Forensics OF

Biological Evidence
Forensics of Death – Presentation, Causes, and Autopsies
Biological Evidences – Sources, Presentation, and Analysis
Sampling and Extraction of DNA in Biological Evidences
Common Forensic Services
Service Function Method
• Alternate light sources (UV light, intense light)
Latent Print • Analysis of friction-ridge details in fingerprints,
• Light comparison microscopes
and footprints, toolmarks in firearms, bullet casings,
• Videospectral Comparator (VSC)
Impressions documents, sometimes in bodies.
• Digital Imaging of the sources of fingerprints
Examination • Visualization, recording, storage, and recovery
• Automated Fingerprint Identification System Database

• Biological Fluids Analysis (Forensic Serology)

• DNA Profiling, analysis, comparison, and storage
• Chemical Serology and DNA isolation
Forensic • Forensic Anthropology (for skeletal remains)
• Polymerase Chain Reaction
Biology • Forensic Entomology (insects in relation to causes
• STR analysis and DNA Index System database
and post mortem interval)
• Forensic Odontology of unrecognizable bodies

• Qualitative and Quantitative analysis of various
Post mortem
organic and inorganic substances and their
toxicology
metabolites
• Analysis of transferred evidence (substances that
Trace Evidence
are exchanged between the assailant and the
Examination
victim in the crime scene) in trace amounts
• Light and Electron Microscopy (single and comparison)
• Chromatography (liquid and gas)
• UV, IR, and Mass spectrometry
• Light and Electron Microscopy (single and comparison)
• Chromatography (liquid and gas)
• UV, IR, and Mass spectrometry
Chemical and Biological Aspects of Death
Stages of Death
Stage Presentation

Puncture/gunshot wounds: continuous bleeding, severe oxygen depravation to several parts of the body; the
points of entry, depth, affected organs/tissues, and the exit wounds are determined.
Blunt-force trauma: severe tissue damage, resulting in bleeding (internal or external hemorrhage) and
malfunction; trauma to vital organs could cause organ failures; usually presents with severe red-to-purple
ante mortem coloration and bleeding of the area where the force was sustained
(before death) Poisoning: traces of poison is usually found in the liver during autopsy; in some cases, there are various
colorations in the mouth and tongue
Asphyxiation: severe oxygen deprivation of multiple organ systems which causes failure; strangulation marks on
the neck. Since this type of murder is slow, transfer evidences are usually more abundant due to the victim’s
resistance and defense.

Dead on the spot – for sudden, very severe forces

agonal period Dead on arrival (to the hospital) – more agonizing, typical in some cases where the victim is bleeding to death.
(point of death) The process is slower, which is more cruel. During the agonal period, the medical personnel priority is to revive
the victim, which sometimes causes body marks that can be misinterpreted in the autopsy.
Chemical and Biological Aspects of Death
Stages of Death
Stage Presentation (PMI are rough estimates)
“Fresh” status (approx. first 2 days after death)
• Temperature continues to decline (algor mortis, about 0.6 to 1.5 oC per minute) due to cessation of the
normal metabolism
• Accumulation of potassium ions in the vitreous humor of the eye.
• Hypostasis, or livor mortis – post mortem lividity; purple to red-purple discoloration of the skin due to
the settling of blood in the blood vessels due to gravity. The skin and mucous membranes appear pale
due to the settling of the blood on the body area near the ground.
“Bloat” status (approx. 2 to 20 days after death)
post mortem • Uncontrolled increase in the population of gut and intestinal microbiota, which starts to consume the
(after death) gut and intestinal tissues. Their metabolism becomes anaerobic due to lack of O2 which is sourced from
the blood.
• Increased presence of detritivores – animals that consume detritus, or decaying matter. Some
detritivores include blowflies (and maggots), cockroaches, termites, earthworms, carrion flies, etc.
“Putrefaction” status (more than 20 days)
• The body enters the stage of advanced decay – the gases start to escape following perforation, causing
the body to collapse in itself. Foul gases start to saturate the immediate environment.
• In some cases, there is formation of adipocere (corpse wax), which may inhibit further decomposition
and DNA of the victim even after many years.
Chemical and Biological Aspects of Death
A person is considered dead after the following observations:
• Lack of breathing, pulse, heartbeat, pain responses, and muscular reflexes
• Maximally dilated pupils, which are also unresponsive to light stimulus
• Absence of spontaneous breathing and blood pulse on the neck and wrists

