Module 3 Lesson 1 Forensic 3

Forensic 3: Forensic Chemistry and Toxicology
Subject: Forensic Chemistry and Faculty:

Instr. JO ANN T. GERADA
Toxicology Instr. JEAN G. BULLO
Course, Year & BS Crim 3 Date:
Sec:
Identification, collection, preservation,
Module No. 3 investigation, presentation, of biological
evidence.
● Blood
Lesson No. 1 ● Semen and other body fluid (Saliva, Urine and etc.)
for DNA
Overview:
In this module, we discuss about the Identification, collection,

preservation, investigation, presentation, of biological evidence.
Module Outcome:
At the end of this module the students must have:
1. Identified biological evidence such as blood, semen and other body fluids.
2. Described the component, properties, and characteristics of biological
evidence as blood, semen and other body fluids.
3. Determined the process of collection, preservation, investigation of
biological evidence as blood, semen and other body fluids.
4. Described the test on biological evidence as blood, semen and other body
fluids.
5. Explained the importance of the study biological evidence in the
investigation as blood, semen and other body fluids.
Take Off (Motivation)
In the conduct of crime scene investigation for crimes of violence and other
crimes, the crime scene processing or the evidence collection, handling and
transportation shall primarily be conducted by the SOCO specialists of
Crime Laboratory. However, in some instances the First Responder or the
Investigator-on-case may have to collect evidence that might
otherwise be destroyed or contaminated if uncollected. In such cases,
the collection should be properly handled and documented. The following
procedures are set as guide not only for the SOCO team but may also apply
to any crime scene investigator in the collection and handling of evidence.
The collection and submission of standard samples for comparison,
however, must be done by the Crime Laboratory.
COMPILED BY: JO ANN T. GERADA AND JEAN G. BULLO

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COLLEGE OF CRIMINAL JUSTICE EDUCATION105
Content Focus: (Content)
General Rules for the Collection and Preservation of Biological

Materials
a. Use protective gloves.
1. If possible, avoid touching individual smears/traces. Remember that

gloves can entail a risk of contamination.
2. Change gloves after handling each kind of material and otherwise

as necessary.
3. Use disposable equipment for preliminary tests and collection of

trace evidence.
4. Cover surfaces where materials are to be placed with protective

paper. Keep victims‟ and suspects‟ clothes separate.
b. Avoid coughing or sneezing on evidence/materials.
c. Packaging of biological materials.
1. Use paper packaging for all biological materials or materials that

are soiled with biological matter. Although plastic bags are useful in
many cases, they cannot be recommended for routine use on account
of residual moisture.
2. Separate outer packages are to be used for trace evidence and for
clothes from persons involved.
3. Do not mix materials/samples from different people, for example

clothes, in the same parcel.
4. Fold the opening of the bag twice and seal with tape. Envelopes should
also be sealed with tape.
d. Special precautions
1. Make an explicit note if a person from whom material has been
collected is suspected of having an infectious disease.
2. Prevent contamination by avoiding all contact between collected
evidence and clothes seized from people.
3. Packages containing collected materials must not be opened until the
examination in the laboratory is to commence. The only exception is

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when moist or wet material must be dried out under normal room
conditions.
A. Blood
Blood left at a crime scene can be analyzed in several ways by a criminal

investigator. Blood typing may provide class evidence because more than one
person has the same blood type. Because white blood cells contain DNA, it is
possible to determine with a high degree of certainty using DNA profiling
whether evidence blood left at a crime scene matches the blood of a suspect
(or victim).
Blood-spatter evidence can also be used to help recreate a crime scene to

validate the information provided by a witness or suspect. By using blood
spatter, it is possible to note the direction from which the blood originated,
the angle of impact, and the point of origin of the blood. Further examination
of the blood drops might indicate if the blood spatter resulted from a high- or
low-velocity impact, indicating the type of weapon used to cause the injury.
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 1980s, forensic serology was a most important part of
lab procedures. With the development of DNA techniques, more time, money,
and significance were placed on 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.
COMPOSITION OF BLOOD
Blood is a circulating tissue consisting of three types of cells: red blood

cells, white blood
cells, and platelets.
These cells are
suspended in a liquid
known as plasma.
Plasma is similar to salt
water in composition. It
carries dissolved
proteins, such as
antibodies, hormones,
and clotting factors, and
nutrients such as
glucose, amino acids,
salts, and minerals.
BLOOD CELLS

