Forensic Serology

What is Serology?
Serology is a branch of science that deals with the determination of the type and
characteristics of blood, blood testing, and the examination of bloodstain, semen, saliva,
and other body fluids that may or may not be involved with DNA typing. The preparation
of testimony through the results obtained via serology and its presentation in a trial by
oral and written testimony delivered by an expert witness is the main job function of a
forensic serologist.

Importance of Serology in Crime Investigation

Blood can be used as circumstantial or corroborative evidence in identifying the
perpetrator of a crime. It can also be used to prove disputed parentage as in a paternity
test, determine the cause of death, or the length of time at which the victim survived an
attack. With the use of visualizing agents, such as luminol and hydrogen peroxide. Blood
stain patterns can also help determine the direction of the escape of the victim or the
assailant, the origin of
the flow of blood, and the approximate time at which a crime was committed. Picture of
whole blood and the separation of its serum.

That Is Blood?
Blood circulates throughout the body and is made up of four elements, namely,
the red blood cells or erythrocytes, the white blood cells or leucocytes, blood platelets,
and plasma. Human blood consists of 65% plasma, of which 90% is water and 10% are
proteins (albumen, globulin and fibrinogen). Blood clots found at the crime scene usually
exhibits a straw-yellow colored liquid called serum. The difference between serum and
plasma is that the latter contains no fibrinogen, which changes to an insoluble form
called fibrin. Blood examination in forensics can include blood typing and testing for the
presence of blood stain in serology, bloodstain pattern analysis, and testing for the
presence of deoxyribose nucleic acid (DNA) in paternity tests.

Blood and Blood Stains

Blood and blood stains are very vital evidence in crimes of violence against
persons. Blood refers to a highly complex mixture of cells, enzymes, proteins and
inorganic substances that can be characterized by way of their chemical reactions. The
presence of these substances makes for its ease to be subjected to laboratory testing for
identification

Components of Blood:
1. Red Blood Cells. These are also known as erythrocytes. They are responsible
for the transport of oxygen throughout the body as well as for collecting and
removal of carbon dioxide from the tissues, transporting it to the lungs for
eventual removal from the body through the lungs. The red blood cells contain
proteins which are specific antigens. Antigens are responsible for blood-type
characteristics and for adverse reactions during blood transfusions. Wrong blood
type will result to adverse antigen reaction and can result to death.
2. White Blood Cells. These are the soldiers of the body and responsible to fight
disease. They are also known as leucocytes. They contain antibodies that have
 been produced through the lifetime of the person and help the individual fight
diseases. They are designed to fight invading organisms to preserve the integrity
of the organism but in abnormal situations may attack itself in a condition known
as autoimmunity.
3. Plasma & Platelets. Plasma is the fluid portion of blood that is composed
principally of water. The platelets are the solid materials suspended in plasma.
This is the component of the blood that is responsible for blood clotting. Blood
clotting is a necessary action to protect an organism from Heading to death. The
protein in the blood called fibrin traps and enmeshes red blood cells and causes
blood clotting to occur.

Blood Types
There are different blood types for different individuals and blood typing can be
helpful information when investigating a crime. The different blood types and their
relative ratios in a given population are as follows:

Ratios of Blood Types .

 O+ 1 in 3 persons
 O- 1 in 15 persons
 A+ 1 in 3 persons
 A- 1 in 16 persons
 B+ 1 in 12 persons
 B- 1 in 67 persons
 AB+ 1 in 167 persons
 AB-1 in 29 persons

Blood Grouping Test

The blood of every human being belongs to one of four blood groups, which are
known as gro ups O, A, B and AB. This blood group cannot be altered by the lapse of time
or presence of a disease. Blood groups are hereditary, and science has proven that the
ABO system is one of the best ways in determining true parentage.

Application of ABO System

Parents (Mother Possible Child Impossible Child

and Father)
blood type
O&O O only A, B, & AB
O&A O&A B & AB
O&B O&B A & AB
A&A O&A B & AB
B&B O&B A & AB
A&B O, A, B, & AB None
O & AB A&B O & AB
A & AB A, B, & AB O
B & AB A, B, & AB O
AB & AB A, B, & AB O

