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Forensic Serology
Chapter 12
Karl Landsteiner
In 1901 he discovered that all humans do not
have the same blood type
He discovered the A-B-O blood group system
This saved millions of lives by preventing
mismatched blood transfusions
Other researchers were able to discover the Rh
factor because of his research
29 years later, they gave Karl a Nobel Prize
Other Blood Factors
The ABO proteins and Rh factor proteins
are not the only proteins on the surface of
blood cells
There are more than a 100 different
surface blood factors
But the ABO factors are most important
for matching a donor to a recipient
Blood and Forensics
No 2 individuals (except identical twins)
can share all the 100+ blood factors
So forensic scientists used this tool to link
blood from a crime scene to an individual
After 1990 however, DNA fingerprinting
has been used instead of blood typing to
identify blood stains and other biological
What is Blood?
Various cell types found in a
liquid matrix called plasma
• Erythrocytes (RBCs)
– Transport O2, CO2
– Stay within blood vessels
• Leukocytes (WBCs)
– Fight infection (immune
– Can move from blood
vessels into tissues
• Platelets (Cell fragments)
– Help with clotting
White Blood Cell
Red Blood Cells
Characteristics of Red Blood Cells
Red blood cells (erythrocytes) are biconcave disks that
contain oxygen-carrying hemoglobin.
Red blood cells discard their nuclei, mitochondria and
most organelles during development and so cannot
reproduce or produce proteins.
In addition to 100s of cell surface proteins, their cell
surfaces have a special class of blood factor proteins called
There are at least 15 different antigens, but the most
important are the ABO and Rh antigens
Role of RBCs
Transport inhaled O2 from lungs to all body cells
Remove CO2 from all body cells and take it back
to lungs for exhalation
When oxygen combines with hemoglobin bright
red oxyhemoglobin results.
Deoxygenated blood or blood containing CO2
(deoxyhemoglbin) is darker.
RBCs are destroyed after about 120 days
Summary of Functions of WBCs
 Leukocytes can squeeze between cells lining walls of
blood vessels by diapedesis and attack bacteria and debris.
 Neutrophils and monocytes are phagocytic, with
monocytes engulfing the larger particles.
 Eosinophils moderate allergic reactions as well as
defend against parasitic infections.
 Basophils migrate to damaged tissues and release
histamine to promote inflammation and heparin to inhibit
blood clotting.
 Lymphocytes are the major players in specific immune
reactions and some produce antibodies (T cells and B cells)
Blood Platelets
Blood platelets are fragments of cells
Platelets help repair damaged blood vessels by
adhering to their broken edges.
Thrombopenia, or low platelet counts increase
bleeding risks
Thrombocytosis or high platelet count may lead to
thrombosis (clotting, bruising, strokes, heart
Blood Plasma
• Plasma is the clear,
straw-colored fluid portion
of the blood.
• Plasma is mostly water,
with a mixture of, amino
acids, proteins,
carbohydrates, lipids,
vitamins, hormones,
electrolytes, and cellular
Hematocrit means the percentage of red blood cells in blood
A blood hematocrit is normally 45% cells and
55% plasma.
Buffy Coat?
Buffy coat is the layer that contains most of
the white blood cells and platelets.
“Buffy” because it is buff (color of naked skin)
in color
The buffy coat is used to extract DNA from
blood - RBCs have no DNA, only WBCs do.
ABO Blood Group
Type A blood has A antigens on red blood cells and antiB antibodies in the plasma.
Type B blood has B antigens on red blood cells and antiA antibodies in the plasma.
Type AB blood has both A and B antigens, but no
antibodies in the plasma.
Type O blood has neither antigen, but both types of
antibodies in the plasma.
Adverse transfusion reactions are avoided by preventing
the mixing of blood that contains matching antigens and
– Adverse reactions are due to the agglutination of red blood cells.
Rh Blood Group
The Rh factor was named after the rhesus
If the Rh factor surface protein is present on red
blood cells, the blood is Rh positive; otherwise it
is Rh negative.
There are no corresponding antibodies in the
plasma unless a person with Rh-negative blood
is transfused with Rh-positive blood; the person
will only then develop antibodies for the Rh
Blood Groups and Transfusions
Antigens and Antibodies
Clumping of red blood cells following
transfusion is called agglutination.
Agglutination is due to the interaction of
proteins on the surfaces of red blood cells
(antigens) with certain antibodies carried
in the plasma.
Only a few of the antigens on red blood
cells produce transfusion reactions.
– These include the ABO group and Rh group.
Agglutination of RBCs
Antibody: a protein that destroys or inactivates
a specific protein antigen. Antibodies are found
in the blood serum
Antibodies are very SPECIFIC to an antigen.
They are designed by the immune system to fit
an antigen.
