Module 3

Body Fluid Evidence

Overview
• This module will explore the significance of body fluid
evidence found at crime scenes. The two main types of
body fluids that may be found at a crime scene or upon
the bodies of the individuals involved with a particular
crime are blood and semen.
Lesson 1

All About Blood

Blood
• Blood is the most common evidence found after a violent crime
or a serious motor vehicle accident. It may be found at the
crime scene, on the perpetrator, on the victim, or it may be
found in more than one of these locations.
• An average human adult has about five liters of blood in the
circulatory system.
Components of Blood
Blood is a complex fluid that has four components, which from
largest to smallest quantity are as follows:
1) Plasma
2) Red Blood Cells
3) White Blood Cells
4) Thrombocytes
(or Platelets)
Plasma
• fluid portion of blood
• comprised mainly of water, dissolved nutrients, minerals
and gases
• makes up 55% of blood
Red Blood Cells
• specialized cells designed to attach to oxygen and facilitate the
transportation of carbon dioxide
• also known as erythrocytes
• Contain hemoglobin (iron-containing protein), which is
responsible for the red color
White Blood Cell
• specialized cells that focus upon the destruction of foreign
antigens (fight disease)
• only type of cell that has a nucleus and therefore can be used
for DNA analysis
• Secrete antibodies to aid immune response
• less numerous as red blood cells
Thrombocytes (Platelets)
▻ specialized cells involved in the clotting of blood
▻ prevent excessive bleeding
Clotting:
▻ When blood clots it separates into a solid dark-red clot and a
clear yellow liquid known as serum
▻ Serum is the liquid that remains after the blood’s proteins have
done their job and the blood clots
▻ Serum and plasma look similar, but plasma is present in
unclotted blood
Clotting
▻ Normal clotting time is 3 to 15 minutes
▻ Can be affected by disease and/or medication
○ Hemophilia
○ blood thinners
▻ The appearance of blood as it clots can be used to estimate the
time of a crime
○ Liquid – a few minutes old
○ Shiny gel – less than an hour
○ Clotted – a few hours
Blood flow during stress
During most violent crimes and
motor vehicle accidents, the
body of every individual
involved is in a state of stress.

This increases the heart rate which Which leads to a release of

leads to an increase in blood large amounts of blood
pressure.
Red Blood Cell Count
▻ A healthy male has about 5.4 million red blood cells per cubic
millimeter of blood
▻ A female has 4.8 million per cubic millimeter of blood.
▻ An average adult has an average of 25 trillion red blood cells!
▻ After about 120 days, a red blood cell becomes weak and
nonfunctional
▻ old red blood cells are broken down by the spleen, liver, and
bone marrow
Red Blood Cell Count and Elevation
• Under low oxygen conditions at high elevations, the human body
produces a greater number of red blood cells.
• Therefore, it is possible for forensic scientists to estimate the
elevation of the location at which an individual was simply by
looking at their red blood cell count.
• For example, Calgary is at a higher elevation (1033 m) than
Edmonton (689 m).
• As a result, an individual who has been in Calgary longer than 5
days has a higher red blood cell count than an individual from
Edmonton
 Determining the Organism that Blood Comes
from
• A red blood cell of birds, reptiles, and lower
vertebrates (i.e., birds, frogs, toads) has a
nucleus
• These cells don’t hold as much hemoglobin as
anucleated cells, and therefore cannot carry as
much oxygen
• Mature red blood cells in humans and other
mammals are anucleated -- they have no nucleus
Blood Typing
• During the early 1900s, the discovery was made that the red
blood cells in one human are not necessarily the same as the red
blood cells in another human
• Blood typing is generally considered “identified” evidence as
people can have the same blood type
• It may become “individualized” if DNA analysis of white
blood cells is possible
Blood Typing
• Three types of protein-markers were discovered on the surface
of red blood cells:
A, B and Rh+
• The protein markers you are born with are inherited from the
genes from your parents
• If you ever require a blood transfusion, the compatibility of the
blood given is very important.
Blood type Compatible with... Incompatible with…