Collection, Handling and Preservation of Blood & Bloodstained Evidence

 For samples that have to be sent to the laboratory, observe the following
procedure:
1. For protection of the investigator and to preclude the possibility of acting as an
agent of contamination, wear surgical gloves in handling blood and bloodstained
evidence.
2. For bloodstained materials, air dry. Do not use direct sunlight or heat.
3. Ensure that during the air drying, it is protected from contamination from
elements i.e. dirt, dust.
4. When dried, put the material inside a paper bag. (A paper bag is preferred
because plastic bags would cause moistening and ruin the evidence)
5. For dried blood on hard surfaces, scrape from the surface of the material using a
clean spatula. Gather the scrapings in a clean piece of paper, in the scrapings and
secure the evidence in an envelope provided for that matter.
6. Mark the bag with initials, the date and exhibit number before fastening it. Do
NOT bag the items if they are not thoroughly dried.
7. Do not fold across the stained area. If it cannot be avoided and you have to fold
the evidence, first cover the stained area with a clean piece of paper then fold in
the evidence.
8. Bloodstained materials should be packaged individually.
9. For liquid or fresh blood, use a clean, disposable medicine dropper to collect and
transfer samples from source to a glass vial containing the preservative or direct
to a glass slide. After the glass slide is smeared, cover with a glass slip and label
accordingly. Alternatively, fresh blood can be collected using sterile gauze to
absorb from the pool of blood at the crime scene.
10. Collect a comparison standard. The comparison standard is 5ml of blood each
taken from the victim and the suspect. Place these in separate vials. Label
accordingly. Add a preservative. The preservative of choice is Sodium Fluoride
(NaF).
11. In case extraction of blood has to be performed, only a qualified physician or a
medical technologist can perform this task. The vials are marked with the donor's
name, doctor's name, the date, exhibit number and other pertinent information.

Issues concerning the Forensics of Blood

1. The following questions must be answered relative to the examination of blood
samples:
 Is it blood?
 From what species did the blood originate?
 If the blood is of human origin, how closely can it be associated to a
particular individual?
2. The determination of blood is best made by means of a preliminary color test.

Screening Test

Name of Test Color Reaction Indicating

presence of Blood
Benzidine Test Intense Blue or Intense green
Phenolphthalein Test (Kastle-Meyer Deep permanganate color
Test)
Guaiacum test (Van Deen’s or Blue
Schonbem’s test)
Luecomalachite Green Test Bluish-Green

3. Wet blood has more value than dried blood because more tests can be run.
 For example, alcohol and drug content can be determined from wet blood
only.
 Blood begins to dry after 3-5 minutes of exposure to air. As it dries, it
changes color towards brown and black.
 4. Blood at the crime scene can be in the form of pools, drops, smears, or crusts.
 Pools of blood obviously have more evidentiary value in obtaining a wet
sample.
 Drops of blood tell the height and angle from which the blood fell. The
forensic science of blood spatter analysis says that blood which fell
perpendicular to the floor from a distance of 0-2 feet would make a circular
drop with slightly frayed edges.
 Drops from a higher distance would have more pronounced tendrils fraying
off the edges as in a sunburst pattern.
 A blood smear on the wall or floor tells the direction of force of the blow.
 The direction of force is always in the direction towards the tail, or smaller
end, of the smear, or spatter. In other words, the largest area of the smear
is the point of origin as in a wave cast-off pattern.
 Blood crusts need to be tested with crystalline methods to make sure it's
blood.
5. Refrigerated red blood cells have a shelf life of about 42 hours, and the serum
containing white blood cells can be refrigerated much longer, almost up to a year.
DNA can be extracted from blood (if white blood cells which always contain a
nucleus are present), and also from sperm, bone marrow, tooth pulp, and hair
roots. Blood, however, is commonly used in DNA testing.

Tests for the Presence of Blood

1. Spot Test
a. Benzidine Test - presumptive test for the presence of blood.
b. Kastle-Meyer Test - drop of phenolphthalein and hydrogen peroxide will
be observed as pink coloration due to the presence of peroxidase activity
of the blood hemoglobin. A positive result from the Kastle- Meyer color test
is highly indicative of blood.
c. Luminol Test - Luminol is a spray reagent used to test for the presence of
blood even if the blood is not visible under ordinary light. This is viewed
under an ultraviolet source.
2. Microcrystalline Test. There are characteristic reactions that result in the
formation of crystals. The formation of crystals is a result of the reaction of the
protein components of the blood with that of the chemical reagent and these are
specific and characteristic for human blood. Crystals formed are examined under
a microscope.
a. Takayama Test - utilizes Takayama Reagent
The Takayama test is one of the micro-crystal confirmatory tests
for the blood that is frequently used in forensic laboratories. It is
also called the Hemochromogen test, and a positive result is
indicated by the pink feathery crystals of pyridine-
hemochromogen. It was first developed in 1912 by Masaeo
Takayama, a Japanese forensic pathologist. Later, Takayama’s
name has become the synonym of hemochromogen crystals.
Reagent for Takayama Crystal Test and Preparation
This is how you prepare the reagent for the test:
1. Take a test tube and pour 5 ml of saturated glucose solution.
2. Add 5 ml of 10% Sodium Hydroxide solution.
3. To this mix, add 5 ml of pyridine solution.
4. Mix well.
5. Now, add 10 ml of distilled water to the beaker and mix well.
The Preparation
 Following is the step-by-step procedure for performing this test in
laboratory.
1. Add 2-3 drops of prepared Takayama reagent.
2. Cover the slide with a coverslip.
3. Observe under a standard microscope.

