of violence.
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Blood Forensics

 When a darkish substance is found at a crime scene, it must first

be determined to be blood.
 There are several tests—presumptive tests used strictly for
screening---that will differentiate between blood and other
substances
Introduction to Blood 10

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.
◼ Red Blood Cells contain the protein
hemoglobin, which carries oxygen.
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 Today, blood typing also includes

different types of enzymes and
proteins that perform specific
activities in the body, which helps
to individualize the blood. (More
than 150 serum proteins and 250
cellular enzymes have been
isolated, as well as many more
antigens.
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What is in Blood

 The plasma is the

liquid part of the
blood, which is 90%
water. The other 10%
contains proteins,
waste products,
gases, and nutrients.

 Plasma makes up
55% of blood.
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Blood Volume

 On average, accounts for 8 % of total

body weight
- 5 to 6 liters of blood for males
 4 to 5 liters of blood for females

 A 40 percent blood volume loss, internally

or/and externally, is required to produce
irreversible shock (death).

 A blood loss of 1.5 liters, internally or

externally, is required to cause
incapacitation
What is in Blood? 16

 The three types of cellular

elements in the blood are:
erythrocytes (red blood cells),
leukocytes (white blood cells), and
platelets.
 Plasma is the other portion that is
in blood.
 Red Blood Cells 17

 Red Blood Cells

transport oxygen on
molecules called
Hemoglobin.
 Erythropoiesis is the
production of new red
cells.
 They are made at 2-3
million a second.
 Average lifespan is 120
days.
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White Blood Cells

 Leukocytes are the mobile units of the body’s immune

system. They are also called white blood cells.
 They defend against the invasion of pathogens.
 They identify cancer cells.
 They remove the body’s litter by phagocytosis.
 They can leave the circulation and go to the
sites of invasion and tissue damage.
 There are five kinds of leukocytes.
 5-10 million/ml of blood in normal person.
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Platelets

 They remain
functional for about
10 days.
 1/3 stored in spleen
 250 million/ml of
blood
 They begin the
clotting to a wound.
Collection equipments : 20
Crime scene investigation tools box - Buccal cell
collectors -FTA cards
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Fast Technology for Analysis of nucleic acids
(FTA) cards
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Collection

 Different methods of collection of human biological evidences from

crime scece as:
 collecting the entire article
 cutting the stains
 swabbing
 scraping dry samples

This depends on the kinds of evidence and it's state

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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?)
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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?)
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The Forensic Serologist

The forensic serologist studies body

fluids such as semen, saliva, and blood
mainly for identification purposes.

Role(s):
•Establishing type and characteristics of
blood
•Blood testing
•Examination of bloodstains
•DNA typing
•Preparation of court testimony &
evidence
Visual examination of the evidence:
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Bloodstain Analysis:
Blood or not?

To determine whether or not blood is

present at a crime scene, forensic
investigators use color or crystalline tests.
In the past, police investigators were
trusted to verify the presence or absence of
blood, but Miller v. Pate (1967) enforced that
physical tests should be completed in the
search for blood. The Benzidine test was
widely implemented until it was discovered
to be a known carcinogen. The current
Kastle-Meyer test, which uses the
chemical, phenolphthalein, operates on the
fact that when phenolphthalein comes in
contact with hemoglobin, it releases
peroxidase enzymes that cause a bright
pink color to appear.
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Bloodstain Analysis:
Blood or not?
To determine whether or not blood is
present at a crime scene, forensic
investigators use color or crystalline tests.
In the past, police investigators were
trusted to verify the presence or absence of
blood, but Miller v. Pate (1967) enforced that
physical tests should be completed in the
search for blood. The Benzidine test was
widely implemented until it was discovered
to be a known carcinogen. The current
Kastle-Meyer test, which uses the
chemical, phenolphthalein, operates on the
fact that when phenolphthalein comes in
contact with hemoglobin, it releases
peroxidase enzymes that cause a bright
pink color to appear.
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Identification of blood and bloodstains:
Luminol
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Luminol

