Forensic serology

Introduction
Forensic serology is the detection, classification and
study of various bodily fluids such as :
Blood
Semen
fecal matter
perspiration, and
their relationship to a crime scene.
A forensic serologist may also be involved
in DNA analysis and bloodstain pattern analysis.
 Serology
Serology – term used to describe a broad range of laboratory tests
using reactions of blood serum and body fluid.
The serology section of a forensic laboratory may deal
with any or all of the following:
blood typing.

characterization of unknown blood.

stain patterns for crime reconstruction.

paternity testing.

semen identification in rape cases.

DNA techniques used for identification.

 Blood terminology

ABO blood groups: based on having 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.
 Cont........

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.
 Blood composition
Blood is slightly alkaline fluid which is mixture of
many components:
Cells
Inorganic substances (salts)
Enzymes
Water
Proteins
that circulates though out the vascular system
carrying nourishment and transporting oxygen and
waste
 Blood cells

The most non-fluid portion of blood consists of red cells

which out number white cells by five hundred to one.

While medical scientist are more interested in white cells,

forensic scientist are more interested in red cells and
secondly with serum.
 Cont........
With serum the analyst can determine the freshness of
blood sample because serum clots several minutes after
exposure to air. ( a centrifuge is necessary to separate
clotted material from the rest of serum)

In serum also found antibodies, which have important

forensic implications.
 History
1901 KARL
LANDSTEINER
First to identify ABO
human blood groups
Nobel prize in 1930 for
work
1937: identified Rh
factor (+ or -)
 Blood typing
Blood typing involves determination of the antigens
present on an individual’s RBCs
The two most common blood typing systems used are the
A-B-O method and the Rh method
type A blood – contain “A” antigen on RBCs
type B blood – contain “B” antigen on RBCs
type AB blood – contains both A and B antigens
type O blood – contain no A or B antigens
Rh+ blood – contain Rh antigen
Rh- blood – no Rh antigen
Cont........
 Blood type basics
Four blood types: A (39%),
B (11%), AB (4%) and O
(46%)

Rh Factor: 83% of
population are positive

Testing for blood type is

done through agglutination
and antibodies
 Blood typing
1. Separately add A and B
antibodies to a few drops of
the blood sample.
2. Look for agglutination.
3. Agglutination will occur
only if the antigens are
present on the RBC.
4. No agglutination with
either = O.
5. Agglutination with both =
AB
BLOODSTAIN PATTERN
ANALYSIS
 Introduction

Definition:
A field of forensic investigation that deals with
the physical properties of blood and the patterns
produced under different conditions as a result of
different forces being applied to the blood, blood
as a fluid follows the laws of physics.
 Cont........
Bloodstain Pattern Analysis is the scientific
study of bloodstains to assist in establishing spatial
and sequential events occurring during and
sometimes after the act of bloodshed.
The diameter and shape of blood splatters, which
reflect the origin and trajectory of external blood
flow in the context of homicide or violent death, in
which the skin surface is disrupted.
 Cont......

The science of bloodstain pattern analysis applies

scientific knowledge from other fields to solve
practical problems. Such as:
Biology
Chemistry
Maths &
physics
 Cont........
Also known as:
Blood spatter Pattern Analysis
OR
Bloodstain Pattern Investigation
(BPA/BPI)
Reconstructing events that must have
happened to produce bleeding.
Requires a BPA specialist.
Bloodstain terminology
Cont........
Cont........
 Blood pattern
reconstruction
Scene pattern Lab results
reconstruction reconstruction
• Stain condition • Genetic marker
typing
• Pattern
• Age determination
• Distribution
• Source determination
• Location • Race determination
• Directionality • Sex determination
 Blood stain evidence may
reveal:
Origin of blood stain.
Distance of blood stain from target.
Direction from which blood impacted.
Speed with which blood left its source.
Position of victim and assailant.
Movement of victim and assailant.
Number of blows and shots.
 Liquid blood
Physical properties:

1.Viscosity

2.Surface tension

3.Specific gravity

Shows projectile motion

Cont........
Cont........
 Characteristics of blood
drop
A blood droplet remains spherical in space until it
collides with a surface.

Once a blood droplet impacts a surface, a blood

stain is formed.