(https://mcq.forensicreader.com/takayama-test/)

b. Teichmann Test - utilizes Teichmann Reagent

A confirmatory test for blood-based on the formation of distinctive
haematin crystals that are viewed under a microscope. The reagents
typically used are sodium chloride and glacial acetic acid. Dr. Ludwig Karl
Teichman discovered a test for the detection of blood in at the test name
as Teichmann Crystal Test.

Basis of the Test

In Teichmann Test hemoglobin converted to haemin crystals which
converted to salt in presence of halogen after then rhombic crystals forms.

Indication for the Presence of Blood

It produced Dark brown Color and rhombic shape if blood is present.
 3. Precipitin Test - A precipitin test is a test used to measure a specific reaction
between antigen and antibody resulting in a visible precipitate. This test is used in
criminology for determining the human or other source of a blood stain. It can be
used to determine whether the blood is of an animal or a human being. Antigen
and antibody in their respective wells move toward each other until a visible
precipitin line in the gel between the wells is formed is a confirmatory test for
human blood.
4. Bloodstain Patterns - Bloodstains and bloodstain patterns are useful for
interpreting and reconstruction of events that occurred during the bleeding
process. Characteristics of the blood pattern will help reconstruct the events and
help shed light to a crime.

Bloodstain Pattern Analysis

Bloodstain pattern analysis (BSPA) refers to the examination of the shapes and
the distribution patterns and locations of bloodstains which can provide an interpretation
of the physical events that gave rise to their origin. Here, the distribution and
appearance of bloodstain and spatters are used to interpret and reconstruct the events
that have occurred to produce the bleeding.

What questions can blood spatter answer?

1. Where did the blood come from?
2. What caused the wounds?
3. From what direction was the victim wounded?
4. How were the victim(s) and perpetrator(s) positioned?
5. What movements were made after bloodshed?
6. How many potential perpetrators were present?
7. Does bloodstain evidence support or refute witness statements?

Characteristics of Blood Spatter

a. Direction of travel: The direction of travel can be determined by the

bloodstain's shape - the pointed end always faces its direction of travel.
b. Surface texture: The harder and less porous the surface, the fewer blood
spatter produced.
c. Origin of blood spatter: Origin in a 2D configuration can be determined by
establishing lines through the long axis of individual bloodstains; the intersection
of the lines represents the point from which the blood originated.
d. Impact angle: A drop of blood striking a surface at a right angle produces a
nearly circular stain - as the angle decreases, the stain becomes more elongated.

Uses of BSPA

a. Show assumptions concerning events and their sequence –Here, the position of
the victim (which may indicate blood smears or blood trails) may show evidence
of the struggle between the suspect and the victim.
b. Confirm or refute statements made by principals in the case. Here, stain patterns
on a suspects/victim's clothing can help verify consistency with the accounts
given by either the witnesses or the suspect.

Characteristics of a Blood Spatter

  Low velocity (5 f/s, 1.5 m/s) e.g., free-falling drops, cast off from weapon
 Medium velocity (25-100 f/s, 7.5-30 m/s) e.g., baseball bat blows
 High velocity (100 f/s, 30 m/s) e.g., gunshot, machinery

Three Categories of Bloodstain

1. Passive Bloodstains - these are drops created or formed by the force of gravity
acting alone.
 Drip-caused by dripping blood
 Drop - created by force of gravity
 Pool - this refers to a change in the shape and direction of a bloodstain due to the
influence of gravity or movement of the object.

Target surface texture

 Bloodstains can be found on various surfaces, such as clothing, carpet, tile,
wood, wallpaper, and so on.
 The type of surface upon which blood strikes can affect the amount of
resulting spatter, including the appearance and size of the blood drops.

Spreads out smoothly

 Blood droplets that strike a hard smooth surface, such as a piece of glass, can
have little or no distortion around the edge.
 Surface texture of the spreading edge is broken by irregular surface.
 Surfaces, such as wood or concrete, are distorted; spines and secondary spatter
may be present.
 Blood droplets that strike linoleum flooring take on a slightly different
appearance; distortion (scalloping) around the edge of the blood droplets may be
found.
2. Transfer
 A transfer bloodstain is created when a wet and bloody surface comes in contact
with a secondary surface.
 A recognizable image of all or part of the original surface may be observed in the
pattern, as in the case of a bloody hand or footwear.

3. Projected/Impact – result from blood projecting to air

 Arterial Spurt/Gush – Bloodstain patterns resulting from blood exiting the body
under pressure from a breach artery.
 Cast-off-Stains – Blood released or thrown from a blood-bearing object in
motion.
 Impact Spatter – Bloodstain patterns created when a blood source receives a
blow or force resulting in the random dispersion of smaller drops of blood. Velocity
affects stain pattern

Semen and Seminal Stain

The examination of semen and seminal stains is an important part in the routine
investigation of sexual offenses like cases of rape, adultery, sodomy, bestiality, and
sexual homicide. Semen and seminal stains contain cells, proteins, and inorganic
substances that make for the ease with which they can be subjected to tests when
obtained relative to a crime.
 Semen is the fluid within the male reproductive tract that may or may not contain
spermatozoa. A condition known as aspermia is when a male has no spermatozoa in his
seminal fluid. Another condition that may be encountered is oligospermia. This is one
whereby males have abnormally low sperm counts or with few spermatozoa.

