Forensic Serology

“Out damned spot! Out, I say

Here’s the smell of the blood still,
All the perfumes of Arabia will not
Sweeten this little hand. Oh, Oh, Oh!”
—William Shakespeare’s Lady Macbeth, in Macbeth

Chapter 10
 Using Blood in Forensics
 Test for blood type
 Provides DNA evidence
 Analyze bloodstains
 Blood spatter patterns
 Livor mortis- pooling of blood in dead
organisms
Chapter 10
 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
Chapter 10
 Serology
Serology is the examination and analysis of body fluids. A
forensic serologist may analyze a variety of body fluids
including saliva, semen, urine, and blood. From 1950 to the
late 1980’s, forensic serology was a most important part of
lab procedures. With the development of DNA techniques,
more time, money, and significance was placed in developing
DNA labs. However, with limited funds and the time required
for DNA testing, most labs still use many of the basic
serology testing procedures.

Chapter 10
 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
Chapter 10
individual
 Role

of blood in the human body
1. Blood is a fluid connective tissue
 2. Transportation
 It transports oxygen, carbon dioxide, nutrients,
wastes, water, ions, and minerals
 3. Regulation
 Hormones (to regulate body functions)
 Heat (to regulate body temperature)
 4. Protection
 Clotting protects against blood loss
Chapter 10
 Provides immunity to many diseases
 What is Blood?
3 Cell Types + Fluids:
• Erythrocytes (Red Blood
Cells)
• Leukocytes (White Blood
Cells)
• Platelets (Cell fragments)
• Plasma
 White Blood Cell

Platelets

Red Blood Cells

Characteristics of Red Blood Cells
 Redblood cells (erythrocytes) are
biconcave disks that contain the
oxygen-carrying protein
hemoglobin.
 Characteristics of Red Blood Cells
 In addition to 100s of cell surface
proteins, their cell surfaces have a special
class of blood factor proteins called
Antigens.

 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
– O2 + hemoglobin = bright red blood

 Remove CO2 from all body cells and take it

back to lungs for exhalation
– CO2 + hemoglobin = dark red blood

 RBCs are destroyed after about 120 days

 Summary of Functions of WBCs
 Leukocytes can squeeze between cells lining walls of
blood vessels and attack bacteria and debris.
 Neutrophils and monocytes are phagocytic (cell killers).
 Eosinophils moderate allergic reactions as well as
defend against parasitic infections.
 Basophils migrate to damaged tissues to promote
inflammation and 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
attacks).
 Blood Clotting
 The blood clots when
the protein FIBRIN
traps and meshes
RBC’s together.
 SERUM is a yellow
liquid that remains
after the RBC’s clot.
 Blood Clotting and Forensics
 The clotting process begins 3-15 minutes from
injury.
– 1st forms dark, shiny, jelly-like mass
– 2nd begins to contract and separate from serum.

 How long ago did the victim begin bleeding?

– A few minutes: Blood is still liquid
– Less than an hour: Blood is shiny, gelantinous, in a
pool.
– Several hours: Blood has separated into clot and
serum
 Hematocrit
Hematocrit means the
percentage of red blood cells
in blood.

Scientists use a centrifuge to

separate the parts of blood
based upon density of the
parts.

 A blood hematocrit is normally 45% cells and 55% plasma.

 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
wastes
 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
 Antigens are on red blood cells and
antibodies are in the plasma.

 Adverse transfusion reactions are avoided

by preventing the mixing of blood that
contains matching antigens and
antibodies.

– Adverse reactions are due to the

agglutination of red blood cells.
 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.
 Rh Blood Group
 The Rh factor was named after the rhesus
monkey.

 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
factor.
 Blood Groups
Type Antigen Antibody Can Give Can Get
Blood To Blood From

A A B A, AB O, A

B B A B, AB O,B

AB A and B Neither AB A, B, O, AB
A nor B

O Neither A and B A, B, O, AB O
A nor B

Universal Donor is O, Universal Recipient is AB.

Agglutination of RBCs
Population Distribution
of Blood Types in the U.S.
Type Percent

O 45

A 40

B 11

AB 4
Relative Frequency of Blood
Types in Human Populations
Population O A B AB
US whites .453 .413 .099 .035
US blacks .491 .265 .201 .043
Chinese .439 .270 .233 .058
Eskimos .472 .452 .059 .017
Armenians .298 .499 .132 .080
Bolivian .931 .053 .016 .001
Indians
• Genotype: describes the allelic pair
combination
• Phenotype: describe the trait/characteristic
expressed
 Genetic Blood Typing
(Genotypes)
Mother

A B O

A AA AB AO
Father
B AB BB BO

O AO BO OO
Transmission of our traits
Allele Blood Type
Combinations (phenotype)

AA + AO A

BB +BO B

AB AB

OO OO
 Paternity Testing
• Human leukocyte antigen (HLA) is used in
paternity testing.
• If a man and child share the HLA markers
there is a 90% probability that the man is
the child’s father.
• The Haptoglobin marker + ABO + HLA
increases the probability to 95%
 DRUG - DETECTION
Hypothesis:
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
system
 The drug can be attached to a carrier molecule that is
a protein
 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 molecule
 We can isolate these antibodies from the animal’s
blood serum
 And use these antibodies to detect the presence of the
drug in human blood or urine
Now how does a criminalist use these
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 THC-
9-carboxylic acid
 Questions for a Criminalist:
1. Is it blood?
2. Is it human blood?
3. If it is human, can it be linked to a
suspect / victim and how closely?
 The Kastle-Meyer Test to ID Blood
(tests for hemoglobin)
 Phenolphthalein = colorless
 Blood+ phenolphthalein + hydrogen
peroxide = bright pink because of
hemoglobin
 This test can yield some false positives
 Certain vegetable matter can produce the
bright pink positive (such as potatoes)
 Death By Potato?
 Although the Kastle-
Meyer 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.
 Moisten them with distilled water and wipe
the bloodstain. A color change to green is
a positive indicator of blood
Bayer Hemastix©
 Luminol
 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 blood
Advantages of using Luminol
 Allows one to detect stains that would not be
ordinarily be visible
 Extremely sensitive - can use it in very dilute
concentrations
 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
 Drawbacks
 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
 So we know its blood – but is it
human?
 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)
 Is it human blood?
 Human Blood  Frog Blood
 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
other. If they bind to each other, they form
a single line of precipitate in between the
wells
 This is a positive for human blood
 DNA TEST
 DNA testing is the ultimate for learning as
much as possible about a blood sample.
 It has made many other tests obsolete.
 Sources
 Dizengoff, La Guardia High School
 Kendall Hunt, Forensics for High School
 Fundamentals and Investigations, Cengage
 Forensic Science, Saferstein
 www.bio-guru.com
 www.cohs.com