Droplets falling from different height, with

different angle, hitting the same surface, will produce
stains with different pattern and shape.
 Conditions affecting
shape of blood droplet

Shape and size of blood spot

Depends mostly on nature of target surface:
Texture (rough or smooth)
Porous or non porous

Size is related to distance fallen:

Standard 50ul drop of blood

There is a little change in spot diameter beyond a fall

distance of 1.2 m.
1. Size of blood drop
 2. Target surface

The harder and less porous the surface,

the less the blood drop will break.
The softer and more porous the surface,
the more the blood drop will break apart.
The pointed end of blood stain shows
the direction of travel.
 Cont........
Smooth surface:

Rough surface:
 3. Angle of impact
The more acute the angle of blood,
the more elongated stain.
90° angle drops are perfectly round
drops.
80° are more elliptical shape.
At about 30° the stain will begin to
produce a tail.
The more acute the angle, the easier
to determine the direction of travel.
Cont........
 Wet vs. dry blood
Wet blood is more significant than dry blood because
scientist can perform more tests in order to investigate
exact crime.
For example alcohol and drug content can be
determined from wet blood only.
Blood is dried after 3-5 mins.
Color changes from deep red to brown to black.
Blood can be categorized into pools, drops, smear or
crusts.
Blood testing by using
different techniques
 Characterization of
bloodstain

1.Is it blood?
2.Which species it come from?
3.If it's human, can it be associated with a
particular individual?
4.Can the sex, age and race of the source of
blood be determined?
 1. Blood or not?

To determine whether or not blood is present at a crime scene

investigators color and crystalline tests are used.
Firstly, benzidine test was used (carcinogenic). Benzidine +blood
stain +hydrogen peroxide= pink color
• Now Kastle-Meyer test is used:
phenolphthalien+bloodstain+hydrogen
peroxide= bright pink color.
• Microcrystalline Test
• Haem forms crystals when reacted with certain
reagents. The most common such reagent is
pyridine, which forms characteristic pink
crystals. The test is carried out on a
microscope slide, with the reagents being
added to the stain under a cover slip, and
crystal formation observed microscopically.
Luminol test (Invisible blood stains)
Luminol, a chemical sprayed on carpets and furniture,
reveals a slightly flourescent light in the dark where
bloodstains are present.
Long dried blood has tendency to crystallize or made to
crystallize with various chemicals.
Luminol is made up in alkaline solution (pH 10.4-10.8)
using sodium carbonate, and sodium perborate (NaBO3.H2O).
The solution is applied as a spray and the presence of blood
produces a bluish luminescence which persists for about 45
seconds.
Luminol test
 Cont........
Instrumental tests:
Chromatographic techniques.
Tests are used practically for several different
purposes including:
1. Confirmation of the nature of visible stains.
2. The detection of non-visible stains. and
3. The enhancement of hard to see stains.
 Cont.........
High performance liquid chromatography (HPLC)
can be used to confirm the identity of blood using the
absorbance of haemoglobin for detection. This method can
also be used to identify the species of origin from variations
in the globin chains, to distinguish foetal haemoglobin from
adult haemoglobin, and to give an estimate of the age of a
bloodstain.
 2. Animal or human
blood?
Precipitation test:
This test involves injecting an animal , usually
a rabbit , with human blood.

Animal's body produces human antibodies.

Extracted from animal's serum.

Antiserum then placed a sample from crime

scene.

Clotting reveals that blood source is human.

 Cont........
Gel Diffusion
Human antibodies and bloodstain are placed
in wells on an agar gel.
If antibodies and antigens move towards each
other and form a line of precipitation, it is
human blood.
Electrophoretic Method
Similar to Gel diffusion except electrical
current is used to move antibodies and antigens
towards each other
 3.Particular individual?
To test a blood sample, for determining a
particular individual forensic investigator must
have adequate and quality blood sample.

Tests:

Blood group testing

DNA finger printing etc.

 4. Age, sex and race?

various color and nitrate tests, and heredity principles are

applied.

No exact determination possible.

Age determination: Clotting and crystallization of blood.

Sex determination: testosterone and chromosome testing.

Race determination: racial genetic markers involving

protein and enzyme tests.
Forensic DNA analysis

PCR and RFLP

Polymerase chain reaction
• The polymerase chain reaction (PCR) is a
biochemical technology in molecular biology
to amplify a single or a few copies of a piece
of DNA across several orders of magnitude,
generating thousands to millions of copies of a
particular DNA sequence.
• As PCR progresses, the DNA generated is itself
used as a template for replication, setting in
motion a chain reaction in which the DNA
template is exponentially amplified.
• A basic PCR set up requires several components and reagents. These
components include:
• DNA template that contains the DNA region (target) to be amplified.
• Two primers that are complementary to the 3' (three prime) ends of
each of the sense and anti-sense strand of the DNA target.
• Taq polymerase or another DNA polymerase with a temperature
optimum at around 70 °C.
• Deoxynucleoside triphosphates (dNTPs, sometimes called
"deoxynucleotide triphosphates"; nucleotides containing
triphosphate groups), the building-blocks from which the DNA
polymerase synthesizes a new DNA strand.
• Buffer solution, providing a suitable chemical environment for
optimum activity and stability of the DNA polymerase.
• Divalentcations magnesium or manganese ions; generally Mg2+ is
used, but Mn2+ can be utilized for PCR-mediated DNA mutagenesis,
as higher Mn2+ concentration increases the error rate during DNA
synthesis
• Initialization step: This step consists of heating the reaction to a
temperature of 94–96 °C (or 98 °C if extremely thermostable polymerases
are used), which is held for 1–9 minutes. It is only required for DNA
polymerases that require heat activation by hot-start PCR.

