FORENSIC SEROLOGY

Courtesy of C. Fanning
 IN 1901, KARL LANDSTEINER ANNOUNCED ONE
 OF THE MOST SIGNIFICANT DISCOVERIES OF
THE 20THCENTURY – THE TYPING OF BLOOD – A
FINDING THAT 29 YEARS LATER EARNED HIM
A NOBEL PRIZE.
 For years, physicians had attempted to transfuse
blood from one individual to another, but their
efforts often ended in failure because the
transfused blood tended to coagulate, or clot in the
body of the recipient, causing instantaneous death.
 Landsteiner was the first to recognize that all
human blood was not the same; instead, he found
that blood is distinguishable by its group or type.

HISTORY of Serology
  OUT OF LANDSTEINER’S WORK
CAME THE CLASSIFICATION
SYSTEM THAT WE CALL THE A-B-O
SYSTEM. NOW PHYSICIANS HAD
THE KEY FOR PROPERLY
MATCHING THE BLOOD OF
a donor to that of a recipient.
One blood type cannot
be mixed w ith adifferent
blood type w ithout
disastrous consequences.
This discovery had
important implications for
blood transfusion and
the millions oflives it
has since saved.

Karl Landsteiner (1868-1943)

  LANDSTEINER’S FINDINGS OPENED A NEW FIELD OF
RESEARCH IN THE BIOLOGICAL SCIENCES. OTHERS BEGAN
TO PURSUE THE IDENTIFICATION OF ADDITIONAL
CHARACTERISTICS
that could further differentiate blood. By 1937, the
Rh factor in blood had been demonstrated and,
shortly thereafter, numerous blood factors or groups
were discovered. More than 100 different blood
factors have been identified. However, the ones in
the A-B-O system are still the most important for
properly matching a donor and recipient for a
transfusion.

Karl Landsteiner
 
UNTIL THE EARLY 1990’S, FORENSIC SCIENTISTS FOCUSED
ON BLOOD FACTORS, SUCH AS A-B-O, AS OFFERING THE
BEST MEANS FOR LINKING BLOOD TO AN INDIVIDUAL.
What made these factors so attractive was that in
theory, no two individuals, except for identical
twins, could be expected to have the same
combination of blood factors. In other words,
blood factors are controlled genetically an d have
the potential of being highly a highly distinctive
feature for personal identification.
 What makes this observation so relevant is
great
the frequency of bloodstains at crime scenes,
especially crimes of the most serious nature:
homicides, assaults, and rapes.

Application to Forensics
  THE ADVENT OF DNA TECHNOLOGY HAS DRAMATICALLY
ALTERED THE APPROACH OF FORENSIC SCIENTISTS
TOWARD INDIVIDUALIZATION OF BLOODSTAINS AND
OTHER BIOLOGICAL EVIDENCE. THE SEARCH FOR
GENETICALLY
controlled blood factors in bloodstains has been
abandoned in favor of characterizing biological
evidence by select regions of our deoxyribonucleic
acid (DNA), which carries the body’s genetic
information. As a result, the individualization of
dried blood and other biological evidence become
a reality and has significantly altered the role that
crime laboratories play in criminal investigation.

Application to Forensics
 NATURE OF BLOOD

•The word blood refers to a highly complex mixture of cells, enzymes,

proteins, and inorganic substances.

•Plasma, which is the fluid portion of blood, is composed principally of

water. The plasma accounts for 55% of blood content.

•Red blood cells (erythrocytes), white blood cells (leukocytes), and

platelets (thrombocytes) are the solid materials suspended in
plasma.

•Blood clots when fibrin traps and enmeshes the red blood cells. If
the clotted material where removed, a pale yellowish liquid known as
serum would be left.

•Antigens, usually proteins, are located on the surface of red blood

cells and are responsible for blood-type characteristics.
 ANTIGENS AND ANTIBODIES

•Red blood cells transport oxygen from the lungs to the

gody tissues and remove carbon dioxide from tissues by
transporting it back to the lungs, where it is exhales. For
reasons unrelated to the red blood cell’s transporting
mission, on the surface of each cell are millions of
characteristic chemical structures called antigens. More
than 15 blood antigen systems have been identified, but the
A-B-O and Rh systems are the most important.

•An individual that is type A has A antigens on his/her red

blood cells, type B has B antigens, AB has both A and B
antigens, and type O has neither A nor B antigens.

Cont.
 ANOTHER IMPORTANT BLOOD ANTIGEN
HAS
been designated as the Rh factor, or D
antigen. People with the D antigen are
said to be Rh positive; those without this
antigen are Rh negative.
 In routine blood banking, the presence or

absence of the three antigens - A, B, and

D - must be determined in testing
compatibility of the donor and recipient.