The “Fresh” status – 0 to 2 days PMI

• Hypostasis – gradual cooling due to the cessation of internal homeostasis
• A pale presentation due to cessation of blood circulation

Livor Mortis, or the settling of blood and Rigor Mortis caused by loss of muscle
Imbibition caused by hemolysis of
other fluids due to the influence of flexibility due to starvation (the body no
blood, which in turn stains the vessels
gravity. This begins approximately 30 longer produces the required ATP both
and the connective tissues.
minutes after death. for the skeletal and smooth muscles)
Chemical and Biological Aspects of Death
The “Bloat” status – 2 days to 20 days PMI
• Clostridia and enterobacteria start to metabolize and consume the gut and intestinal matter.
• The metabolism is anaerobic since the body no longer supplies oxygen. Product gases (like CH4, CO2, and H2S) evolve as part of
the anaerobic respiration, which causes a foul odor.

Accumulation of detritivores in the early
stages of the bloat, such as blowfly
maggots and cockroaches.
Bloating is a result of the buildup of
gases within the body due to several
bacterial anaerobic processes. As the
body is consumed by microorganisms, it
starts to perforate, releasing the foul
gases.
Facial bloating at its late stages may
already obscure some causes, like
asphyxiation or poisoning, due to
discolorations in the eyes, neck, and
tongue.
Chemical and Biological Aspects of Death
The “Putrefaction” status – 20 days to 50 days PMI
• Also known as advanced decomposition status – increases the difficulty of autopsy
• Due to the perforation of the skin tissues, water and gases start to escape, causing collapse of the body
• The increase in the seepage in bodily fluids and gases attract more insects
• Exposed parts are colored black (black putrefaction)
• Depending on the conditions, the body may undergo mummification, adipocere preservation, or butyric fermentation

Mummification happens when the When conditions remain anaerobic at

release of water during putrefaction is
rapid, which kills the microbes and
thus inhibiting further decomposition
When conditions remain ideal for
elevated temperatures and high
several bacteria, including the
humidity, adipocere (grave wax) forms.
Clostridium, butyric acid
It also preserves the remains for a long
fermentation occurs, which usually
time.
preceeds skeletonization.
Cause of Death - Autopsy

Incision Injuries from a knife Stab injuries are usually from Blunt-force trauma injuries, Gunshot injuries in the head
in the limbs. the chest and abdomen areas in the head (close contact)

3rd degree burn of almost Death by corrosion – ingestion Ligature strangulation (used a Cherry-red coloration due to
100% of the body of conc. H2SO4 strangulation object) marks CO poisoning
Crime Scene Investigation
Principles of Crime Scene Reconstruction
Principle Definition

Locard’s Exchange If a crime is committed and there was contact, a cross-transfer of materials or impressions between the
(Edmond Locard) victim and the assailant is very likely to happen.

Steno’s Superposition In a crime scene, objects or impressions that are placed on top of each other must be chronologically
(Nicolas Steno) placed. The older material must be deposited or placed underneath the newer material.

Disruptions in the natural strata are indicative of significant events, and can be used to determine when
Steno’s Lateral Continuity
the disruption occurred. It is useful when describing the directions used by either the assailant or victim
(Nicolas Steno)
at the event of the crime.

A partial evidence (a piece of cloth, paper, wire) can be considered as physical evidence if the rest of it is
Principle of Association found to be in possession of the assailant. All tranfer evidences are viable under this principle as well.
This is the principle behind toolmark/impression analysis

This principle states that the preceding events should only be the logical causes of the event of interest.
Principle of Chronology
This is important in determining the real cause of death or injury to the victim.
Crime Scene Investigation
Analysis of the Crime Scene
• The aim for the analysis is to build a story on how the crime happened based on all the presented evidence.
• There are several guiding principles of crime scene analysis – superposition, continuity, association, exchange, and chronology.