Antiserum: blood serum in which there are
specific antibodies
Agglutination: the clumping together of red
blood cells by the action of an antibody
Precipitin: an antibody that reacts with its
corresponding antigen to form a precipitate
Secretor: an individual who secretes his or her bloodtype antigen(s) in body fluids. Approximately 80 percent
of the population are secretors
Enzyme: a type of protein that acts as a catalyst for
certain specific reactions
Iso-enzymes: multiple molecular forms of an enzyme,
each having the same or very similar enzyme activities
Antibodies or Agglutinins
Proteins that are present in the serum
responsible for ensuring that the only blood
cells that can survive in a person are cells
of the correct blood type
Type O Blood
Possessed by people whose genotype is
both parents passed on the O gene
have no antigens
these cells can be introduced into a person with
Type A or Type B because these cells are not
attacked by the antibodies these people possess
have both a & b antibodies
can only have other O type cells mixed with this
Relative Frequency of Blood
Types in Human Populations
US whites
US blacks
Since foreign substances in blood are identified
and then isolated by the immune system, it
should be able to detect the presence of drugs
and other chemicals.
This hypothesis is incorrect. The immune system
only creates antibodies and launches attacks
against foreign proteins (either free proteins or
ones bound to cells) and not against other
chemical compounds.
We can fool the immune
The drug can be attached to a carrier molecule that is a
This drug-protein combo can be injected into an animal
such as a rabbit or rat (no human volunteers)
The animal’s immune system will create antibodies that
are specific to the shape of this drug-protein combo
We can isolate these antibodies from the animal’s blood
And use these antibodies to detect the presence of the
drug in human blood or urine
Polyclonal Antibodies
Usually when the immune system produces
antibodies, they are a mixture of
immunoglobulin molecules designed to
recognize and bind to many different sites on an
antigen molecule or different epitopes. In other
words, they are made against a specific antigen,
but each recognizing a different epitope on the
antigen. These antibodies are called Polyclonal
Polyclonal antibodies are not
very specific or reliable
Because the animal makes so many different
kinds for ONE antigen, the antibodies may not
be consistent and some antibody molecules may
not work very well.
Monoclonal Antibodies
Monoclonal antibodies are those that are not
only specific to one antigen, but specific an
exact epitope on that antigen.
In other words, it is a collection of identical
antibodies that bind to a single antigen site or
How are polyclonal antibodies made?
1. Inject the animal with the protein
(antigen) and wait for a period of time for
the animal to make polyclonal antibodies
2. Then collect the animal’s serum which
will contain the polyclonal antibodies.
How are monoclonal antibodies made?
1. Inject the animal with the protein (antigen)
2. Remove the spleen and isolate spleen cells
(the spleen produces the antibodies)
3. Fuse the antibody-producing spleen cells with
tumor cells that were already growing in
culture (these are now called hybridoma cells)
4. Grow the hybridoma cells in culture and isolate
the cells that are producing the antibody of
your choice.
5. These cells will be making antibodies that bind
to only ONE epitope on the antigen. The cells
can be maintained indefinitely.
6. The antibodies are therefore called monoclonal
Now how does a criminalist use these
monoclonal antibodies?
To detect drug presence in blood, urine, etc.
A.K.A. EMIT assays (bio jargon for test)
EMIT stands for Enzyme-multiplied
Immunoassay Technique
EMIT is most often used to test for marijuana
(THC) metabolites in urine.
One of the primary THC metabolites is THC9-carboxylic acid
How EMIT works
Let’s suppose you’re checking someone’s urine for the
presence of THC-9-carboxylic acid (from THC or
Take urine sample to be tested
Add monoclonal antibodies for THC-9-carboxylic acid
to the urine
The antibodies will immediately bind to any THC-9carboxylic acid molecules present in the urine
Then add enzyme-labeled THC-9-carboxylic acid
molecules to the urine
Any antibodies that did not bind to THC-9-carboxylic
acid prior to this step (extra antibody molecules),
will bind to the enzyme-labeled THC-9-carboxylic
One can now measure the amount of unbound or
unused enzyme-labeled THC-9-carboxylic acid to get
a value of THC originally present in the urine.
Questions for a Criminalist:
Is it blood?
Is it human blood?
If it is human, can it be linked to a
suspect / victim and how closely?
Color tests to identify human blood
The Benzidine color test was used for
years to identify human blood
But benzidine was identified as a
carcinogen, so nowadays the
phenolphthalein test is performed instead.
The phenolphthalein test is also known as
the Kastle-Meyer color test
Both, the benzidine and the Kastle-Meyer
test look for the presence of hemoglobin in
the sample.
The Kastle-Meyer Test
When blood, phenolphthalein and
hydrogen peroxide are mixed, the
hemoglobin in the blood will cause the
normally colorless phenolphthalein to a
bright pink color
This test can yield some false positives
Certain vegetable matter can produce the
bright pink positive (such as potatoes)
Death By Potato?