A+, A-, O+, O- B protein markers

A+

A- A-, O- B and Rh+ protein markers

B+ B+, B-, O+, O- A protein markers

B- B-, O- A and Rh+ protein markers

AB+ AB+, AB-, A+, A-, B+, B-, O+, O- None

AB- AB-, A-, B-, O- Rh+ protein markers

O+ O+, O- A and B protein markers

O- O- A, B and Rh+ protein markers

Blood Types in North America
• Certain percentages of any given population have certain blood
types. For example, in North America, 42% have blood type A,
about 46% have blood type O, 9% have blood type B, and
blood type AB is the least common at 3%.
• Blood type identification does not by itself prove that a blood
sample belongs to a particular suspect because others may also
have the same blood type. However, it helps to shorten the list
of suspects.
Blood Enzymes & Proteins
• A number of unique enzymes and proteins in human blood can
help to further identify the source of blood evidence.
• Just as each blood type is found in a certain percentage of the
population, so too are these unique blood enzymes and
proteins.
• Finding one or more of these enzymes or proteins using various
chemical tests helps forensic experts to prove further the
identity of an individual
• However, the enzymes degrade quite quickly, so a fresh blood
sample would provide a better likelihood of finding enzymes
Blood Types and Anti-serum
• Chemical substances called ‘anti-serums’ determine blood
type
• The three types of antiserums are antiserum A, antiserum B,
and antiserum Rh+
• Each identifies one of the three protein-markers that exist on
the surface of red blood cells (A, B, Rh+)
Determining Blood Types
• To determine the blood type of a sample, antiserum is added
• if an agglutination reaction occurs (precipitate forms causing
cloudiness) a positive test for the that protein-marker has
occurred
 Example 1
Three separate small samples of the blood being tested are created.
-A drop of antiserum A is added and mixed into sample #1.
-A drop of antiserum B is added and mixed into sample #2.
-A drop of antiserum Rh+ is added and mixed into sample #3.
-Each sample is mixed gently for 1 to 3 minutes.
-Cloudiness is observed in sample #1. The formation of a precipitate in
sample #1 indicates a positive agglutination reaction has occurred.
-No reaction has been observed in sample #2 and #3.
Conclusion: The unknown blood
sample is type A-.
 Example 2:
Three separate small samples of the blood being tested are created.
-a drop of antiserum A is added and mixed into sample #1.
-a drop of antiserum B is added and mixed into sample #2.
-a drop of antiserum Rh+ is added and mixed into sample #3.
-each sample is mixed gently for 1 to 3 minutes.
-cloudiness is observed in all three samples. The formation of a
precipitate in samples #1, #2 and #3 indicates a positive agglutination
reaction has occurred in all three samples.
Conclusion: The unknown blood
sample is type AB+.
Lesson 2
Blood Evidence
 Identifying Blood Evidence
• If trace evidence is suspected to be blood, it must be confirmed
• Often criminals think that they have sufficiently cleaned up all
blood left behind, but end up missing trace amounts of blood
spatter.
• Forensic investigators are aware of this and as a result one of
the first things they will often do at a secured crime scene is
bring in powerful lighting equipment to illuminate the area.
• Dried blood will appear a deep burgundy or brownish-black in
color
The Phenolphthalein Test
• A Phenolphthalein Test will be done when there is a suspicious
stain or mark that may be blood
• The chemical formula for phenolphthalein is C2020H1414O44 and it is
an acid-base indicator
• Phenolphthalein is colourless in a neutral or acidic solution but
pink in a basic solution
• Blood is a basic solution, and will therefore turn bright or dark
pink during a phenolphthalein test
• When blood, phenolphthalein , and hydrogen peroxide mix,
hemoglobin (oxygen-carrier) causes the peroxide to react with
the phenolphthalein, thereby producing the pink colour
The Phenolphthalein Test
• Advantages
• fast - results are immediate
• very sensitive – it can detect blood at 1 part to 5 million

