FORENSIC SCIENCE
Serology
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What is Serology?
• Serology is the study
of serums such as
blood, saliva, semen
and sweat.
• A forensic serologist
applies this
information to
criminal activity and
the law.
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What is Blood?
• Blood is a very
complex mixture of
cells, enzymes,
proteins and inorganic
substances.
• The fluid portion is the
plasma.
• Solids are suspended
in the plasma.
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Characteristics of Blood
• Blood plasma is made primarily of water and accounts for
55% of the total amount of blood content.
• Three different types of cells are suspended in the plasma.
These cells are:
• Erythrocytes are the red blood cells and are responsible for
carrying oxygen.
• Leukocytes are the white blood cells and are responsible
for “cleaning” the system of foreign invaders.
• Thrombocytes are the platelets and are responsible for
blood clotting.
• Blood serum is the liquid that separates from the blood
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when a clot is formed.
Blood Questions
• When blood is found at a crime scene,
several questions must be answered:
• 1. Is it blood?
• 2. If so, is it human or animal?
• 3. If human, what type of blood is it?
• 4. How did the blood fall or spatter?
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1st Question – Is it blood?
• A crime scene investigator
cannot presume that every
red stain or liquid is blood.
• Ketchup, spaghetti sauce
and red jello may all be
mistaken for blood.
• The investigator will
perform presumptive tests
at the scene to determine
if the substance is blood.
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The Presumptive
Blood Tests
Two of the common Presumptive Tests are color tests.
1. Kastle-Meyer -phenolophthalein and hydrogen
peroxide are mixed together and the blood’s
hemoglobin will cause the formation of a deep
pink color. Several household items will yield a
false positive for a color test. For example,
potatoes and horseradish yield a false positive.
However, since these are unusual in a crime scene
setting the Kastle-Meyer is a reasonable
indication of blood.
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The Presumptive
Blood Tests
2. Hemastix – the strip is moistened with distilled
water and then dipped into the substance. A green
color indicates blood.
A third option is luminol.
3. Luminol reacts with blood to produce light instead
of color. Luminol detects blood even if the area
has been cleaned.
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It is blood, but is it human blood?
• At the lab, the technician will need to
determine if the blood is human or animal.
• First, the technician will complete a
microscopic examination.
• The red blood cells of mammals do not
have a nucleus. This is an important
distinction.
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Non-Mammalian Animal Blood
Frog Blood
• Larger nucleic red
blood cells that
undergo cell division
• No platelets
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Human Blood
• Numerous non-nucleic red
blood cells--5 to 6 million
per mm3
• Larger but less numerous
white blood cells 5 to
10,000 per mm3
• Tiny, cellular fragments
350 to 500,00 per mm3
called platelets
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It is blood, but is it human blood?
• The next test that the technician will conduct is the
precipitin test.
• In this test, the blood is injected into a rabbit;
antibodies are formed; rabbit’s blood is extracted
as an antiserum; the antiserum is placed on sample
blood. It will negatively react with human
proteins.
• This test is very sensitive and requires only a
small amount of blood.
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It is human blood, but what type?
Around 1900, Karl Landsteiner discovered that there
are four different kinds of human blood based on
the presence or absence of specific antigens found
on the surface of the red blood cells.
In 1940 Landsteiner and Weiner reported the
discovery of the Rh factor by studying the blood
of the Rhesus monkey. 85% of Caucasians, 94%
of Black Americans and 99% of all Asians are Rh
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positive.
Blood Type Terminology
• ABO blood groups--based on having an A, B, both or
none of the factors on the red blood cell
• Rh factor--may be present on the red blood cell; positive
if present and negative if not
• Antigen--a substance found on a red blood cell
• Antibody--a substance that reacts with an antigen
• Agglutination--clumping of red blood cells; will result if
blood types with different antigens are mixed
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Blood Genetic Terminology
• Genotype--letters that represent the traits; ie, AA,
AO, BO, BB, AB and OO
• Phenotype--words that describe the traits
• Heterozygous--different alleles for the same trait;
ie: AO, BO, AB
• Homozygous--the same alleles for the trait; ie:
AA, BB, OO
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Blood Typing
• A blood type has antigen A and will agglutinate
with B.
• B blood type has antigen B and will agglutinate
with A.
• AB blood type has antigen A and B and will not
agglutinate with either A or B.
• O blood type has neither antigen A or B and will
agglutinate with either.
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Blood Reactions to Antiserum
REACTION
Anti-A Serum
Agglutination
Anti-B Serum
No agglutination
No agglutination Agglutination
Agglutination
Agglutination
No agglutination No agglutination
BLOOD TYPE
Type A
Type B
Type AB
Type O
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Blood Typing
Reactions to
Anti-Serum
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Blood Groups
Only certain blood types can donate or receive blood from other blood
types. If the wrong blood is transfused, the person may die.