• Denaturation step: This step is the first regular cycling event and consists
of heating the reaction to 94–98 °C for 20–30 seconds. It causes DNA
melting of the DNA template by disrupting the hydrogen bonds between
complementary bases, yielding single-stranded DNA molecules.

• Annealing step: The reaction temperature is lowered to 50–65 °C for 20–40

seconds allowing annealing of the primers to the single-stranded DNA
template. Typically the annealing temperature is about 3-5 degrees Celsius
below the Tm of the primers used. Stable DNA-DNA hydrogen bonds are
only formed when the primer sequence very closely matches the template
sequence. The polymerase binds to the primer-template hybrid and begins
DNA formation.
• Extension/elongation step: The temperature at this step depends on the
DNA polymerase used; Taq polymerase has its optimum activity
temperature at 75–80 °C, and commonly a temperature of 72 °C is used
with this enzyme. At this step the DNA polymerase synthesizes a new DNA
strand complementary to the DNA template strand by adding dNTPs that
are complementary to the template in 5' to 3' direction, condensing the 5'-
phosphate group of the dNTPs with the 3'-hydroxylgroup at the end of the
nascent (extending) DNA strand. The extension time depends both on the
DNA polymerase used and on the length of the DNA fragment to be
amplified. As a rule-of-thumb, at its optimum temperature, the DNA
polymerase will polymerize a thousand bases per minute. Under optimum
conditions, i.e., if there are no limitations due to limiting substrates or
reagents, at each extension step, the amount of DNA target is doubled,
leading to exponential (geometric) amplification of the specific DNA
fragment.

• Final elongation: This single step is occasionally performed at a

temperature of 70–74 °C for 5–15 minutes after the last PCR cycle to
ensure that any remaining single-stranded DNA is fully extended.

• Final hold: This step at 4–15 °C for an indefinite time may be employed for
short-term storage of the reaction.
 Reverse transcriptase PCR
(RT-PCR)

This PCR was designed to amplify RNA sequences

(especially mRNA) through synthesis of cDNA by
reverse transcriptase (RT).
Subsequently, this cDNA is amplified using PCR.
This type of PCR has been useful for diagnosis of
RNA viruses, as well as for evaluation of
antimicrobial therapy
 Real time PCR

To quantify the number of copies of nucleic acids

during PCR
Intercalating agents such as SYBR Green are
fluorochromes dramatically increase the fluorescence
by binding to a double-stranded DNA .
Thus, the increase of DNA in each cycle reflects a
proportional increase in the emitted fluorescence.
 Multiplex PCR

It is a modification of polymerase chain reaction in

order to rapidly detect deletions or duplications in a
large gene.

This process amplifies genomic DNA samples using

multiple primers and a temperature-mediated DNA
polymerase in a thermal cycler
 Nested PCR:
• Increases the specificity of DNA amplification, by
reducing background due to non-specific
amplification of DNA. Two sets (instead of one pair)
of primers are used in two successive PCRs. Nested
PCR is often more successful in specifically
amplifying long DNA fragments than conventional
PCR, but it requires more detailed knowledge of the
target sequences
 Hot-start PCR:
• A technique that reduces non-specific amplification
during the initial set up stages of the PCR. It may be
performed manually by heating the reaction
components to the melting temperature (e.g., 95°C)
before adding the polymerase. Hot-start/cold-finish
PCR is achieved with new hybrid polymerases that
are inactive at ambient temperature and are instantly
activated at elongation temperature.
 Colony PCR
• the screening of bacterial (E.Coli) or yeast clones for
correct ligation or plasmid products. Selected
colonies of bacteria or yeast are picked with a sterile
toothpick or pipette tip from a growth (agarose) plate.
This is then inserted into the PCR master mix or pre-
inserted into autoclaved water. PCR is then conducted
to determine if the colony contains the DNA fragment
or plasmid of interest
 Allele-specific PCR:
• A diagnostic or cloning technique which is based on single-
nucleotide polymorphisms (SNPs) (single-base differences in
DNA). It requires prior knowledge of a DNA sequence, including
differences between alleles, and uses primers whose 3' ends
encompass the SNP. PCR amplification under stringent
conditions is much less efficient in the presence of a mismatch
between template and primer, so successful amplification with an
SNP-specific primer signals presence of the specific SNP in a
sequence.
 Application of PCR in
forensics
Genetic basis of diseases with sudden
death can be investigated.
Forensic molecular pathology involves
application of molecular biology in medical
science to investigate the genetic basis of
pathophysiology of diseases that lead to
deaths ("molecular autopsy").
 Cont........