It is important to analyze superposition
of objects in the crime scene. When
chronology is applied, it helps
determine if the scene was staged or
not.
The lateral continuity of disturbances
(caused by footprints, dragging, etc.)
should be analyzed to generate a path of
the assailant or victim from and towards
the crime scene.
Blood on the faucet tap. Principle of
association requires that the blood be
tested for DNA, while the print made
by the blood should be obtained for
matching.
Crime Scene Investigation
Analysis of the Crime Scene
• Collection of every possible evidence for the benefit of forensic investigations (mostly for crime scene reconstruction)
• Photograph of the evidence location and measurements
• On-Site tests for presence of blood and other trace substances
• Complete description of the evidence
➢ direct, circumstantial, or testimonial; physical, chemical, or biological; macroscopic or microscopic

• The usual evidences collected:

• Photographs of blood splatter, if there are any
• Impressions (fingerprints, palm prints, footprints, shoe prints, tool marks, etc.)
• Physical evidences (bullet casings, glass shards, fibers, dirt, etc.)
• Chemical evidences (poisons, drugs, gunshot residues, luminol glow, etc.) – determined by tests
• Biological evidences (bodily fluids, skin tissues, hair, insects, plant material, etc.)
➢ bodily fluids - blood, semen, saliva, stomach fluid, cerebrospinal fluid
➢ Tissues - skin tissues, connective tissues found in the hair roots, dandruff (but hard to find)

• Analysis of the time, nature, and cause of death

• Analysis of the post mortem interval (PMI)
• Stabbing, incising, hacking, gunshot, blunt-force trauma, crushing trauma, asphyxiation, suffocation, burn, etc.
Crime Scene Investigation
Some Sampling Opportunities per Cause of Death in Murder Cases
Cause of Death Sampling and Evidence Opportunities in the Crime Scene
• The wound pattern could indicate the distance of the gunshot and strength of the gun used.
Gunshot • The position of the body in the crime scene could indicate several clues
• High-velocity blood splatter pattern indicates wounds from high-speed projectiles
• If there are signs of struggle, there may be a lot of transfer evidences (suspect’s hair and skin tissue) can be
Stabbing, Incision, found in the victim’s hands and nails
strangulation • Low-velocity blood splatter patterns, trail patterns, etc. These crimes usually leave a lot of blood.
• Several impressions may be found (fingerprints, footprints) in cases where the murder was not planned
• Some pieces of the weapon (surface paint, shards, chips) may be found in the wounds of the victim
• Weapon impressions are more obvious for these cases
Blunt Force
• Low-velocity blood splatter patterns
Trauma
• Defense injuries in the arms/hands may also contain the same transfer evidences
• Impressions of the suspect may also be available in the scene
• Orally-ingested poisons usually affect the upper digestive tract. If not, there is liver damage
Poisoning • CO poisoning has a unique presentation (cherry red coloration in the body)
• HCN poisoning has a unique presentation (bitter almond smell, bright red coloration in the body)
Characteristics of Evidences and Analysis Approach
Types of Evidence
Type Definition Examples
Characteristics of an Evidence that is same as that of a very large Type of gun (pistol, rifle)
Class Characteristics
number of samples Type of shoes (leather, rubber, running)
Characteristics of an Evidence that is still the same as that of a
Brand of rubber shoes and sole design
Specific Characteristics large number of samples, but is can be considered as subsets of
Caliber and brand of a certain pistol/rifle
class characteristics, which reduces population size
Characteristics of an Evidence that are present only on a certain Wear patterns on the soles of the shoes
Unique Characteristics group of objects or a single object; can be considered a subset of Firing pin pattern made by the gun on its bullet
specific characteristics casing
In many texts, “specific characteristics” are still class characteristics.
Approaches on Testing and Analysis
Type Definition Examples
Compares a certain characteristic of evidence from a
Comparative Comparison of firing pin patterns on a casing found in the crime
standard sample or another evidence; may require an
Analysis scene vs. the casing test fired by the investigator from the same gun
exemplar, control or comparison standard
Identifies specific patterns and interprets it based on Fire patterns; Blood patterns; questioned documents; autopsy;
Interpretation
previous experiences/events or statistics behavioral analysis
Chemical tests for the presence of blood; analysis of poison
Laboratory Conducts standard chemical and physical tests both
concentration based on post-mortem concentration; compositional
Analysis for qualitative and quantitative analysis
analysis of trace evidences using SEM-EDX; DNA test
Crime Scene Investigation
DNA Sampling and Extraction from Various Sources
Source Summary of Sampling Methodology
• Blood collection is usually exemplary. Blood is collected from possible suspects and the victim.
• First step is the lysing of the erythrocytes. which leaves the nuclei and mitochondria of the other cells intact and
Blood removes haemoglobin from the red cells. Lysis buffer (SDS, proteinase K, EDTA) is added before centrifugation.
• Separate DNA from proteins and cellular debris by salting out and centrifugation.
• Purify DNA using concentrated ethanol.
• Semen is usually collected through a rape kit (a sperm sampling + preservation kit) or a simple vaginal swab.
• Lysis agent (SDS, proteinase K, EDTA) to disintegrate the “female fraction” into the supernatant.
• Since spermatozoa is more resistant to lysis than blood cells, the next step involves centrifugating the sample along
Semen
with a surfactant (SDS, proteinase K, EDTA, dithiothreitol) to release the nuclei and mitochondria.
• Separate DNA from proteins and cellular debris by salting out and centrifugation.
• Purify DNA using concentrated ethanol
• Skin samples are usually obtained in small quantities but are easily sampled when found.
Soft Tissues • Hair samples must include a root where the connective tissues are located, to be able to sample DNA.
from skin, • For bone samples, the soft tissues are usually collected in the marrow.
urine, and • Soft tissue samples are mixed with the lysis buffer and centrifugated, just like blood samples.
perspiration • Separate DNA from proteins and cellular debris by salting out and centrifugation.
• Purify DNA using concentrated ethanol.
Polymerase Chain Reaction (PCR) is used if the DNA sample size is too low for RFLP analysis
Forensic Analysis
DNA Sampling and Extraction from Various Sources