Vocabulary:
 Aspermia – Aspermia is the complete lack of semen with ejaculation, which is
associated with infertility.
 Azoospermia – is the medical condition of a man whose semen contains
no sperm.
 Oligospermia - males have abnormally low sperm counts or with few
spermatozoa. A male’s sperm count is considered lower than normal if they
have fewer than 15 million sperm per milliliter of semen.

What is a Semen?

Semen refers to the body fluid produced by the male sex organ, per ejaculation.
Each ml contains 100 million or more spermatozoa. The fresh ejaculate semen has a
gelatinous, sticky consistency, and tends to become more fluid when exposed. It has two
parts-the seminal fluid (consisting of cellular elements including spermatozoa) and
epithelial cells and crystals composed of choline and lecithin.

What are Spermatozoa?

Spermatozoa (sperm) are the male sex cells that carry a man's genetic material.
They are so tiny that they can't be seen without a microscope. In a healthy man, one
ejaculation usually contains between 40 million and 600 million sperm. Sperm have an
oval head, a short middle, and a long tail. They move by whipping their tails. A sperm
fertilizes a woman's egg (ovum) by breaking through the membrane that surrounds the
egg. Sperm develop in a man's testicles. They are added to semen before a man
ejaculates. (https://myhealth.alberta.ca/Health/Pages/conditions.aspx?hwid=tp10397)
Each sperm cell carries a copy of the male's genetic information in the form of DNA
(deoxyribonucleic acid).

Forensic Characterization of Semen

A large number of criminal cases handled by a forensic laboratory often involve

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

The forensic examination of semen follows a two-step process:

a. Location of the semen; and
b. Conduct of tests to prove the identity of the one who produced it.

Collection, Handling, and Preservation of Semen and Semen-Stained Evidence

1. When clothes or fabric are washed, traces of semen are lost. That is why it is
imperative that the suspect's apparel is seized. The earlier that these are
recovered, the greater are the chances of testing for the presence of semen and
seminal stains.
2. Air dry the material on clean paper.
3. Put the dried material inside a paper bag. Do not bag material if not thoroughly
dried. Ensure that there is no friction against the stain.
4. Do not fold or roll over the stain. If the material has to be folded, cover the
stained area with a clean piece of paper and fold in the material.
5. Materials that have semen stains should be treated individually. Separate packing
should be observed.
6. Fluid semen should be placed in a test tube. It may be preserved using a few
drops of 10% formalin during hot weather to avoid putrefaction.
7. Collect a comparison standard from both the suspect and victim.

Collection of Rape Case Evidence

In collecting semen evidence for cases involving rape, the following precautions
must be considered:

1. A rape victim must be subjected to a medical examination. During such

examination, possible forms of evidence that may be collected include the
following: pubic hairs, blood, vaginal swabs, any piece of clothing, urine, and
fingernail scrapings.
2. Outer/undergarments must be placed in separate paper bags with the proper
evidence tag; the victim must be made to stand on a piece of paper as
possible forms of trace evidence are collected.
 3. A suspect must also undergo a medical examination, during which clothing,
hair, penile swab, and blood samples are collected for comparison.
4. Objects found at the scene of the crime should be submitted to the laboratory
with proper documentation.

Additionally, non-motile sperm can still be collected from a living female for up to
three to six days.

Non-Motile Sperm - Non-motile sperm are sperm cells that are unable to move. Non-
motile sperm cannot rhythmically swing the tail to move in a forward direction
due to a structural or functional defect of the sperm.

Forensic Examination of Semen and Seminal Stain

1. Wet Specimen – Here, a drop of the fluid is placed on a glass slide to

which a few drops of distilled water are added. This is then examined
under a high-powered microscope to determine the presence of sperm
cells or spermatozoa. A spermatozoon has a head and a thin flagellate tail.

2. Dry Specimen
a. Physical examination – This includes visual examination; dry
semen has a stiff starchy feeling when deposited on a piece of
clothing. It may have a slight deepening of color (grayish white
sometimes yellowish) and disappearance of odor. It also exhibits
bright bluish fluorescence under UV light.
b. Chemical examination
 Florence test - This test was discovered by Dr.Florence in
the year 1886. When Florence reagent
(PotassiumIodide+Iodine+Water) is applied to the slide it
produces rhomboidal shape dark crystals of choline
periodide. Similarly, any tissue or biological material
containing sufficient high choline concentration would give
positive Florence Test. According to Davis and Wilson, if
swab is collected within one day of sexual act, choline can
be detected. At the same time if swab is taken after 14
hours of intercourse, there is a possibility of negative result.
(http://epgp.inflibnet.ac.in/epgpdata/uploads/epgp_content/
S000016FS/P000699/M011534/ET/
1516257369FSC_P12_M8_e-text.pdf)

Basis – Choline is detected in this method

Choline - is a water-soluble nutrient found in various bodily

fluids, including semen. It is a component of many biological
substances, including cell membranes and certain enzymes.
Choline's presence in semen is a natural part of its
composition.