Type
Antigen
Antibody
Can Give
Blood To:
Can Get
Blood From
A
A
B
A, AB
O, A
B
B
A
B, AB
O,B
AB
A and B
Neither
A nor B
AB
A, B, O, AB
O
Neither
A nor B
A and B
A, B, O, AB
O
Population Distribution
of Blood Types in the U. S.
Type
Percent
O
45
A
39
B
12
AB
4
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Typing of Dried Blood Stains
Absorption-elution technique




Antiserum is placed on the blood stain. Antibodies
combine with the specific antigens.
Unreacted serum is washed off the bloodstain.
Stained material is heated to 56 degrees C, breaking the
antibody-antigen bond. This process is known as
elution.
Known red blood cells are added. Agglutination will
occur if antigens present on the added RBC’s were also
originally on the stained material.
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Differentiating Within Blood Types
The determination that the blood type is O +
eliminates 55% of the population. In order to
narrow down the possibilities within a specific
blood type, the blood is tested for certain enzymes.
Enzymes are proteins that speed up or slow down
chemical reactions. Enzymes that exist in
different forms are called polymorphic and can be
broken down into their separate proteins called
iso-enzymes.
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





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
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Commonly Used
Blood Enzymes and Proteins
Adenosine deaminase
Adenylate kinase
Carbonic anhydrase II
Erythrocyte acid phosphatase
Esterase D
Glucose-6-Phosphate dehydrogenase
Glyoxylase U
Group-specific component
Haptoglobin
Peptidase A
Phosphoglucomutase
6-Phosphogluconate dehydrogenase
Transferrin
AD
AK
CA II
EAP
EsD
G6PD
GLO I
Ge
Hp
Pep A
PGM
6PGD
Tf
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PGM--phenotypes
Electrophoresis can separate the protein
components into these 10 iso-enzymes.
-
+
1-
1+ 2-
2+
2-1-
2+1- 2-1+ 2+1+ 2+2- 1+1-
Three of these are most common PGM 1+, PGM 2-1+ and PGM 2
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EAP--phenotypes
Determined by the intensity of the fluorescence.
The darker the band the more it fluoresced.
B
C
A
A
BA
B
CB
C
CA
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The final question, how did the
blood fall or spatter?
A group of blood spatter
specialists deal with
the physical properties
of blood and the
patterns produced
under different
conditions as a result
of various forces being
applied to the blood.
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The final question, how
did the blood fall or spatter?
• Blood, as a fluid, follows the laws of
physics. It is not influenced nor affected by
race, gender, or age of the one bleeding.
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BLOOD DROPLET
Characteristics
• A blood droplet will remain spherical in
space until it drops onto a surface
• Once a blood droplet impacts a surface, a
bloodstain is formed.
• A droplet falling from the same height,
hitting the same surface at the same angle,
will produce a stain with the same basic
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shape.
BLOOD DROPLET
Volume
• Is approximately 0.05 cc
• Is not the same for all blood droplets--from
0.03 cc to 0.15 cc
• Is directly dependent upon the surface or
orifice from which it originates
• The impact area is called the target.
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CONDITIONS EFFECTING
BLOODSTAIN SHAPE
•
•
•
•
Height of the droplet
Size of the droplet
Angle of impact
Velocity at which the blood droplet left the
original surface
• Texture of the target surface
– On clean glass or plastic--droplet will have smooth outside edges
– On a rough surface--will produce scalloping on the edges
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Questions Answered by
Blood Spatter Interpretation
• The distance between the target surface and
the origin of blood at the time of blood shed
• The point(s) of origin of the blood
• Movement and direction of a person or an
object
• The number of blows, shots, etc. causing
the bloodshed and/or the dispersal of blood.
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Questions Answered by
Blood Spatter Interpretation
• Type and direction of impact that produced
the bloodshed
• The position of the victim and/or object
during bloodshed
• Movement of the victim and/or object after
bloodshed
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Bloodstain Terminology
• Angle of impact--angle at which blood strikes a
target surface.
• Bloodstain transfer--When a bloody object comes
into contact with a surface and leaves a patterned blood
image on the surface.
• Backspatter--blood that is directed back toward its
source of energy.
• Cast-off--blood that is thrown from an object in motion
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Bloodstain Terminology
• Contact stain--general term referring to bloodstains
caused by contact between a wet, blood-bearing surface
and a second surface which may or may not have blood on
it
– Transfer--image is recognizable and may be
identifiable with a particular object
– Swipe--wet blood is transferred to a surface which did
not first have blood on it
– Wipe--a non-blood bearing object moves through a
wet bloodstain, altering the appearance of the original
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stain
Bloodstain Terminology
• Directionality--relates to the direction a drop of blood
traveled in space from its point of origin
• Terminal velocity--the greatest speed to which a free
falling drop of blood can accelerate in air. It is dependent
upon the acceleration of gravity and the friction of the air
against the blood--approximately 25 feet/second.
– High velocity--greater than 100 feet/second; gives a
fine mist appearance
– Low velocity--5 feet/second or less
– Medium velocity--5 to 25 feet/second.