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Each blood cell performs a different function. Red blood cells (erythrocytes)
carry respiratory gases, mainly oxygen and carbon dioxide. The hemoglobin in
red blood cells is an iron-containing protein that binds to oxygen in the lungs
and transports the oxygen to cells in all the tissues in the body. Hemoglobin in
red blood cells is also responsible for the red color in blood. White blood cells
(leukocytes) fight disease and foreign invaders. Platelets (thrombocytes) aid
in blood clotting and are involved in repairing damaged blood vessels. Plasma
is the fluid portion of the blood (55 percent). Cells (45 percent) composed of
Erythrocytes are red blood cells. They are responsible for oxygen
distribution. Red blood cells are most numerous; 5 to 6 million per mm3.
Leukocytes are the white blood cells; they are responsible for “cleaning” the
system of foreign invaders. White blood cells are larger and less numerous;
5,000 to 10,000 per mm3. Thrombocytes or platelets are responsible for
blood clotting. Platelets are tiny, cellular fragments; 350,000 to 500,000 per
mm3. Serum is the liquid that separates from the blood when a clot is formed.
HISTORY OF DNA PROFILING
In 1982, white blood cells were used as a source of DNA by Dr. Alec Jeffreys
to produce the first DNA profile. The first legal case involving DNA evidence is
described in a novel entitled The Blooding by Joseph Wambaugh. Today, DNA
profiling or DNA fingerprinting is widely accepted.
BLOOD TYPING
Blood typing is less expensive and quicker for analyzing blood evidence than
is DNA profiling. Because many different people share the same type, this
blood evidence is considered to be class evidence. By typing the blood found
at a crime scene, it is possible to link a suspect to a crime scene or to exclude
a suspect. However, matching blood types does not prove guilt because many
people share the same blood type.
Discovery of Blood Types
In 1900, Karl Landsteiner found that blood from one person did not always
freely mix with blood from another person. Instead, clumping might occur,
which could result in death. The presence or absence of particular proteins
found embedded within the cell or plasma membranes of red blood cells
determine a person’s blood type. In 1901, Landsteiner described the A and B
proteins found on the surface of red blood cells. Other red blood cell proteins,
such as the Rh factor, were later identified. The presence or absence of these
cell-surface proteins gives rise to our present system of blood typing. An
antibody reaction test is used to identify each blood type.
Paternity Determination by Blood Type Chart Calculator

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Blood type can be used to disprove paternity only in some cases. In other
words, the non-father's blood type may be the same or be of a type where
either man could be the father based solely on the blood type but not in the
results of a DNA Paternity test.
The ABO blood type charts below can be used to predict the possibilities of
paternity. The charts below can assist you to determine either:
1. The ABO blood type of the child when the blood type of the
father and the mother are known (top chart), or
2. The ABO blood type of the father if the blood type of the child
and the mother are known (bottom chart).
ABO Blood Type Calculator
ABO blood types can be complicated to understand. This is mostly due to the
fact that the 'O-type' antigen is masked by the presence of an A- or
B-type antigen. This is explained in the following chart, showing the
different the genotypes that make up the blood types.
Genotype (DNA) Blood Type
AO or AA A blood type
AB AB blood type
BO or BB B blood type

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OO O blood type
For example, two O blood type parents can produce a child with only O blood
type. Two parents with A blood type can produce a child with either A or O
blood types. Two parents with B blood type can produce a child with either B
or O blood type. One parent with A and another with B can produce a child
with A, B, AB or O blood types. If one parent has A and another has AB, they
can either produce a child with A, B or AB blood types. If one parent has A
and another has O, they can either produce a child with A or O blood types.
Rh Positive (Rh+) and Negative (Rh-) Blood Types
The Rh (+/-) factor is inherited separately from

the ABO blood types. Similarly, to the masking
effect of the O gene in ABO blood types, the Rh
negative (Rh-) gene is also masked by the
presence of a Rh positive (Rh+) genotype.
Therefore, a person may have a Rh + blood type
and can still have an Rh - gene (See the chart
below). Furthermore, 2 parents with Rh + blood
types can have a child with Rh - blood type.
BLOOD SPATTER
When a wound is inflicted and blood leaves the body, a blood-spatter pattern
may be created. A single stain or drop of blood does not constitute a spatter.
Instead, a grouping of bloodstains composes a blood-spatter pattern. This
pattern can help reconstruct the series of events surrounding a shooting,
stabbing, or beating.
HISTORY OF BLOOD-SPATTER ANALYSIS
In 1894, Pitoroski wrote the earliest reference to blood-spatter analysis. In

1939, Balthazard was the first researcher to analyze the meaning of the
spatter pattern. In 1955, blood-spatter evidence was used by the defense in
the Sam Shepard case, helping to exonerate him. In 1971, Dr. Herbert
MacDonnell used blood-spatter analysis as a tool in modern forensic
examinations. Today, blood-spatter evidence is used to explain events at a
violent scene.
Bloodstain Pattern Analysis:
Is the examination of the shapes, locations, and distribution patterns of

bloodstains, in order to provide an interpretation of the physical events which
gave rise to their origin.

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Based on the premise that all bloodstains and bloodstain patterns

are characteristic of the forces that have created them.
The determinations made from bloodstain patterns at the scene or
💧
from the clothing of principals in a case can be used to:
Confirm or refute assumptions concerning events and their sequence:
Position of victim. (standing, sitting, lying) Evidence of a struggle.
💧 (blood smears, blood trails)

Confirm or refute statements made by principals in the case:
Are stain patterns on a suspects clothing consistent with his reported
actions?
Are stain patterns on a victim or at a scene consistent with accounts
given by witnesses or the suspect?
Properties of Blood
■ Blood Volume: On average, accounts for 8 % of total body weight

⮚ 5 to 6 liters of blood for males
⮚ 4 to 5 liters of blood for females
⮚ A 40 percent blood volume loss, internally or/and externally, is
required to produce irreversible shock (death). A blood loss of 1.5
liters, internally or externally, is required to cause incapacitation.
■ Surface Tension
The elastic like property of the surface of the liquid that makes it tend to
contract, caused by the forces of attraction between the molecules of the
liquid. The cohesive forces tend to resist penetration and separation.
💧 Categories of Bloodstains
Categories Visual Immage Division
TRANSFER
BLOODSTAINS 💧 Contact bleeding
A transfer bloodstain is 💧 Swipe or Smear
created when a wet, 💧 Wipe
💧 Smudge
bloody surface comes in
contact with a secondary
surface.