Procedure:
 A few drops of a watery solution of the stain are
extracted and taken on a slide and a drop of Florence
reagent (8%) W/V solution of Iodine in water
containing 5% W/V of Potassium Iodide) is poured &
allowed to mix slowly under a cover slip.
 Dark brown crystals of choline periodide, generally
needle-shaped, formed within a few minutes.

 Barberio's test- using picric acid as a crystallizing agent,

this results in the formation of spermine picrate crystals,
 which appear as slender, yellow-tainted rhomboid needles
with obtuse angles that may sometimes appear as ovoid
crystals.
Barberio’s test was invented by Barberio in the year
1905. When the questioned stain is allowed to react with
picric acid it leads to the formation of yellow needle-shaped
spermine picrate crystals, including the presence of seminal
stain.
(http://epgp.inflibnet.ac.in/epgpdata/uploads/epgp_content/
S000016FS/P000699/M011534/ET/
1516257369FSC_P12_M8_e-text.pdf)

Basis – Detection of spermine

Spermine - is a polyamine involved in cellular metabolism

that is found in all eukaryotic cells. Spermine is the chemical
primarily responsible for the characteristic odor of semen.

 Acid phosphatase (Dr. Sidney Kaye) (Presumptive) -

This test identifies the presence of acid phosphatase, which
produces an orange-red pigment
The AP test detects the enzyme acid phosphatase
that is secreted from the prostate gland. However, this test
is only presumptive because acid phosphatase is found in
other bodily fluids. To perform the test, a drop of the
reagent sodium alpha-napthyphosphate is added to the
presumptive stain followed by a drop of fast blue B reagent.

Positive Results:
 If acid phosphatase is present, a purple color will
appear within a short period of time (30 seconds).
Weak stains may take between 1 to 3 minutes.
 A positive reaction indicates the presumptive
presence of seminal fluid.

(https://en.wikipedia.org/wiki/Forensic_serology)

Note that the Florence and Barberio tests are based on the
formation of characteristic crystals that are observed under a
microscope, while the acid phosphatase indicates the presence of
acid phosphate enzyme, which indicates human origin.

3. Microscopic examination- this test identifies sperm cells or

spermatozoa, which indicate that the semen is of human origin:
spermatozoa die as the semen dries. In this test, the rapid stirring of a
fabric-stain-water mixture will transfer a small amount of sperm into the
water. A drop is then taken, dried, stained, and visualized under a high-
powered microscope.
 Stained Sperm under the Microscope

Microscopic Problems

Some problems may arise in the examination of sperm using a microscope due to
several factors, which are listed below.

 Some sexual crimes may involve males who have an abnormally low sperm
count-this condition is called oligospermia.
 Sperm are very brittle when dried and tend to easily disintegrate. They are
also difficult to remove from cloth material.
 Some males may suffer from aspermia, a condition where there is no sperm in
their seminal fluid, which is due to a vasectomy.
 DNA typing is a method employed by forensic scientists to identify individuals
by their respective DNA profiles. DNA profiles are encrypted sets of numbers
reflecting a person's DNA makeup. These can also be used as the person's
identifier. It can be used in, for parental testing and criminal investigation.
 Biogical examination (Farnum 1901) - This test is used to differentiate human
from animal seminal fluid. It is also used to detect spermato precipitins.

The Prostate-specific antigen (Confirmatory)

Prostate-specific antigen (PSA) is a glycoprotein produced in the prostate gland in

males and secreted into the seminal fluid. The prostate gland is a male reproductive
organ responsible for producing seminal fluid, which is a component of semen (Fluids and
Cells, enzymes, or proteins). Its main function is to liquefy the seminal fluid, helping
sperm swim more freely. This high amount makes PSA a useful marker in forensic
science for the detection of even small amounts of seminal fluid.

The advantages of a PSA determination are:

 Prostate-specific antigen (PSA) or p30 is an accepted marker for detecting semen

in criminal cases.
 The PSA test is not presumptive like the Acid Phosphatase (AP) Semen Detection
Test.
 The detection of PSA is possible in cases where no spermatozoa can be found (for
example vasectomized or azoospermic men).
 PSA can be recovered at detectable concentrations in 30-year-old semen.
 Semen samples can show positive PSA results even at a high dilution (dissolve in
a liquid) (Dilution factor of 1:200,000).
 PSA is detectable in post-ejaculate urine and male urine from adult men and can
be detected in the urine of eleven-year-old boys.
LDH ISOENZYME METHOD – detect spermatozoa. Seminal stains are extracted with 1
ml of water. 0.25 ml of clear extract is mixed with (0.25 ml of 40% W/V) of sucrose. 0.1
ml of this mixture is subjected to vertical polyacrylamide gel electrophoresis.
Electrophoresis is carried out in refrigerators for 150 minutes using a current of 5 ml
Isoenzyme bands are revealed by staining.