To establish the filiations of a person

Paternity testing

To obtain evidence from minimal samples of

saliva, semen or other tissue debris
 RFLP

The DNA sample is broken into pieces (digested)

by restriction enzymes and the resulting restriction
fragments are separated according to their lengths
by gel electrophoresis.
In addition to genetic fingerprinting RFLP was an
important tool in genome mapping, localization of
genes for genetic disorders, determination of risk for
disease, and paternity testing
Cont........
 FORENSIC DNA ANALYSIS

Short Tandem Repeats (STRs)

 Three generations of
DNA testing

RFLP DQ-alpha Automated STR

AUTORAD TEST STRIP ELECTROPHEROGRAM
Allele = BAND Allele = BLUE DOT Allele = PEAK
 STRs

•Short tandem repeats

•Describes a type of DNA polymorphism in which:
–a DNA sequence repeats
–over and over again
–and has a short (usually 4 base pair) repeat unit
•A length polymorphism -- alleles differ in their
length
3 repeats: AATG AATG AATG
4 repeats: AATG AATG AATG AATG
5 repeats: AATG AATG AATG AATG AATG
6 repeats: AATG AATG AATG AATG AATG AATG
 Application in forensics

Individual identification possible

People differ in length at these loci
Genetic fingerprinting
Paternity, maternity analysis

Person 1 ..GCCAGCTAGCTAGCTAGCTAGCTAGCTTTCAT..
Person 2 ..GCCAGCTAGCTAGCTAGCTAGCTTTCAT..

Person 3 ..GCCAGCTAGCTAGCTAGCTAGCTAGCTAGCTT..
1 2 3 4 5 6 7
 Steps to develop SSR markers
• Construct small‐insert clone library
• Screen it by hybridizing labelled oligo (with SSR
motif of interest)
• Sequence positive clone
Design primers in single copy regions flanking SSR repeats
such that the amplified fragmentswill be > 50 bp and < 350 bp
• Identify size polymorphism on PAGE gels.
Automated STR Test
 Basic steps in analysis

Extraction:
Separates DNA from sample

Amplification or PCR:

Amplifies small portions of DNA (STR regions)

Separation:

Separates amplified fragments according to size.

Crime Scene Samples &
Reference Samples

•Extract and purify DNA

Differential extraction in sex

assault cases separates out
DNA from sperm cells
 Extract and Purify DNA

•Add primers and other reagents

 PCR Amplification

•DNA regions flanked by

primers are amplified

Groups of amplified STR products are labeled

with different colored dyes
(blue, green, yellow)
The ABI 310 Genetic Analyzer:
SIZE, COLOR & AMOUNT
 ABI 310 Genetic Analyzer:
Capillary Electrophoresis
•Amplified STR DNA injected
onto column
•Electric current applied

•DNA pulled towards the

positive electrode

•DNA separated out by

size:
Detector
–Large STRs travel slower Window
–Small STRs travel faster

•Color of STR detected and

recorded as it passes the
detector
 Capillary Electrophoresis

Sample will have one or two peaks at each loci.

 Resources
•Books
–‘Forensic DNA Typing’ by John M. Butler (Academic Press)
•Internet
–Applied Biosystems Website: http://www.appliedbiosystems.com/ (see human
identity and forensics)
–Forensic Bioinformatics Website: http://www.bioforensics.com/
–STR base: http://www.cstl.nist.gov/biotech/strbase/ (very useful)
•Scientists
–Larry Mueller (UC Irvine)
–Simon Ford (Lexigen, Inc. San Francisco, CA)
–William C. Thompson (UC Irvine)
–William Shields (SUNY, Syracuse, NY)
–Marc Taylor (Technical Associates, Ventura, CA)
–Keith Inman (Forensic Analytical, Haywood, CA)
•Testing laboratories
–Technical Associates (Ventura, CA)
–Indiana State Police (Indianapolis, IN)
•Other resources
–Forensic Bioinformatics (Dayton, OH)