If the DNA sample is too low for an RFLP analysis, a Polymerase Chain
Reaction (PCR) is done to increase the DNA sample size.

Steps:
• The DNA is denatured to separate the strands (90-95OC).
• The sample is annealed at about 60oC - 65oC to allow primers to
attach to the separated strands.
• The temperature is increased to 70oC to allow elongation. This is the
optimum temperature of the thermophyllus aquaticus polymerase
(Taq polymerase) enzyme.

The step may be repeated several times depending on the amount of

DNA initially present.
Forensic Analysis
DNA Sampling and Extraction
from Various Sources

DNA is generally analyzed using a

Restriction Fragment Length Polymorphism
(RFLP)

This technique uses various restriction

enzymes to cleave the DNA at specific
recognition sites to produce fragments that
are then separated either by molar mass,
electric charge, or both.

Identical DNA samples must produce

identical fragments when cleaved by the
same restriction enzyme.
General Steps in DNA Analysis

Gel
electrophoresis
of the cleaved
fragments for
comparison.
Obtain DNA from the
sample by extraction

PCR is used to Addition of restriction enzymes

Gel Electrophoresis
increase sample size (differs from one species to another)
General Steps in DNA Analysis
Epithelial cells are easier to lyse than
spermatozoa. Even after addition of the
SDS and proteinase K, sperm cells will not
lyse and will stay at the residue.

Addition of DTT will enable lysis of the

sperm cells.

Both DDT and SDS can be removed from

the DNA solution by either Organic
Solvent Extraction (P-Cl-A reagent) or by
Solid-Phase Extraction (SPE) using silica as
a gel matrix.
Proteinase K, SDS, and DTT are all removed by phenol-chloroform-alcohol (P-Cl-A).
Phenol-chloroform-Alcohol reagent: phenol:chloroform:isoamyl alcohol (25:24:1)
General Types of Cellular DNA
Nuclear DNA
Type Mitochondrial DNA (mtDNA) Y – Chromosomal DNA (yDNA)
(nDNA, also autosomal DNA)
Location Mitochondria Cell nucleus (for eukaryotes) Y- sex chromosome
Base pair counts 16,500 base pairs 3.3 billion base pairs 59 million base pairs
Inheritance From Mothers Both parents (50-50) From Fathers
• Low differentiation between
mothers • Unique fingerprint of the • Low differentiation between
• Can be extracted even if individual, can be compared fathers
Advantages sample is very old to both biological parents • Can be used to compare
• Can be used to compare • Easier to extract and analyze lineage even between distant
lineage even between distant due to size generations
generations