This method gives a specific biochemical detection of spermatozoa in semen in

the presence of Vaginal Fluid, Blood, Nasal Secretion, Saliva & Urine.
(http://www.forensicindia.com/pgteaching/semen&seminalfluid.htm).

Hair and Fibers

What is Hair?

Hair refers to slender, threadlike outgrowths of the epidermis, which forms a

distinct type of body covering in mammals. Only mammals have true hair; in fact, even
apparently hairless mammals, such as the rhinoceros, elephant, and armadillo, have
hairs around the snout, at the tip of the tail, and behind each scale, respectively. When
the individual hairs are fine and closely spaced, the coat of hair is called fur, when they
are soft and matted together, they are called wool. Meanwhile, coarse, stiff hairs are
called bristles, and when these are pointed, they are called quills as in the porcupine and
hedgehog.

In the field of forensic science, hair is one of the most common types of trace
evidence found at any crime scene. Next to teeth and bones, hair is decay-resistant.
Although it cannot fully provide conclusive evidence, when used with other details, it has
been proven to have an essential role in criminal investigation. This is because hair
transfer may occur during physical contact between the victim and the suspect. Hair may
fall out under conditions that the suspect is not aware of and unable to guard against it.
When properly identified, hair can be used to identify either the suspect or the victim.

Biology of Hair

 It is composed of keratin (protein), which is also the primary component of fingers

and toenails. In humans, keratin is primarily known for its role in forming the
structure of hair, nails, and the outer layer of the epidermis (the skin's outermost
layer). It is resistant to water, heat, and other environmental factors, making it a
crucial part of our body's defense mechanisms against physical damage and
pathogens.
 It is produced from the hair follicle (pores), which is developed in humans during
fetal development; no new follicles are produced after birth. Follicles are
structures within the skin that are responsible for producing hair.
 Hair color is mostly the result of pigments or chemical compounds reflecting
certain wavelengths of visible light. Hair gets its color from a pigment called
melanin, which is produced by specialized cells called melanocytes located at the
base of hair follicles.
o There are two main types of melanin that contribute to hair color
 Eumelanin: This type of melanin is responsible for black and brown
hair colors. It comes in two subtypes: black eumelanin and brown
eumelanin. The ratio of these two subtypes and their distribution
within the hair shaft determine the shade of brown or black.
 Pheomelanin: This type of melanin is responsible for red and
yellow hair colors. Red hair contains primarily pheomelanin with
 very little eumelanin, while yellow hair typically has a combination
of both.
 Hair shape (round, oval, or kinky) and texture (curly or straight) are heavily
influenced by genes. The physical appearance of hair can also be influenced by
nutritional status and intentional alteration (heat curling, straightening, perms,
coloring, etc.).

The body area (head, arm, leg, back, and so on) where a hair strand originated can
be determined based on the color, length, size, shape, and other physical characteristics
of a sample. In testing hair evidence for DNA, the root must be present.

Hair Structure

Three Principal Parts of the Hair

1. Root Bulb – The shape of the

root bulb determines whether or
not the hair was pulled by force.
2. Hair shaft – contains the most
information about the hair.
3. Tip – would show if the hair was
cut, burned, or it has/had split
ends.

Parts of the Hair Shaft

1. Medulla – the central canal of the hair shaft; may be continuous, fragmented,
interrupted, or absent.
2. Cuticle – the outer surface of the hair when viewed under a microscope, it
appears to be composed of scale-like flakes, each overlapping others much like
the scale of a fish.
3. Cortex – contains the hair’s color pigment.
The Medullary Index

Forensic science investigators have determined the medullary index of hair, which
is the diameter of the medulla relative to the diameter of the hair, and is expressed as
the following fraction:

Diameter of medulla

Medullary Index = --------------------------------

Diameter of hair

Human hair has a medullary index of less than 1/3, while that of animals is usually ½ or
more.

Determination of Human Hairs

The body area from which a hair sample originated can be determined through
general morphology. The size, length, shape, stiffness, color, curliness, and microscopic
appearance all contribute to the determination of body area. Pigmentation and medullar
appearance also influence body area identification. Hair strands that exhibit microscopic
characteristics shared by different anatomical areas are called body hairs, which include
hairs found on the lower abdomen, upper legs, and back. There is also a wide range of
variations in head and pubic hairs; thus the majority of work in forensics has focused on
comparing and differentiating hairs from head and pubic regions.