• Disadvantages
• Certain vegetable products (horseradish, potatoes, etc.)
can cause the same reaction to occur
• Does not distinguish between species
The Luminol Test
• At a crime scene, large pools of blood are
easy to spot, but what if all the blood has
been cleaned up by the assailant?
• Blood that has been wiped away can be
detected using a powerful chemiluminescent
chemical solution that contains luminol
• Luminol is just as sensitive as
phenolphthalein as it can detect blood at 1
part to 5 million.
The Luminol Test
• Investigators use luminol at crime scenes where blood loss is
suspected but not visible.
• Luminol is sprayed over a large area in near-total darkness so
that any reaction is obvious.
• Officers then photograph and/or videotape the results, using a
flash technique that picks up the glow while making the area
around it visible on film
• When luminol comes into contact with iron pigment (in red
blood cells), it reacts and produces a greenish-blue light
• Spraying hydrochloric acid on the sight first speeds up the
reaction
The Luminol Test
Advantages
• non-destructive - after spraying it at a crime scene other
chemical tests such as the phenolphthalein test can still be
performed to confirm that it is blood
• works well even after a long period of time has caused the
blood to age and decompose
Disadvantages
• Luminol will degrade over time (the light will fade)
The Luminol Test
• Luminol can point the way to further evidence or give
indications how events may have unfolded.
• For example, if Luminol reacts positively by producing a glow
upon a carpet, pulling back the carpet may reveal the floor
soaked with more blood that could be tested further.
• Like phenolphthalein, luminol can not distinguish between the
blood from humans or blood from other animals because both
contain hemoglobin
 Lesson 3
Blood Spatter Evidence
Blood Spatter Evidence
Information determined from blood spatter evidence includes the
following:
▻ Origin(s) of blood
▻ Type of weapon that produced the blood spatter
▻ Direction of impact that produced the blood spatter
▻ Position of victim(s) and/or suspect(s) involved
▻ Movement and direction of victim and/or assailant after the
initial assault
▻ Number of impacts the victim received
Free-Falling Blood
▻ Passive bleeding
▻ Blood will ‘free-fall’ when the gravitational force exceeds the
surface tension of the blood and causes it to separate from its
source
▻ Surface tension causes the blood to fall as a spherical droplet
▻ When free-falling blood hits a horizontal surface at an angle of
90°, it creates a circular pattern
▻ As the angle of impact deviates from 90°, the stain becomes
elongated and more ellipse shaped
Free-Falling Blood
▻ The greater the distance blood free-falls, the greater the size of
the stain pattern and the more cast-off residue
▻ Diameters of drops falling from higher than 7 feet don’t
significantly increase
▻ The outside portion of a blood droplet dries before the inner
portion, leaving a peripheral ring stain
Projected and Impacted Blood Spatter
• When a force (internal or external) acts upon an exposed source
of blood, the blood is broken into many small droplets by the
energy applied
• The greater the impact or force, the smaller the size of the blood
droplets and the greater the number of blood droplets produced
• For example, getting hit several times by a human fist (medium
velocity force) leaves a few fairly large blood droplets as
opposed to being struck by a bullet (high velocity force) that
leaves many small blood droplets
Projected and Impacted Blood Spatter
▻ Considers the velocity at which the impacting object strikes the
blood source as well as the velocity at which the blood leaves
the blood source when it is struck
▻ Separated into low, medium, and high velocity patterns
Classifying Spatters by Velocity
Low Velocity Spatters
▻ Occur when an object moving less than five feet per second
strikes a surface
▻ Impact results in fairly large spatters, typically four millimeters
or greater in diameter
▻ Produced by cast-off blood, arterial bleeding (gushing spurts),
and gravitational force
Classifying Spatters by Velocity
Medium Velocity Spatter
▻ Occurs when objects move between 5 and 100 feet per second
▻ Smaller spatters with drops between 1 and 4 millimeters in
diameter
▻ Come from impacts with blunt or sharp objects and expirated
blood
High Velocity Spatter
▻ Result when an object strikes the victim at a speed faster than
100 feet per second
▻ Spatter is small, usually less than 1 mm in diameter, and
appear as mist-like stains
Did you know?
▻ Expirated blood describes blood a victim expels from his/her
lungs or airways.
▻ Each time the victim exhales, blood sprays from his/her mouth
and nose.
Classifying Spatters by Velocity
▻ Impact spatter – typically occur with beatings, stabbings,
gunshots, or any other circumstance where a foreign object
impacts the victim
▻ Projection spatter – results from arterial bleeding, cast-of
blood, and expirated, or exhaled, blood
▻ Combination spatters – include impact and projection spatter
Investigating Blood Spatter
▻ Directionality – the course the blood drop followed
▻ The pointed end of a bloodstain faces the direction of travel
▻ Angle of Impact– the slant at which the blood drops strike the
surface
▻ Measured using a protractor or a computer program
▻ The angle of the surface is considered 0°, and dropping straight
down (vertically) is 90°
Investigating Blood Spatter
▻ Point of Convergence – a 2D representation of the point where
lines tracking the pathways of two or more spatters meet,
indicating the general location from which the bloodstains were
projected
Investigating Blood Spatter
▻ Point of Origin – a 3D representation of the point where lines
tracking not only the pathways but also the angles of impact of
two or more spatters meet, indicating the general spatial
location of the blood source
○ Investigators can find the point of origin by stretching
strings along the angle of impact of each stain
○ Shows the position of the victim or suspect when the stain-
producing event took place
Investigating Blood Spatter
▻ Void Pattern – an absence of blood pattern in an area where
you’d otherwise expect to see them
▻ Often the void indicates where the attacker stood because his
body prevented the blood from splattering on the surfaces
behind him
 Lesson 4
Other Body Fluid Evidence
This lesson contains information about
sexual assault/rape.
Secretors
▻ If a person is a secretor, the antigens, antibodies, proteins, and
enzymes in their blood are also present in their other body
fluids