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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.
PASSIVE
BLOODSTAINS 💧 Drops
Passive Bloodstains are 💧 Drip patterns
drops created or formed
by the force of gravity 💧 Pools
acting alone. 💧 Clots
TARGET SURFACE
TEXTURE
● Bloodstains can occur
on a variety of ✔ Blood droplets
surfaces, such as that strike a
carpet, wood, tile, hard smooth
wallpaper, surface, like a
clothing, and the list piece of glass,
goes on…… will have little or
no distortion
● The type of surface around the
the blood strikes edge.
affects the amount of
resulting spatter, ✔ Blood droplets
including the size and that strike
appearance of the linoleum flooring
blood drops. take on a slightly
different
appearance.
Notice the
distortion
(scalloping)
around the edge
of the blood
droplets.
✔ Surfaces such as
wood or
concrete are
distorted to a

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larger extent.
Notice the spines
and secondary
spatter
LARGE VOLUMES
OF BLOOD
Patterns created by same
volume of blood, from Dripped Blood
same source to target
distance)
Spilled Blood
PROJECTED
BLOODSTAINS
Projected bloodstains are ✔ Arterial Spurt /

created when an exposed Gush
blood source is subjected
to an action or force, Bloodstain
greater than the force of pattern(s) resulting
gravity. from blood exiting
the body under
(Internally or externally pressure from a
produced) breached artery:
The size, shape, and ✔ Cast-off Stains

number of resulting stains
will depend, primarily, on
the amount of force ✔ Impact Spatter
utilized to strike the blood
source. Blood stain patterns
created when a
blood source
receives a blow or
force resulting in

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the random
dispersion of
smaller drops of
blood.
This category can

be further
subdivided into;
▪ Low Velocity
Gravitational pull
up to 5 feet/sec.
Relatively large
stains 4mm in
size and greater.
▪ Medium Velocity
Force of 5 to 25
feet/sec.
Preponderant
stain size 1 to
4mm in size
▪ High Velocity
Force of 100
feet/sec. and
greater.
Preponderant
stain size 1mm
in size and
smaller. Mist like
appearance
BLOOD-SPATTER ANALYSIS

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Given blood-spatter patterns, it is possible to determine the direction the

blood was traveling, the angle of impact, and the point of origin of the blood.
Blood-spatter patterns can help determine the manner of death, based on the
blood
velocity.
Table of blood-spatter parameters.
DIRECTIONALITY OF BLOODSTAINS
When a droplet of blood strikes a surface perpendicular (90 degrees) the

resulting bloodstain will be circular. That being the length and width of the
stain will be equal. Blood that strikes a surface at an angle less than 90
degrees will be elongated or have a tear drop shape.
Directionality is usually obvious as the pointed end of the bloodstain (tail)

will always point in the direction of travel.
IMPACT ANGLE DETERMINATION
ANGLE of IMPACT is the acute angle formed between the direction of the
blood drop and the plane of the surface it strikes.

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By utilizing trigonometric functions it’s possible to determine the impact angle

for any given blood droplet. By accurately measuring the length and width of
a bloodstain, the impact angle can be calculated using the SIN formula below
SIN Φ = opp (a)

hyp (c)
SIN Φ = Width (a) 1.5cm

Length (c) 3.0cm
SIN Φ = 0.5
Φ = SIN -1 0.5
= 30 degrees
POINT OF CONVERGENCE AND ORIGIN DETERMINATION
The common point, on a

2-dimensional surface, over
which the directionality of several
bloodstains can be retraced.
Once the directionality of a group
of stains has been determined,
it's possible to determine a
two-dimensional point or area for
the group of stains.
THREE-DIMENSIONAL POINT OF ORIGIN

DETERMINATION USE OF THE COMPUTER
FOR POINT OF ORIGINCALCULATIONS
WITH IMPACT ANGLE CALCULATIONS.
The Collection and Preservation of Blood
Type of evidence/ Procedure Packing/storage

samples
Blood on removable Remove the whole Put each piece of
materials object material in a paper
packaging and seal with

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tape. If the material is

wet or moist, pack each
piece of material in a
plastic bag sealed with
tape. Open and allow to
dry on arrival at the
police station. Send to
the PNP CL in wrapping
paper or an envelope.
Pools of blood Collect blood on some Put the swabs in the
swabs. In the case of swab wrapper or a
larger accumulations of folded piece of paper.
blood, take several Package in an envelope,
samples from various store dry and cool.
places.
Blood in Water Collect on several Put the swabs in the
swabs. swab wrapper or a
folded piece of paper.
Package in an envelope,
store dry and cool.
Collect water with a Pour the water into a
clean pipette or clean, dry test tube with
syringe. a cork or a vacuum tube
with a purple cork.
Dry blood If possible, cut away Put each sample in a
part of the surrounding paper bag or envelope.
material. Keep dry and cool.
If this is not possible, Put the swabs in the
moisten a swab with swab wrapper or a
water. Rub it until it folded piece of paper.
becomes dark Package in an envelope,
brown/red or until the store dry and cool.
swab absorbs all the
blood.
B. Semen
What is Semen? Semen is a

mixture of various fluids that carry
live spermatozoa to the female
ovule for fertilization. A fertile
semen sample holds tens of
millions of spermatozoa per
milliliter and can provide useful
DNA evidence. The volume of