Sources of Human Hair

1. Head Hairs – Head hairs are often the longest hairs on the human body. They
have a uniform diameter and a cut tip. These are subject to more alteration than
 hairs from other body areas through the use of hair dyes, rinses, permanents,
frosts, and other chemical applications. Environmental alterations can also be
determined, which result from wind dryness, excessive exposure to sunlight, and
other conditions. Given that these hairs are affected by chemical and
environmental conditions, it is recommended that head hair samples must be
obtained from the suspect and victims as soon as possible. Head hair samples
obtained years after a crime are generally not suitable for meaningful comparison
purposes. It is also recommended that investigators collect at least 25 to 100 full-
length hair strands from different areas of the scalp with the use of one's fingers
and or tweezers.
2. Pubic Hairs – Pubic hairs are also routinely compared in a forensic laboratory
examination. These are not subject to much change over time as head hairs, and
as such, a sample taken a year after a crime may still be suitable for meaningful
comparison. It is recommended that a known pubic hair sample be obtained as
soon as possible after a crime and should contain at least 25 full-length hair
strands taken from different areas of the pubic region. In terms of appearance,
pubic hairs are generally coarse (rough) and wiry. They exhibit diameter
variations (also called "buckling") and often have a continuous to discontinuous
medulla. These hairs may have tapered, abraded, or cut tips.
3. Facial Hairs – Facial hairs are more commonly called mustache hairs or beard
hairs. These have a wide medulla and a razor-cut tip, and have a coarse
appearance, and can have a triangular cross-section. Heavy shouldering or
troughs in the hair can be observed under magnification. In an investigation, the
presence of facial hairs on the clothing of a suspect or victim may help establish
contact between these individuals. Although these hairs can be compared
microscopically, the significance of the association may not be as great as those
obtained from head or pubic hair samples.
4. Limb Hairs – Hairs from the legs and arms are called limb hairs. These are
shorter in length, have an arc-like shape, and are often abraded or tapered at the
tips. The medulla in limb hair is traced to discontinuous, and then the pigment is
generally granular. Although limb hairs are not routinely compared in a forensic
laboratory, they can differ in appearance between individuals. However, these
differences are not considered sufficient for meaningful comparison purposes.
Nevertheless, the presence of leg or arm hairs on items of evidence may help
corroborate other investigative information.
5. Fringe Hairs – These originate from the neck, sideburns, upper leg, abdomen,
and back. They are deemed not suitable for significant comparison purposes and
have little evidentiary significance.

Racial Determination

A human can be associated with a particular racial group on the basis of

established models for each group. Forensic examiners differentiate between the hairs of
people with Caucasoid (European ancestry). Mongoloid (Asian ancestry) or Negroid
(African ancestry) origin, all of which exhibit microscopic characteristics that are distinct
from the others. However, racial determination from the microscopic examination of
head hairs from infants can be difficult, and hairs from individuals of mixed racial
ancestry may possess microscopic characteristics attributed to more than one racial
group. Nevertheless, the identification of race is most useful as an investigative tool, and
can also be an associative tool when an individual's hair exhibits unusual racial
characteristics.

1. Caucasoid (European) – Hairs of this origin have a fine to medium coarseness.

These are generally straight or wavy in appearance and possess colors ranging
 from blonde to brown-black. The hair shafts of Caucasian hairs vary from round to
oval and have fine to medium-sized pigment granules that are evenly distributed.
2. Mongoloid (Asian) – Hairs of this origin are straight, regularly coarse, and
circular, with a wider diameter than the other racial groups. The cuticle is usually
significantly thicker than that of the Negroid and Caucasian hairs, and the medulla
is continuous and wide. The hair cortex of this type of hair contains pigment
granules that are generally larger in size than those of Caucasian hairs, often
appearing to be grouped in patchy areas within the shaft. Mongoloid hair can
have a characteristic reddish appearance due to its pigment.
3. Negroid (African) – Hairs of this origin are curly or kinky, have a flattened
cross-section, and can appear wavy, curly, or coiled. Negroid pigment granules
are larger than those found in Mongoloid and Caucasian hairs and are grouped in
clumps with varying sizes and shapes.

Laboratory Analysis of Hair

Purposes of Examination

The examination of hair is conducted for the following purposes:

1. to determine whether the hair in question originated from an animal or

human being and the comparison of questioned and known hairs; and
2. to ascertain whether two or more individuals could have come into contact
or whether one or more individuals could have come into contact with an
object.

The examination of this associative evidence is useful in crimes of violence, such

as sexual assault, homicide and aggravated assault, where physical contact is likely to
have occurred. Other crimes, such as burglary and armed robbery, typically involve the
recovery of debris and articles of clothing that may contain hairs that, in turn, can be
used to identify the suspects.