-Red blood cells contain hemoglobin

(Hb) – the protein responsible for
transporting oxygen
-Each Hb contains four iron (Fe)
containing hemes

Heme structure
Preservation and storage: 31
Cardboard box allows simple and safe drying and
storage of swabs
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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?)
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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

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

Benzidine
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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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Phenolphthalein (Kastle-Meyer) test :

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Phenolphthalein (Kastle-Meyer) test :
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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

1. Rub stain with moist swab

2. Add TMB
3. 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

1. Rub stain with moist swab

2. Add TMB
3. 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
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Presumptive Screening Tests
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 The first test is simply the use of a powerful light

moved across every surface of a crime scene. That
yields possible traces for visual inspection.
 It is used by crime scene investigators to locate
traces of blood, even if it has been cleaned or
removed.
 The iron present in any blood in the area causes the
chemical reaction that leads to the blue
luminescence revealing the location of the blood.
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 Any glow detected may be documented by a long

exposure photograph as the glow only lasts 30
seconds.
 If nothing is seen, but there is reason to suspect
blood had been present, a chemical called luminol
is sprayed across the scene because it reacts to
blood by making it luminescent. It only takes about
five seconds.
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• Luminol also can point the way to other evidence or

how events might have unfolded at a crime scene. If it
is sprayed on a carpet and produces a glow, officers
might peel back the rug to reveal a floorboard soaked
with blood. It might also show the direction in which a
criminal walked or dragged a body, or whether the
victim was shot from behind or above.
–There is one problem with this test: luminol can
destroy the properties of the blood that investigators
need for further testing.
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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
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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?)
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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

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?)
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Blood Antigens

-On the surface of red blood cells are chemical structures

(proteins) called antigens
-The presence of antigens allow a living system to
recognize foreign biological substances
-Antigens also impart specific characteristics to the red
blood cells. These characteristics can be partly used to
identify individuals
-Human red blood cells have more than 15 different
antigen types that have been discovered
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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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Precipitin Test
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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

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

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?)
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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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Blood Typing

-Blood typing involves determination of the antigens present on an

individual’s RBCs

-The two most common blood typing systems used are the A-B-O
method and the Rh method
-type A blood – contain “A” antigen on RBCs
-type B blood – contain “B” antigen on RBCs
-type AB blood – contains both A and B antigens
-type O blood – contain no A or B antigens
-Rh+ blood – contain Rh antigen
-Rh- blood – no Rh antigen
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Introduction to Blood

ABO blood groups found on outside of cell

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Blood Typing Example

-A sample of unknown blood is mixed with three anti-sera samples:

Tube 1 (Anti-A): No reaction
Tube 2 (Anti B): No reaction
Tube 3 (Anti Rh): Cloudy reaction

-In terms of the A-B-O and Rh systems, what type blood is the
sample?

-The ABO antigens are not confined to RBC; 80% of Americans are
secretors, found on other cells
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Blood Typing Example

-A sample of unknown blood is mixed with three anti-sera samples:

Tube 1 (Anti-A): No reaction
Tube 2 (Anti B): No reaction
Tube 3 (Anti Rh): Cloudy reaction
O positive
-In terms of the A-B-O and Rh systems, what type blood is the
sample?
-The ABO antigens are not confined to RBC; 80% of Americans
are secretors, same antigens found on other cells
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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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Blood Typing

*Rh factor is determined by the

presence of another antigen, the D
antigen.

*People having the D antigen are

Rh positive; those not having the
antigen are Rh negative
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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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Blood Typing

 After determining that the blood is human,

analysts can go ahead and determine
blood type with an ABO test, and then work
on the gender of the person from whom the
blood came.
 These days (from 1985), DNA technology has
replaced the tests for specific enzymes and
proteins.
 It's more accurate to match DNA from a
blood sample at a crime scene to a source
than to draw up an entire blood profile.
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