▻ The protein markers on their red blood cells (i.e. A, B and Rh+)
can also be found in high concentrations in their saliva, semen,
tears, sweat, and vaginal secretions

▻ Approximately 80% of North Americans are known as

secretors.
Secretors
▻ The same anti-serum test that is used to identify blood type can
be used on a secretor’s body tissues yielding the same results
▻ This means that if a body fluid other than blood has been left
behind at a crime scene, there is a high probability that it can
be typed and the blood type of the person who left behind this
fluid can be determined
Sex-Related Crimes
▻ One of the most disturbing crimes that can occur involves a
person being forced to engage in sexual activity against their
will

▻ The majority of all sex-related crimes (ie. rape/sexual assault,

molestation, pedophilia and other indecent acts) are committed
by men

▻ forensic investigators look for semen left behind by a suspect

during the investigation of sex-related crimes
Basic Information about Semen
▻ During sexual activity, when the male reaches his peak of
sexual arousal, the penis may ejaculate semen – an average of 3
– 5 mL
▻ Seminal fluid (semen) is comprised of water, mucous, various
minerals, and a small amount of fructose (energy source for
sperm)
▻ On average, sperm can survive in the vagina for up to 6 or 7
days, the rectum for 2 to 3 days, and in the mouth for less than
24 hours; though remnants of sperm may remain longer in
homicide victims
Basic Information about Semen
▻ If semen is found on material, and the material is protected
from extremes of temperature, harsh chemicals, and other
unfavorable environmental conditions, dried semen stains can
remain identifiable and usable for DNA analysis for many years
Stats
▻ In 2003 over 23,000 sexual assaults were reported to police
across Canada.
▻ Women raped by someone they know are less likely to go to the
police than women who are sexually assaulted by a stranger
▻ Half of all violent crimes, including sexual assault, involve
alcohol consumption by the perpetrator, victim or both
▻ An estimated 94% of sexual assaults are never reported to
police
Collection of Semen from a Crime Scene
▻ When investigators arrive at the scene of a sex-related crime, the
victim is taken to a hospital for an extensive physical examination
(known as a ‘rape kit’) by a physician and nurse with specialized
training