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ejaculate is anywhere between 2-6 milliliters, with roughly 100-150 million

spermatozoa in each mL. Because of illness, drug use, or injury, the sperm
count may be lower.
PARTS OF SEMEN LOCATION OF SEMEN AND

SEMEN STAIN AS EVIDENCE
The main constituents of semen: 1. Under clotting
A. Solid components 2. Clothing
1. Sperm 3. Skin
2. Miscellaneous solids, like skin 4. Air
cells 5. Vagina
B. Fluid component 6. Rectal contains of the victim
● Seminal plasma 7. Around the genitals
The solid fraction is primarily the spermatozoa themselves, plus any
fragments thereof and miscellaneous skin cells shed along the way.
Seminal plasma contains proteins, salts, organics (including flavins which
are the source of its UV fluorescence, and choline), and some cellular
material.
The components originate from several sources, including seminal vesicles

and the prostate gland. The prostate is the source of the enzyme acid
phosphatase (AP) and the protein prostate specific antigen, or p30
protein.
Exploitable semen for analysis can be detected up to:

• 31 hours in the mouth
• 44 hours in the anus
• 110 hours in the rectum
• 10 days in the vagina
• 19 days in the cervix
Common Semen Tests
The easiest and least intrusive to the evidence is the Alternate Light
Source. UV light and the ALS detect seminal stains by phosphorescence
and luminescence of the various materials in the semen.
Also very easy is the presumptive test for the enzyme, acid phosphatase
(AP). Many animal and vegetable tissues have AP, but it is a very high

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concentration in semen. Both of these are extremely easy to use in the

field or at the lab, and both are looking at fluid portion, not solids.
Alternate Light Source
Alternate Light Source is our first

step. It works on the principle
that most items will have
luminescent qualities at certain
wavelengths of light, and a band
pass filter allows only that
wavelength to reach our
eyes/the camera.
Using ALS
The alternate light source is the least destructive technique we have

(other than plain white light). Use it to save yourself time and effort
locating stains. Be sure you understand how it works and why it works
before going to court. Remember, while we examine the items with ALS,
we might mark it lightly with a sharpie, since once were in regular light,
we may not be able to see the stain any longer.
When using ALS, the flavins and bacteria in seminal fluid cause the glow.
Drops and stains can have a distinctive ring at the outer perimeter, caused
by the migration of the fluid out through the material or on the surface.
Smears are still bright, but without the ring. Best in UV light or range just
above.
Testing for Semen using Acid Phosphatase
Testing for Semen using Acid Phosphatase ALS examination is obviously

the first step. However, all stains that fluoresce are not necessarily semen
and not all semen stains will fluoresce. Acid Phosphatase testing is easy,
fast, and inexpensive. If you have lab capabilities, you can actually mix
your own from reagent powder. But for field-testing, there are several kits.

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The Chemistry Behind Acid Phosphatase
The AP spot test contains sodium-α-naphthyl phosphate and o-dianisidine

(Brentamine Fast Blue). If acid phosphatase is present in a sample and a
drop of the reagent is added, the enzyme catalyzes the sodium-α-naphthyl
phosphate producing free naphthyl. That reacts with o-dianisidine
producing a purple-colored compound.
Are There False Positives?
Of course, there are false positive. Feminine products, fecal stains, plants,
pregnant women and prepubescent girls are all potential intereferences
with the test. But remember, semen is a heterogeneous fluid and a single
stain will contain various levels of acid phosphatase, P30, and sperm.

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Since AP can be found in lower levels in other bodily fluids, it’s important
to view the test immediately and report it accurately.
Time Frame for Testing
How long would you expect to find the following in the vagina after
intercourse? Acid Phosphatase is detectable for approximately 72 hours
after intercourse and sperm can be found approximately 3 to 5 days
depending on activity after the assault.
Testing Other Items

How long would you expect to find AP in a dried stain, such as clothing or
bedding?
AP may survive for many years in a dried state; however, environmental

conditions (heat, moisture, bacterial growth) could affect the survival rate.
There are four examinations for seminal stains or seminal fluid in

the form of stains namely:
1. Physical Examination
2. Chemical Examination
a. Florence Test b. Barberio’s Test c. Acid-phospahtase Test
3. Microscopic Examination
4. Biological Examination
C. Saliva
Saliva is a biological secretion inside the mouth that is primarily a digestive

aid as the salivary amylases break down the starches in our food. It
contains urea, glucose, progesterone, various traces of acids, amino acids,
creatinine, and more than 1,000 different proteins. No specific test exists
for its detection, although the presence
of alpha-amylase strongly supports the
identification of saliva.
Additionally, saliva is a carrier of cells for

genotyping. Cells might be found on
stamps, food, drinking glasses,
cosmetics, pillows, bite marks on skin,
and so forth.