Antigens and Antibodies

 BLOOD TYPING
•Serum is important because it contains proteins known
as antibodies. The fundamental principal of blood typing is that for
every antigen, there exists a specific antibody. Each antibody
symbol contains the prefix anti-, followed by the name of the antigen
for which is it specific.

•The serum-containing antibody is referred to as the antiserum,

meaning a serum that reacts against something (antigens).

•Antibodies are normally bivalent – that is, they have two reactive
sites. This means that each antibody can simultaneously be
attached to antigens located on two different red blood cells. This
creates a vast network of cross-linked cells usually seen as
clumping or agglutination.
 SEROLOGY

•The term serology is used to describe a broad scope of

laboratory tests that use specific antigen and serum antibody
reactions.

•The identity of each of the four A-B-O blood groups can be

established by testing the blood with anti-A and anti-B sera.
 SEROLOGY ANTIGEN-ANTIBODY REACTION

•The concept of specific antigen–antibody reactions has

been applied to immunoassay techniques for the detection
of drugs of abuse in blood and urine.

Red blood cells containing A Red blood cells containing B

antigens do not combine with B antigens are agglutinated or
antibodies. clumped together in the presence
of B antibodies.
 
LET’S LOOK A LITTLE MORE CLOSELY AT THIS PHENOMENON. IN NORMAL BLOOD,
ANTIGENS ON RED BLOOD CELLS AND

antibodies coexist without destroying each other

because the antibodies present are not specific toward
any of the antigens. However, suppose a foreign serum
added to the blood introduces a new antibody. This
results in a specific antigen-antibody reaction that
immediately causes the red blood cells to link together,
or agglutinate.

Serology Antigen-Antibody Reaction

  NATURE HAS TAKEN THIS SITUATION INTO ACCOUNT,
BECAUSE WHEN WE EXAMINE THE SERUM OF TYPE
A BLOOD, WE FIND ANTI-B AND NO ANTI-A.

Similarly, type B blood contains only anti-A,

type O blood contains both anti-A and anti-B,
and type AB blood contains neither anti-A or
anti-B. The antigen and antibody
components of normal blood are summarized
in the table given to you in your lab….
Lab #1: Training Lab: Blood Types

Serology Antigen-Antibody Reaction

  SEROLOGY INVOLVES A BROAD SCOPE OF LABORATORY TESTS
THAT USE SPECIFIC ANTIGEN AND SERUM ANTIBODY REACTIONS.
 An antibody reacts or agglutinates only with its specific
antigen. The concept of specific antigen-antibody
reactions has been applied to techniques for detecting
abused drugs in blood and urine.
 Every red blood cell contains either an A antigen, a B
antigen, or no antigens. The type of antigen on one’s red
blood cells determines one’s ABO blood type. People
with type A blood have A antigens on their red blood
cells, those with type B blood have B antigens, and those
with type O blood have no antigens on their red blood
cells.

Quick Review
IMMUNOASSA
Y
TECHNIQUES
{ Radioimmunoassay and EMIT
  THE CONCEPT OF A SPECIFIC ANTIGEN-ANTIBODY REACTION IS
FINDING APPLICATION IN OTHER AREAS UNRELATED TO
BLOOD TYPING. MOST SIGNIFICANTLY, THIS APPROACH HAS
BEEN EXTENDED TO THE DETECTION OF DRUGS IN BLOOD
AND URINE.
 A ntibodies that react with drugs do not
exist
naturally; however, they can be produced in animals
such as rabbits by first combining the drug with
a protein and injecting this combination into the
animal.
This drug-protein complex actsas an antigen
stimulating the animal to produce antibodies.

Immunoassay Techniques
 INJECT INTO
Drug Protein Carrier RABBIT
attached to drug
Rabbit produces
The recovered blood serum of the animal now antibodies nearly
contains antibodies that are specific or nearly specific to the drug.
specific to the drug originally attached to the
protein carrier.
Currently, thousands of individuals regularly submit to urinalysis for the
presence of abused drugs. These individuals include military personnel,
transportation industry employees, police and corrections personnel, and
subjects requiring pre-employment drug screening. Immunoassay testing for
drugs has proven quite suitable for handling the large volume of specimens
that must be rapidly analyzed for drug content on a daily basis.
 IMMUNOASSAY

•A number of immunological assay techniques are commercially

available for detecting drugs through antigen-antibody reaction.

•One such technique, the enzyme-multiplied immunoassay

technique (EMIT), is used by toxicologists because of its speed
and high sensitivity for detecting drugs in urine.

•In a typical EMIT analysis, antibodies that will bind to a specific

drug are added to the subject’s urine.