Although the KastleMeyer test can give a
false positive in the
presence of certain
plant matter, it is
unlikely that this plant
matter would be
present at a crime
scene and be
mistaken for blood.
Other blood detection tests
Doctors use Hemastix© strips to detect
blood in urine
 But these strips can be used at a crime
scene to detect fresh or dried blood.
 One simply moistens them with distilled
water and wipes the bloodstain
 A color change to green is a positive
indicator of blood
Bayer Hemastix©
Luminol is a chemical that exhibits chemiluminescence,
with a striking blue glow, when mixed with an
appropriate oxidizing agent. It is a white to slightly
yellow crystalline solid that is soluble in water and most
polar organic solvents.
Usually, a solution of hydrogen peroxide (H2O2) and a
hydroxide in water is used as the activator.
In the presence of a catalyst such as an iron compound,
the hydrogen peroxide is decomposed to form oxygen
and water:
What do you see with luminol?
It glows a bright blue in the dark, when it comes in contact with bloo
Advantages of using Luminol
Allows one to detect stains that would not be
ordinarily be visible
Extremely sensitive - can use it in very dilute
This allows the CSI to spray large areas with it
It does not interfere with DNA, so a CSI can
collect samples for DNA analysis even after it
was sprayed with luminol.
Bloody Footprints that were wiped
Luminol glows even in the
presence of certain other
fluids – semen, feces,
bleach, tonic water, etc.
Luminol glows in the
presence of certain
vegetable matter - Mr.
Potato head strikes again!
Thanks to Quinine
Crystal Tests
Less sensitive than color tests
 They involve the crystallization of certain
components of blood when certain
chemicals are added
 Takayama test and Teichman test
 Not used as much by CSI
So we know it’s blood – but is it
 The
Precipitin Test
– Rabbits injected with human blood
– They make antibodies in their serum
– They are bled and the serum recovered
– The serum is called human antiserum,
because it will react to human antigens
– It will cause coagulation when mixed
with human blood
Precipitin Test
(Human blood)
(Human antiserum
made in rabbits)
Gel Diffusion Test
Antigen (blood) and antibodies (human
antiserum from rabbits) are placed in
separate wells in a gel.
 They are forced to move towards each
 If they bind to each other, they form a
single line of precipitate in between the
 This is a positive for human blood
DNA testing is the ultimate
 It has made most other tests for
determining if the blood is human,
Analyzing Bloodstain Patterns
Surface Texture
Direction of travel
The harder and less porous the a surface, the less
spatter results
The pointed end of a bloodstain always faces the
direction of travel
Impact angle
If the strike angle is 90deg then the stain is circular.
As the strike angle increases, the stain becomes
more and more elongated
Origin of blood spatter
If you draw straight lines through the long axis of
several blood stains, the point where the lines
converge is the origin of the blood spatter
Types of Blood Stain Patterns
1. Passive Bloodstains
Passive stains are drops
created or formed by the
force of gravity acting
This category can be
further subdivided to
Passive drops on various surfaces
• Blood droplets that strike a hard smooth surface, like a
piece of glass, will have little or no distortion around
the edge.
• Blood droplets that strike a rough surface have a
slightly different appearance. They tend to be
distorted, scalloped or spined at the edges.
Passive: Dripped Blood
patterns created by a volume of blood, from
same source to target distance (repeated drops
onto same spot.)
2. Projected Bloodstains
• Projected bloodstains are created when an
exposed blood source is subjected to an action
or force, greater than the force of gravity.
(Internally or Externally produced)
• The size, shape, and number of resulting stains
will depend, primarily, on the amount of force
utilized to strike the blood source.
• This category can be further subdivided to
– Arterial Gush or spurt
– Cast-off stains
– Impact spatter (medium or high velocity)
Arterial gush or spurt
• Bloodstain pattern (s) resulting from blood
exiting the body under pressure from a
breached artery.
Cast-off Stains
• Blood released or thrown from a blood-bearing object
in motion (Knives, bludgeons)
Projected blood through a syringe
Impact Spatter
• Blood stain patterns created when a blood
source receives a blow or force resulting in
the random dispersion of smaller drops of
• This category can be further subdivided
– Low Velocity
– Medium Velocity
– High Velocity
Low Velocity Impact Spatter
•Gravitational pull up to 5 feet/sec.
•Relatively large stains 4mm in size and greater
Medium Velocity Impact Spatter
• Force of 5 to 25 feet/sec.
• Preponderant stain size 1 to 4mm in size
High Velocity Impact Spatter
Force of 100 feet/sec. and greater
Preponderant stain size 1mm in size and smaller
Mist like appearance
3. Transfer Bloodstains
• A transfer bloodstain is
created when a wet,
bloody surface comes in
contact with a secondary
• A recognizable image of
all or a portion of the
original surface may be
observed in the pattern,
as in the case of a
bloody hand or footwear.