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Bloodstain Pattern
• Terminal Velocity
• Directionality
• Angle of Impact
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Blood Stain Patterns
The shape of a blood stain:


Round--if it falls straight down at a 90 degree
angle.
Elliptical--Blood droplet elongates as the angle
decreases from 90 to 0 degrees. The angle can
be determined by the following formula:
width
=
sine of the impact angle
length
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IMPACT
• The more acute the angle of impact, the more
elongated the stain.
• 90 degree angles are perfectly round with 80
degree angles taking on a more elliptical shape.
• At about 30 degrees the stain will begin to
produce a tail.
• The more acute the angle, the easier it is to
determine the direction of travel.
• The greater the height the drop falls the larger the
spatter.
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Height of the Droplet and Spatter
Size
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Angle of Impact and Spatter Shape
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Blood Stains
• The harder and less porous the
surface, the less the blood drop
will break apart.
• The softer and more porous the
surface, the more a blood drop
will break apart.
• The pointed end of the blood
stain faces the direction the stain
is traveling.
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Area of Intersection
The location of the blood
source can be determined
by drawing lines the
various blood droplets to
the point where they
intersect. This is the
blood’s origin.
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Area of Convergence
The area of convergence
is the point of origin;
the spot where the
“blow” occurred.
It is determined by
drawing a line from the
area of intersection
straight up to where to
where the angle of impact
would intersect
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CRIME SCENE
What evidence
can you see in
this crime
scene? What
story does the
scene tell?
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Bring In The Dogs!
The dog locates
human scent. A
closer look
shows that the
ashes have
human remains
and clothing.
In addition,
look closely at
the rocks on the
next slide.
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Blood Evidence
• Class evidence for blood would include blood
type. If you can determine the DNA you would
have individual evidence.
• Blood stain patterns are considered circumstantial
evidence in a court room. Experts could argue
many points including direction of stains, height
of the perpetrator, position of the victim, left/right
hand, whether the body was moved, etc.
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Another Body Fluid - Sperm
• Among the smallest and most
highly specialized cells in the
human body.
• Has a head and a tail
• Contains 23 chromosomes with
the genetic material found in the
head
• Males release 2.5 to 6 milliliters
of seminal fluid per ejaculation
with approximately 100 million
sperm per milliliter.
Magnified 400X
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Semen
Determination of Seminal Fluid
- Suspect stain may have stiff, crusty appearance
- Acid phosphatase color test- presumptive test
– the presence of acid phosphatase, the enzyme
secreted by the prostate gland into the seminal
fluid, will turn purple when sodium alpha
naphthylphosphate and Fast Blue B solution are
placed on it.
– It will also fluoresce under UV light when it
comes in contact with 4-methyl umbelliferyl 49
phosphate.
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Detection and Collection of Semen
Sample
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Semen (cont.)
Determination of Seminal Fluid
• Prostate Specific Antigen (PSA) or P30--unique to
seminal plasma
– P30 is isolated and injected into a rabbit where
antibodies are produced (anti-P30)
– The stain extract is placed in one well of an
electrophoresis plate and the anti-P30 in the opposite
well. The electric is applied and the antigens and
antibodies move toward each other. The formation of a
precipitation line between the wells shows the presence
of P30 in the sample stain. It must be seminal fluid. 53
Microscopic Examination of
Sperm
• A microscopic
examination will also
determine if the
sample is semen
• The presence of
spermatozoa confirms
that the sample is
semen
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No spermatozoa?
• The male could have oligospermia – a low
sperm count
• The male could have aspermia – no sperm
due to a vasectomy
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Collection of Rape Evidence
• The Medical Examination: Collection of Physical
Evidence
The following types of physical evidence should be
collected during the medical examination for laboratory
analysis:
• HAIR (both pubic and head hair)
– Cannot individually identify a person unless the root is
present;
– Does provide strong collaborative evidence individual
was at the crime scene;
– Can be distinguished between human and animal;
– May be able to determine race; and
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– May be able to establish the part of the body from
which the hair came.
Collection of Rape Evidence (cont)
• FIBERS
– Limited value due to mass production of clothing; and
– In rare cases, can be used to deduce individual
identification with a high degree of certainty.
• BLOOD
– Can be distinguished as to human or animal; and
– DNA analysis can provide individual identification.
• FINGERNAIL SCRAPINGS
– Used to collect DNA evidence for individual analysis
and identification.
• VAGINAL, RECTAL AND ORAL SWABS AND
SMEAR
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– Used to collect DNA evidence for individual analysis
and identification
Secretors
• 80% of people are considered secretors. Their
blood-type antigens are found in high
concentration in their body fluids such as
saliva, semen, vaginal secretions and gastric
juice. If you are a secretor, you will have a
higher concentration of A and B antigens than
does your blood!!
• With the advent of DNA, the secretor evidence
is not as important as it once was.
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