▻ the nurse/physician will collect as much biological material (i.e.

semen, blood, skin, head hair, pubic hair) as possible from the
victim’s body

▻ The areas of the victim’s body that are focused upon during a rape
kit examination include clothing, skin surface, fingernails, pubic hair,
vulva (external genitalia), vagina (internal genitalia), and the rectum
Collection of Semen from a Crime Scene
▻ After any foreign biological materials have been collected, the
evidence is transferred to a DNA analysis laboratory for a DNA
profile (also called a DNA fingerprint).

▻ This examination is obviously not a pleasant experience for the

already traumatized victim; however, the evidence that can be
obtained from a rape kit can help lead to the apprehension of the
perpetrator responsible for this violent crime
Collection of Semen from a Crime Scene
‘Fast Blue B’ test
▻ A special protein called acid phosphatase (AP) is found only in
secretions from the prostate gland

▻ When it reacts with the ‘Fast Blue B’ chemical, it turns from blue to
deep purple

▻ A drawback is that certain fruit and vegetable juices (ex. watermelon

and cauliflower), some fungi, contraceptive creams, and even vaginal
fluid can give a false-positive AP test
Collection of Semen from a Crime Scene
Microscopic Analysis
▻ sperm cells are unique in appearance because they are the only
human body cells with long flagella (tails)
▻ Magnification of 100x or higher is needed
Identification Tests
Prostate-specific antigen (PSA)
▻ If no sperm are seen, the examiner will test for PSA

▻ Involves an antigen-antibody reaction that is quick and simple

Identification Tests
▻ The completion of a DNA analysis of a rape kit is time-
consuming and expensive because it must be done by a highly
trained forensic expert.
▻ Becoming a DNA analysis expert takes a minimum of six years
of education; consequently, few of these skilled scientists are
available
▻ In Canada, DNA analysis for all police departments is
conducted by 5 RCMP labs across the country (Vancouver,
Regina, Winnipeg, Ottawa, and Halifax)
Backlog in the Analysis of Rape Kits
▻ Due to the increased volume of specimens sent in for analysis, a
backlog in the completion of DNA analysis of rape kits in
Canada (and across the world) exists

“DNA analysis takes too long and is

limited to only the most important cases.
Thus, its full potential for public safety is
not being realized.”
Office
Office of
of the
the Auditor
Auditor General
General of
of Canada:
Canada: April
April 2000
2000
Report.
Report. Royal
Royal Canadian
Canadian Mounted
Mounted Police:
Police: Services
Services for
for
Canada's
Canada's Law
Law Enforcement
Enforcement Community.
Community.
Paul Bernardo
The DNA analysis backlog came to attention of the Canadian
public after a judicial inquiry into the Paul Bernardo murder case
in Ontario in 1996. The inquiry found that the DNA analysis of
several rape kits took over two years to complete, during which
time Paul Bernardo committed four more sexual assaults and two
murders.

Shortly after the DNA analysis was finally completed, Bernardo

was arrested and charged with several counts of sexual assault and
two counts of first-degree murder
Stats
The average turnaround time for DNA analysis by RCMP laboratories
was 101 days.
The RCMP completed only 15 percent of its DNA cases within 30 days.
As of October 1999, the DNA analysis backlog was 633 cases.
Wide variations in workload contribute to the DNA analysis backlog.
One laboratory had an average backlog of 28 cases per scientist, while
another had 13 cases per scientist.
At the end of 2004, five years after the Auditor General’s report was
released, a DNA analysis backlog still existed at the RCMP laboratories
across Canada--a backlog of 648 DNA analysis tests.