Nuclear DNA fingerprinting is the routine analysis for crime scene investigations. Other types of DNA are employed depending on
the availability of the persons involved and the status of the biological evidences.
Blood Patterns in the Crime Scenes
Since the blood easily stains surfaces and is a liquid, its presentation patterns in the crime scene can be very
useful in analyzing the events of a crime.
Classification of Bloodstain Patterns Based on Impact Angle:

Passive Stains are stains from
drops, flows, and pools. These
stains were influenced purely by
gravity. The angle of impact is
nearly perpendicular to that of
the surface.
Transfer Stains are stains imprints
made from getting in contact with an
existing bloodstain or source, and then
getting in contact with another
surface. Instead of impact angle, the
wipe/drag direction is considered.
Impact/Projected Stains are stains
made by blood propelled by a certain
force before reaching a surface. The
angle of impact is usually 30-70
degrees with respect to the surface.
Blood Patterns in the Crime Scene
Types of Bloodstain Pattern Based on Velocity:

Low-Velocity Blood Patterns Medium-Velocity Blood Patterns High-Velocity Blood Patterns

• 5 ft/s and below
• Droplet diameter ≥ 4mm
• Gravity influence is high
• Drips, bleeding after injury
• 5-25 ft/s, looks like a splash
• Droplet diameter 1mm to 4mm
• Direction of stain is evident
• Blunt forces, stabbing, traffic
incidents
• At least 50 ft/s, looks like a splash
• Droplet diameter < 1mm
• Direction of stain is evident
• Gunshot and explosion
Blood Patterns in the Crime Scene
Types of Bloodstain Pattern Based on Directionality and Movement:

Cast-Off Patterns
• Linear, medium-to-high velocity
• This is due to blood released from the
weapon after being swung into motion

Drip Trail Patterns Arterial Spurt Patterns

• Linear, Passive to low- • Linear, Passive to low-
velocity velocity
• This is due to blood • This is due to blood
from a bleeding person spraying out from a large
while walking/moving, artery due to pressure
from a bloody weapon caused by the heart
held while moving, or pump
any other blood- • The darkest spurts are
drenched object being usually the starting point
moved from one place of these patterns
to another.
Blood Patterns in the Crime Scene
Types of Bloodstain Pattern Based on Directionality and Movement:
Pool Patterns
• Nonlinear, usually occupies a
large area, darkest at the source
of the blood.
• Indicates massive bleeding at a
single point for a significant
period of time.
Drag/Swipe Marks
• Linear, transfer-type of stains
• Indicates that the bleeding
person has been moved at a
direction indicated by the mark
• Sometimes, false drag patterns
are caused by an attempt to
clean the crime scene
Void Patterns
• Negative Patterns (without blood)
• Indicates a previous presence
before it was moved elsewhere.
• Also indicates movement, violates
Steno’s Law of Lateral Continuity
Blood Patterns in the Crime Scene
Types of Blood Patterns that might be misinterpreted in the Crime Scene

Expectorate Blood Pattern Angled Satellite Stains from a parent

Blood-to-Blood Drip Pattern Drip stain
• Nonlinear patterns that came
• Blood drops on top of a small from the mouth or nose. • If the scene is altered, the parent
pool of blood, creating a splash stain may be absent and these
• May be misinterpreted as a very small stains may be
• Might be misinterpreted as a cast-off stain
medium-to-high velocity pattern misinterpreted as medium
• Density of the spatter and velocity.
• The angle and direction must be analysis of saliva must be done
considered • Luminol test can be done for a
more complete picture.
Blood Patterns in the Crime Scene
Void Patterns and Possible Crime Scene Alteration
• Void patterns are important indications of crime
scene alteration.
• A weapon/object must leave a void pattern if
blood was sprayed to its location before it was
moved.
• It can also give a clue on the type of weapon used,
which can be compared to the autopsy results
• For example, in possible pistol suicide cases, a void pattern
must be present in the hands of the victim as a result of a
blowback spatter, or a high velocity spatter opposite to the
direction of the bullet.
• The void pattern must also be at the correct hand. However,
handedness is not a definitive indication of a foul play.