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Saliva is secreted from three sets of glands; the sublingual, submandibular,

and parotid.
Screening for saliva is based on detection of high levels of amylase in the

sample. It is not a confirmatory test as amylase is found in other body
fluids.
What’s in Your Spit?
Your spit contains mostly water, but bacteria, skin cells from the inside of the
mouth (buccal cells), and the substance we test for – α-amylase – is also
present. This enzyme helps break down carbohydrates, but can vary widely
between people.
Studies have also suggested that vegetarians and people from cultures that
consume a high carbohydrate diet may have higher levels of amylase than
others. In a completely non-scientific local experiment, two CSIs who came
from Central American and East Indian heritages and ate often of their
cultures’ foods (corn, beans, breads, rices, etc) had stronger reactions in
testing than a Caucasian CSI who was avoiding starchy carbohydrates and
eating primarily meats, nuts, and vegetables.
Saliva and the ALS
Saliva visually represents with soft edged white spots, sometimes less intense
than other stains because of fewer solids.
Saliva under UV

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Testing for Saliva
We’re going to look at a lab technique for the RSID-Saliva kit. Be careful when
deciding to do this test though, since case information may indicate sending
an item off for DNA analysis might be better. The RSID Saliva test is specific
for human salivary α-amylase. No cross-reaction has been observed with
blood, semen, urine, vaginal secretions, or menstrual blood. RSID test is kind
of like a pregnancy test. You’re going to get two stripes for positive, or one
for negative.

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Lateral Flow Immunochromatographic Strip Tests for the

Identification of body Fluids Using the RSID-Saliva
Test First specific, non-enzymatic tests for salivary amylase. No observed

cross reaction with blood, urine, sweat, semen, domestic animals, exotic
species, body fluid mixtures. Minor, but consistent signals seen with breast
milk and fecal samples. Fast: Test results in 10 minutes post extraction.
Sensitive: Detects as little as 50 ml of saliva – stated detection limit: 1 ml. No
High Dose Hook effect observed: little or no dilution required, less possibility
of false negatives. Efficient: Assay procedure integrated in DNA-STR analysis
RSID-Saliva should be evaluated exactly 10 minutes after the addition of

sample.
A visible red line at the Control (C) position only, indicates a negative result.
No α-amylase detected.
Visible red lines at both the Control (C) and Test (T) positions, indicate a
positive result. Α-amylase detected. A visible red line at the Test (T) position
only, indicates a failed test. Test failure, no conclusion possible.
No cross reactivity has been observed with saliva from the following animals
and pets: dog, opossum, guinea pig, woodchuck, cow, domestic cat, domestic
rabbit, tokay gecko, cuckoo, mongoose, chameleon, domestic pig, llama,
sheep, horse, goat, grey gull, ferret, hedgehog, skunk, lion, tiger, rhinoceros,
marsh snake, Sykes monkey, Capuchin monkey, tamarin, and marmoset.
A positive signal was obtained from the saliva of gorilla.
The Collection and Preservation of Semen or Saliva

samples
Semen or saliva on Remove the whole Put each piece of
removable object. material in a separate
materials paper bag. Unpack on
arrival at police station
and dry at room
temperature. Send to
the PNP CL in paper
bags.
Moist semen or Collect part of the Put the swabs in the
saliva sample on some swabs. swab wrapper or a

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folded piece of paper.

Package in an envelope.
Store dry and cool.
Draw off liquid with a Pour the liquid in a
clean pipette or clean, dry test tube with
syringe. a cork or a vacuum tube
with a purple cork.
Store in a refrigerator.
Send to the PNP CL by
refrigerated transport.
Semen in condom Close the condom with Keep in a refrigerator
a clip. and send to the Crime
Lab as soon as possible
by refrigerated
transport.
Dried semen or saliva If possible, cut out part Put each sample in a
of the surrounding paper bag or envelope.
material. Store cool and dry.
Otherwise, moisten a Air dry and put the
swab with water. Rub it swabs in the swab
until it is saturated. wrapper or a folded
piece of paper. Package
in an envelope. Store
dry and cool.
D. Urine (Biological Evidence In Forensic Science)
Urine is the by-product of the cellular metabolism in humans. Urine is

yellow-straw colored fluid produced by the process of urination. It is excreted
out from the body by an opening called urethra. It follows a pathway from
kidney to urinary bladder through ureters.
On an average urine production is around 1.4 Liters per day by per person. It
depends from person to person on the various factors like weight, health,
state of hydration and environmental factors.
CONSTITUENTS OF URINE
Urine is an aqueous solution of greater than 95% water, with a minimum of

these remaining constituents, in order of decreasing concentration:
● Urea 9.3 g/L
● Chloride 1.87 g/L
● Sodium 1.17 g/L
● Potassium 0.750 g/L
● Creatinine 0.670 g/L

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● Other dissolved ions, inorganic and organic compounds (proteins,

hormones, metabolites)
EXAMINATION OF URINE
1. Physical Examination
Characteristics Normal Abnormal
Change in color is indicative of some kind

Pale to dark
Color of disease or effect of some drugs or
yellow in colour
medicine
Cloudy urine can be caused by factors

like pus, blood, sperm, bacteria, yeast, or
Clarity Clear
a parasite infection, such as
trichominiasis
Some food, vitamins, and antibiotics can

Odor Bad odor cause urine to have a different odor. A
fruity odor may be caused by diabetes.
high specific gravity means very

concentrated urine. (loss of fluids, sugar
Specific
1.005-1.030 or protein)
Gravity
low specific gravity means diluted urine
(Kidney problem)
between Low pH can be caused because of health

pH
4.6–8.0 ailments
Protein None Health ailments
Ketone None diabetes, starvation or eating disorders
2. Visual Examination
A suspected urine stain may fluoresce pale yellow or pale blue when viewed
under long and short-wave UV light. Safety eyeglasses, which absorb
ultraviolet radiation, must be worn when viewing material for fluorescence.
3. Microscopic Analysis