•Other immunoassay procedures are also available, such as

radioimmunoassay (RIA), which uses drugs labeled with
radioactive tags.
 IMMUNOASSAY TECHNIQUE.

enzyme-multiplied
EMIT has
gained widespread popularity among
toxicologists because of its speed and
high sensitivity for detecting drugs in
urine.

EMI
T
 ANTIGEN-ANTIBODY
REACTION

•When an animal, such as a rabbit or mouse, is injected with an

antigen its body will produce a series of different antibodies, all
of which are designed to attack some particular site on the
antigen of interest.
 ANTIGEN-ANTIBODY
REACTION

•This collection of antibodies is known as polyclonal antibodies.

•Alternately, a more uniform and specific collection of antibodies

designed to combine with a single antigen site can be
manufactured.

•Such antibodies are known as monoclonals.

Characterization of
Bloodstains
FORENSIC
 FORENSICS OF
BLOOD

•The criminalist must be prepared to answer the following

questions when examining dried blood:

1. Is it blood?
2. From what species did the blood originate?
3. If the blood is of human origin, how closely can it be
associated to a particular individual?

•The determination of blood is best made by means of a

preliminary color test.
 TESTING FOR
BLOOD

•A positive result from the Kastle-Meyer (phenolphthalein) color test

is highly indicative of blood. Hemoglobin causes a deep pink color.
Courtesy of C. Fanning

•Alternatively, the luminol test is used to search out trace amounts

of blood located at crime scenes.

•Luminol produces light (luminescence) in a darkened area.

Luminol with
false positive
(bleach)

Courtesy of C. Fanning Courtesy of C. Fanning

  FOR MANY YEARS, THE MOST COMMON TEST WAS THE
BENZIDINE COLOR TEST, BUT BECAUSE BENZIDINE HAS BEEN
IDENTIFIED AS A KNOWN CARCINOGEN, ITS USE HAS
GENERALLY BEEN DISCONTINUED AND REPLACED WITH THE
KASTLE-MEYER TEST.
 Both the benzidine and the Kastle-Meyer color tests are
based on the observation that blood hemoglobin possesses
peroxidase-like activity. Peroxidases are enzymes that
accelerate the oxidation of several classes of organic
compounds when combined with peroxides. For example,
when a bloodstain, phenolphthalein reagent, and hydrogen
peroxide are mixed together, oxidation of the hemoglobin in
the blood produces a deep pink color.

Benzidine and Kastle-Meyer

 Field investigators have
found Hemastix strips

{
a presumptive
useful
test for field
blood. Designed as
a urine dipstick test for
blood, the strip can be
moistened with distilled
water and placed in contact
with a suspect bloodstain.
The appearance of a green
color indicates blood.

Hemastix
 Another important presumptive
identification test for blood is the
luminol test. Unlike the benzidine
and Kastle-Meyer tests, the reaction

{
of luminol with blood produces light
rather than color. By spraying
luminol reagent onto a suspect item,
investigators can quickly screen large
areas for bloodstains. The sprayed
objects must be located in a
darkened area while being viewed for
the emission of light (luminescence);
any bloodstains produce a faint blue
glow.

Luminol and Bluestar

 The identification of blood can be made
more specific if microcrystalline tests
are performed on the material.

{
Several tests are available; the two
most popular ones are the Takayama
and Teichmann tests. Both depend on
the addition of specific chemicals to
the blood to form characteristic
crystals containing
derivatives. Crystal
hemoglobin
tests are far less
sensitive than color tests for blood
identification and are more susceptible
to interference from contaminants that
may be present in the stain.

Microcrystalline Test
 ONCE THE STAIN HAS BEEN CHARACTERIZED AS BLOOD, THE
SEROLOGIST DETERMINES WHETHER THE BLOOD IS OF HUMAN OR
ANIMAL ORIGIN. THE STANDARD TEST IS THE PRECIPITIN TEST.

PRECIPITIN TESTS ARE BASED ON THE FACT THAT WHEN ANIMALS
(USUALLY RABBITS) ARE INJECTED WITH HUMAN BLOOD,
ANTIBODIES FORM THAT REACT W ITH THE
INVADING

human blood to neutralize its presence. The investigator can

recover these antibodies by bleeding the animal and isolating
the blood serum, which contains antibodies that specifically
react with human antigens. For this reason, the serum is known
as human antiserum. In the same manner, by injecting rabbits
with the blood of other known animals, virtually any kind of
animal antiserum can be produced. Antiserums are
commercially available for humans for human s and for a
variety of commonly encountered animals – for example, dogs,
cats, chickens and deer.