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Normal Urine – White blood cells in very less quantity. Bacteria, yeast cells ,
parasites absent.
Abnormal Urine – Red blood cells in the urine indicate kidney or bladder
injury, kidney stones, a Urinary Tract Infection (UTI).
4. CHEMICAL TEST
Detection of urine stain is based on detection its constituents like urea,

amines, phosphates, sulphates, etc.
● Urea in a suspected ‘urine’ stain can be detected with urease-
bromothymol paper. If present, urea would be enzymatically
decomposed to ammonia and carbon dioxide by urease. This liberated
ammonia will shift the pH. It will change the color from yellow to blue.
5. CONFIRMATORY TEST
A. CREATININE TEST
This test is also called Jaffé color test. In this test, picric acid is used to
convert creatinine under alkaline condition to form creatinine picrate. Picric
acid is strong oxidizing agent. Creatinine is getting oxidized which is produced
by muscle metabolism. Amount of urine excreted in urine is proportional to
muscle mass of individual.
Procedure
1. Extract the stained material and blank sample.
2. Put one drop of picric acid in both the samples.
3. Add 5% of sodium hydroxide solution in the stain.
4. Observe the color change. The blank sample turns yellow color and the
stained sample turns red to brown color change.
The red to orange to brown color crystal indicates the presence of

creatinine in the urine.

23
B. TEST FOR UREA NITRATE
Urea nitrate is produced in one step by reaction of urea with nitric acid. This
crystal test indicates the presence of urea in the given sample.
Procedure:
● Take the drop of sample on the glass slide.
● Put 2-3 drops of conc. nitric acid. Prevent overflow.
● Cover the slide with coverslip.
● Observe the slide under the microscope.
Observations:
Appearance of rhombohedral stacked crystals of urea nitrate which are
colorless or transparent indicates the presence of urine in the sample.
C. Detection of Indican
1 ml of resorcinol reagent is added to the small quantity of the extracted stain
then the 1ml of cupric bromide solution is added mixed and whole mixture is
extracted with amylacetate. The red color of the crystal indicates the
presence of Indican.
D. UA/UN Ratio
Uric Acid and Urea Nitrogen ratio multiplied by 20 in human urinary stain is
between 1 and 4, in other stains and animal urine its <1 or >4.
E. Tamm Horsfall protein or Uromodulin

24
Urine contains a glycoprotein called Tamm Horsfall protein, produced

exclusively by renal tubular epithelial cells within the distal loop of Henle. This
can be detected by Radioimmunoassay.
THIN LAYER CHROMATOGRAPHY

Various methods for the detection of urea for urine stain identification have
been described above. Thin-layer chromatography is also used to detect both
urea and creatinine in an effort to make the test more specific for urine, and
also additional components of urine on TLC plates can be used for the same
purpose.
URINE DRUG TESTING

Urine is very frequently tested for the presence of drug in a person’s system.
Urine testing is evasive, quick, cost effective and can be screened for
metabolites of parent drug. Drugs of the category glucocorticosteroids, beta
blockers, stimulants, diuretics, barbiturates can be detected in urine.
FORENSIC IMPORTANCE OF URINE AS AN EVIDENCE

It can be found in the cases of Strangulation, violent cases, poisoning cases,
sexual assault etc. It can be used to analyze the presence of drugs, proteins,
alcohol and poisons.
The Collection and Preservation of Urine

samples
Urine Collect in a plastic Keep in a refrigerator.
bottle or other suitable Send to the PNP CL by
container. refrigerated transport.
Reference Collect 20 ml in two Keep the tubes in a
samples for drug/ test tubes with screw refrigerator.
alcohol analysis caps.
The Collection and Preservation of Body Fluids

samples
Mouth samples Take samples from the Put the swabs in the
oral mucous membrane swab wrapper or a
by rubbing two swabs folded piece of paper.
against the inside of

25
the mouth, teeth and Package in an envelope.

top and bottom of the Store dry and cool.
tongue.
Dry smears of Collect with swabs, Air dry and put the
blood, saliva (licks, moistened with sterile swabs in the swab
kisses, bites, spit etc.) water or tap water. wrapper or a folded
and semen piece of paper. Package
in an envelope. Store
dry and cool.
Vaginal samples Collect samples on Put the swabs in the
swabs, two swabs each swab wrapper or a
from at least three folded piece of paper.
different places, e.g. Package in an envelope.
introitus, cervix and Store dry and cool.
anterior fornix.
Anal sample Collect samples on Put the swabs in the
swabs from the anus swab wrapper or a
and rectum. folded piece of paper.
Package in an envelope.
Store dry and cool.
Penis sample Collect the sample on Put the swabs in the
two swabs moistened swab wrapper or a
with sterile water or tap folded piece of paper.
water. Package in an envelope.
Store dry and cool.
Finger rub Sampling Rub the cuticles and Put the swabs in the
of a suspected finger tops with one swab wrapper or a
perpetrator moistened swab for folded piece of paper.
each hand. Package in an envelope.
Store dry and cool.
Samples for drug/ 10 ml of venous blood Keep the tubes in a
alcohol analysis in a vacuum tube (with refrigerator.
a grey stopper) and 20
ml of urine in two test
tubes with screw caps.
DNA typing from Preferred alternative: Keep the tubes in a

Living persons (must venous blood in a refrigerator.
be taken by Dr/nurse) vacuum tube (with a
purple stopper).
DNA typing from Second alternative: A Air dry and put the
Living persons (must saliva sample is taken swabs in the swab
be taken by Doctor or with two swabs that wrapper or a folded
nurse) are rubbed against the piece of paper. Package

26
oral mucous in an envelope. Store

membrane. dry and cool.
Third alternative: about Place in a folded piece
10 hairs with pulled out of paper and insert in
roots. an envelope.
DNA typing from Blood in a tube. Keep the tubes in a
Dead persons (must refrigerator and send to
be taken by doctor or the PNP CL by
nurse) refrigerated transport.
1 cm3 muscle sample Place tissue samples in
or 10 hair roots. If plastic containers. To be
putrefaction has set in, frozen if they are not
take a 1 cm3 bone sent to the PNP CL the
marrow sample. same day.
Take Action (Analysis/Synthesis)
Protective Measures:
● Use protective gloves to minimize contamination risk.
● Change gloves after handling different materials.
● Utilize disposable equipment for preliminary tests and trace evidence
collection.
● Cover surfaces with protective paper and separate clothes of victims
and suspects.
●
Avoid Contamination:
● Refrain from coughing or sneezing on evidence/materials.
Packaging of Biological Materials:

● Prefer paper packaging for biological materials or those with biological
matter.
● Use separate outer packages for trace evidence and clothes.
● Avoid mixing materials/samples from different individuals.
● Seal bags and envelopes with tape.
Special Precautions:
● Note if material source has suspected infectious disease.
● Prevent contact between collected evidence and seized clothes.
● Delay opening packages until laboratory examination, except for drying
moist material.
Blood Analysis:
● Blood typing offers class evidence; DNA profiling provides more
individualized information.
● Blood-spatter analysis aids crime scene reconstruction.

27
● Serology examines body fluids and proteins.
Blood Composition:
● Blood comprises red blood cells, white blood cells, platelets, and
plasma.
● Each cell type serves a specific function in oxygen transport, immunity,
and clotting.
DNA Profiling History:

● DNA profiling first developed in 1982 using white blood cells.
● Widely accepted today for forensic purposes.
Blood Typing:
● Blood typing is quicker and less costly than DNA profiling.
● Useful for linking/excluding suspects but not proving guilt.
Rh Blood Types:
● Rh factor inherited separately from ABO blood types.
● Rh- gene masked by Rh+ genotype.
Blood-Spatter Analysis:
● Patterns help determine direction, angle of impact, point of origin.
● Categories include transfer, passive, and projected bloodstains.
● History dates back to the late 19th century.
Bloodstain Pattern Analysis:

● Interprets shapes, locations, and distribution of bloodstains.
● Useful for confirming or refuting events and statements.
Properties of Blood:
● Blood volume is about 8% of body weight.
● Surface tension affects bloodstain appearance.
Bloodstain Categories:
● Transfer bloodstains from contact with a surface.
● Passive bloodstains due to gravity.
● Projected bloodstains from external/internal forces.
Collection and Preservation of Blood:
● Different procedures for various types of blood samples.

● Proper packaging and storage to ensure integrity.
Semen:
● Semen is a complex mixture of fluids carrying spermatozoa for
fertilization. It contains solid components (sperm and skin cells) and

28
fluid components (seminal plasma). Semen can be detected up to

several days after deposition, with various tests available, including
Alternate Light Source (ALS) and Acid Phosphatase testing, both
targeting the fluid portion.
Saliva:
● Saliva is a biological secretion containing enzymes and proteins, with
alpha-amylase being a key component. Its detection supports
identification and genotyping of individuals. Detection methods include
ALS, radioimmunoassay, and lateral flow immunochromatographic strip
tests (RSID-Saliva). These tests are used to determine the presence of
saliva, especially for crime scene evidence.
Urine:
● Urine is a byproduct of cellular metabolism, rich in water and various
solutes. It can be crucial in cases involving poisoning, strangulation,
and sexual assault. Physical, visual, and chemical examinations, along
with microscopic analysis, help identify urine stains. Urea and
creatinine tests, thin-layer chromatography, and urine drug testing are
common methods for detecting urine.
The document also emphasizes the importance of proper collection and

preservation of these bodily fluids as evidence, detailing procedures for
different scenarios.
References:
Bertino, A. J. (2012). Forensic science: Fundamentals and investigations 2012

update. Cengage Learning.
Bloodstain pattern analysis tutorial bloodstain pattern analysis is. (n.d.).

SlideToDoc.com - one of the largest repository of presentations.
https://slidetodoc.com/bloodstain-pattern-analysis-tutorial-bloodstain-
pattern-analysis-is/
Canadian Children's Rights Council - Conseil canadien des droits des enfants.
(n.d.). Blood type CHART_CHILD father mother-paternity determination
by blood type - parents & child. Canadian Children's Rights Council –
Conseil canadien des droits des enfants, Child rights Canada, Paternity
testing, Paternity Fraud, Child rights and DNA testing.
https://canadiancrc.com/paternity_determination_blood_type.aspx
Intro to Forensic Science. (n.d.). courseresources.mit.usf.edu - /.