Precipitin Test
 SEVERAL
TECHNIQUES HAVE BEEN


devised for
performing precipitin tests on
bloodstains. The classic method
is to layer an extract of the
bloodstain on top of the human
antiserum in a capillary tube.
Human blood, or for
that matter, any protein of
human origin in the extract,
reacts specifically
with antibodies present in
the antiserum, and
indicated by the formation of
a cloudy ring or
band at the interface of the two
liquids.
Precipitin Test
  ANOTHER METHOD, CALLED GEL DIFFUSION, TAKES
ADVANTAGE OF THE FACT THAT ANTIBODIES AND ANTIGENS
DIFFUSE OR
move toward one another on a plate coated with a
gel medium from a natural polymer called agar.
The extracted bloodstain and the human antiserum
are placed in separate holes opposite each other on
the gel. If the blood is human, a line of precipitation
forms where the antigens and antibodies meet.

Gel Diffusion
  SIMILARLY, THE ANTIGENS AND
ANTIBODIES CAN BE INDUCED
TO
move toward one another
under the influence of an
electrical field. In the
electrophoretic method, an
electrical potential is applied to
the gel medium; a specific
antigen-antibody reaction is
denoted by a line of precitation
formed between hole
the containing the
bloodthe hole containing
and extrac
Gel Diffusion the human antiserum. t
The precipitin test is very sensitive and requires only a small
amount of blood for testing. Human bloodstains dried for 10
– 15 years and longer may still give a positive precipitin
reaction. Even extracts of tissue from mummies four to five
thousand years old have given positive reactions with this
test. Furthermore, human bloodstains diluted by washing in
water and left with only a faint color may still yield a positive
precipitin reaction.
 ONCE IT HAS BEEN DETERMINED THAT
THE BLOODSTAIN IS HUMAN, AN

EFFORT MUST BE MADE TO ASSOCIATE
OR

disassociate the stain with a particular individual. Until the mid-1990’s, routine
characterization of bloodstains included the determination of A-B-O types;
however, the widespread use of DNA profiling or typing has relegated this
subject to one of historical interest only.

Precipitin Test
 RECAP

• The precipitin test uses antisera normally derived from

rabbits that have been injected with the blood of a known
animal to determine the species origin of a questioned
bloodstain.

• Once it has been determined that the bloodstain is of

human origin, an effort must be made to associate or
dissociate the stain with a particular individual.

• DNA analysis has allowed forensic scientists to associate

blood to a single individual.
 A-B-O VS. DNA

• Prior to the advent of DNA typing, bloodstains were

linked to a source by A-B-O typing and the
characterization of polymorphic blood enzymes and
proteins.

• This approach has now been supplanted by the newer

DNA technology.

• DNA analysis has allowed forensic scientists to associate

blood and semen stains to a single individual.
 HEREDITY AND
PATERNITY

• The transmission of hereditary material is accomplished by

means of microscopic units called genes, located on
chromosomes.
• Alternative forms of genes that influence a given
characteristic (such as eye color or blood type) are known as
alleles.
• Paternity testing has historically involved the A-B-O blood
typing system, along with blood factors other than A-B-O.
• Currently, paternity testing has implemented DNA test
procedures that can raise the odds of establishing paternity
beyond 99 percent.
 TESTING FOR SEMINAL
STAINS

• Many of the cases sent to a forensic laboratory

involve sexual offenses, making it necessary to
examine exhibits for the presence of seminal stains.

• The best way to locate and at the same time

characterize a seminal stain is to perform the acid
phosphatase (an enzyme secreted into seminal fluid)
color test.
A purple color indicates acid phosphatase

enzyme.
 TESTING FOR SEMINAL
STAINS
Semen can be unequivocally identified by either the presence
of spermatozoa or of p30, a protein unique to seminal
plasma.

Forensic scientists can successfully link seminal material to

an individual by DNA typing.
 RAPE
EVIDENCE

• The rape victim must undergo a medical examination as

soon as possible after the assault.

• At that time the appropriate items of physical evidence

including clothing, hairs, and vaginal and rectal swabs
can be collected for subsequent laboratory examination.

• All outer and undergarments should be carefully

removed and packaged separately in paper (not plastic)
bags.

• Bedding, or the object upon which the assault took place,

may also be carefully collected.
 RAPE
EVIDENCE

• If a suspect is apprehended within 24 hours of the assault,

it may be possible to detect the victim’s DNA on the
male’s underwear or on a penile swab of the suspect.

• Items routinely collected from the suspect include all

clothing, pubic hair, head hair, penile swab, and a blood
sample or buccal swab for DNA typing.

• The forceful physical contact between victim and assailant

may result in a transfer of such physical evidence of
blood, semen, saliva, hairs, and fibers.