29
https://courseresources.mit.usf.edu/~test/lecture/files/pdf/cas_sample.
pdfb PHILIPPINE NATIONAL POLICE. (2011). FIELD MANUAL ON
INVESTIGATION OF CRIMES OF VIOLENCE AND OTHER CRIMES.
Philippine National Police. https://pnp.gov.ph/images/Manuals
_and_Guides/DIDM/Field-Manual-on-Investigation-of-Crimes-of-Violenc
e-and-other-Crimes.pdf
Urine as a biological evidence in forensic science. (2020, May 27). Forensics

Digest. https://forensicsdigest.com/urine-as-a-biological-evidence-in-
forensic-science/

30
Self-check (Assessment)
Name:______________________________Yr. Sec.________ Date:________

Subject: ______________________ Module no.___________Period: ______
Answer the following questions:
1. Discuss the general rules and precautions for the collection and
preservation of biological materials at a crime scene. Highlight the
importance of using protective gear, avoiding contamination, and
proper packaging methods. Provide examples of different biological
materials and how they should be collected, packaged, and stored.
Explain why it is crucial to maintain the integrity of biological evidence
throughout the entire process, from collection to laboratory
examination. Discuss how these procedures contribute to maintaining
the chain of custody and ensuring the reliability of forensic analysis.
2. Examine the significance of blood evidence in criminal investigations

and the various techniques used for its analysis. Discuss the
importance of blood typing and DNA profiling in identifying suspects
and victims. Describe the different types of bloodstain patterns and
their role in reconstructing crime scenes. Highlight the history and
evolution of bloodstain pattern analysis as a forensic tool. Provide
examples of how bloodstain patterns can reveal information about the
nature of a crime and the events that took place.

31
3. Explain the forensic significance of body fluids as evidence in criminal

investigations. Provide examples of cases where different body fluids
have played a crucial role in solving crimes.
4. Discuss the methods and techniques used for detecting and analyzing
body fluids in forensic investigations. Highlight the advantages and
limitations of these methods.

32

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Forensic 3: Forensic Chemistry and Toxicology

Subject: Forensic Chemistry and Faculty:

In this module, we discuss about the Identification, collection,

Take Off (Motivation)

COMPILED BY: JO ANN T. GERADA AND JEAN G. BULLO

Content Focus: (Content)

General Rules for the Collection and Preservation of Biological

a. Use protective gloves.

1. If possible, avoid touching individual smears/traces. Remember that

2. Change gloves after handling each kind of material and otherwise

3. Use disposable equipment for preliminary tests and collection of

4. Cover surfaces where materials are to be placed with protective

b. Avoid coughing or sneezing on evidence/materials.

c. Packaging of biological materials.

1. Use paper packaging for all biological materials or materials that

3. Do not mix materials/samples from different people, for example

COMPILED BY: JO ANN T. GERADA AND JEAN G. BULLO

Blood left at a crime scene can be analyzed in several ways by a criminal

Blood-spatter evidence can also be used to help recreate a crime scene to

Serology is the examination and analysis of body fluids. A forensic serologist

Blood is a circulating tissue consisting of three types of cells: red blood

COMPILED BY: JO ANN T. GERADA AND JEAN G. BULLO

HISTORY OF DNA PROFILING

Discovery of Blood Types

Paternity Determination by Blood Type Chart Calculator

COMPILED BY: JO ANN T. GERADA AND JEAN G. BULLO

ABO Blood Type Calculator

Genotype (DNA) Blood Type

COMPILED BY: JO ANN T. GERADA AND JEAN G. BULLO

Rh Positive (Rh+) and Negative (Rh-) Blood Types

The Rh (+/-) factor is inherited separately from

HISTORY OF BLOOD-SPATTER ANALYSIS

In 1894, Pitoroski wrote the earliest reference to blood-spatter analysis. In

Bloodstain Pattern Analysis:

Is the examination of the shapes, locations, and distribution patterns of

COMPILED BY: JO ANN T. GERADA AND JEAN G. BULLO

Based on the premise that all bloodstains and bloodstain patterns

The determinations made from bloodstain patterns at the scene or

💧 (blood smears, blood trails)

■ Blood Volume: On average, accounts for 8 % of total body weight

COMPILED BY: JO ANN T. GERADA AND JEAN G. BULLO

COMPILED BY: JO ANN T. GERADA AND JEAN G. BULLO

Projected bloodstains are ✔ Arterial Spurt /

The size, shape, and ✔ Cast-off Stains

COMPILED BY: JO ANN T. GERADA AND JEAN G. BULLO

This category can

COMPILED BY: JO ANN T. GERADA AND JEAN G. BULLO

Given blood-spatter patterns, it is possible to determine the direction the

Table of blood-spatter parameters.

When a droplet of blood strikes a surface perpendicular (90 degrees) the

Directionality is usually obvious as the pointed end of the bloodstain (tail)

IMPACT ANGLE DETERMINATION

COMPILED BY: JO ANN T. GERADA AND JEAN G. BULLO

By utilizing trigonometric functions it’s possible to determine the impact angle

SIN Φ = opp (a)

SIN Φ = Width (a) 1.5cm

POINT OF CONVERGENCE AND ORIGIN DETERMINATION

The common point, on a

THREE-DIMENSIONAL POINT OF ORIGIN

The Collection and Preservation of Blood