Hair Microscopy

The examination of human hairs in the forensic laboratory is typically conducted

using a comparison microscope. It is not considered a conclusive test because it is
difficult to establish a statistical probability for a particular association due to the lack of
reliable quantitative assessments of the microscopic characteristics present in hair.
However, this can still help eliminate a suspect from the crime scene. The comparison
microscope has two compound light microscopes connected by an optical bridge, which
allows for the simultaneous viewing of questioned hairs and known hairs. Here, a glass
microscope slide containing a questioned hair or hairs is positioned on the stage of one
microscope, and a glass microscope slide containing known or reference hairs is
positioned on the stage of the other microscope. This allows the hair examiner to
compare the microscopic characteristics of known and questioned hairs in one field. The
range of magnification used is approximately 40x to 400x.

Methods of Microscopic Analysis of Hairs

1. The parallel mounting technique refers to the method of affixing the samples
to the slide before the coverslip is placed. Here, the mounting medium should not
dissolve the adhesive in the wet mounting. Typical adhesives used today include
gum tragacanth, gum Arabic, vinyl acetate glue, and cyanoacrylate (i.e., "crazy
glue"). Tacking points are at the border of the end of the cover slip.
 a. The dry mount is convenient in terms of preparation. However, the
degree of curl and twist cannot be observed through this method owing to
the constraints in the mounting process. Prior to wet mounting, it may be
useful to study the exterior texture and the overall color of the hair.
Usually, several hairs are placed in parallel on a slide, so that their color
and texture can be compared easily. Here, samples can be fixed with
melted Kronig cement. Usually, several hairs are placed in parallel on
slide, so that their texture and color can be compared easily. The dry
mounting technique is nothing fancier than the words it describes.
Samples can be fixed with melted Kronig cement, but it is unnecessarily if
the cover slip can be fixed firmly on the slide
b. The wet mount is essential in hair analysis because of the refractive
index of the Canada Balsam, a special resin used to prepare slides, which
is close to that of the keratin in the hair. Wet mount can show the interior
structure of the hair, such as inclusion and pigment granules. The
refractive index of the mounting medium plays a significant role in viewing
internal details, and the reagent to be used must be chosen carefully.

The wet mount is essential in hair analysis because of the refractive

index of Canada Balsam – a special resin used to prepare slides – is close
to that of the keratin in the hair. If the dry mount provides a view of
texture and color, the wet mount provides the interior structure of the hair,
such as inclusion and pigment granules. Because the refractive index of
the mounting medium plays the most significant role in viewing internal
details, it is suggested that if other reagents than Canada Balsam is used,
it is chosen carefully.

Prior to performing a microscopic examination, take note of any foreign materials

adhering to the hair sample. Perform the following tests:

 Note the color of the hair.

 Take the actual measurement of the hair sample.
 Note its characteristics: Is it soft, Stiff, or Wiry?
 Using a microcaliper, measure its diameter.
 Note the overall condition of the hair: is it burned, is the tip present, or is the root
is intact.

Fibers & Threads

In such incidences as homicide, assault and sexual offenses involving personal

contact and struggle, fibers and threads can be inadvertently transferred between the
clothing of a suspect and victim. Fiber transfers can also occur between people and their
environment (e.g. carpeting, upholstery or bedding). A force of impact such as in the
case of hit-and-run, the contact between the victim and the vehicle may result to fabric
impressions, fibers, threads, or pieces of clothing left on the vehicle. An intruder entering
a broken window may leave fibers on the jagged glass or screen edges. Ropes and
cordage from a crime scene can be compared with known samples collected from a
suspect.

Types of Fibers

1. Natural fibers - Those occurring in nature

a. Cellulose - Those, such as cotton, linen, and jute, that come from plants.
b. Protein - Those, such as wool or silk, that come from animals.
2. Synthetic fibers - Man-made.
a. Nylon - made from petroleum.
b. Polyester - made from coal and petroleum product.
Important Reminders

1. Per evidence are generally small in nature and thus must be protected from
potential loss.
2. Due to their microscopic nature, several methods must be resorted to in order to
ensure thorough collection such as the use of magnification tools and efficient
alternate light sources.
3. Use recovery techniques that are least intrusive but practical such as picking
scraping or vacuuming.
4. Wrap pieces of evidence to protect any adhering files from being lost.
5. Collect comparison standards from possible sources of fiber transfer.
6. All items must be sealed and labeled for identification.

Fiber Identification Methods

One method of fiber identification is by comparison. Comparison of fibers will

reveal if they are from the same source or not. The weave pattern of fabrics can also be
used for comparison. If a torn fabric is obtained, that piece can be matched up with a
weave pattern and irregularities from a tear edge from another fabric. It is also possible
to match cordage (ropes strings) tears and cuts if the ends are not too distorted. Fabric
impressions may be found in a number of situations. The impression of a victim's
garment may remain on the flat surface of a vehicle in an accident investigation.

Other than comparison there are also different tests which could be used for the
identification of textile fibers such as:

1. Physical Tests
a. Staining Test – Fibers will react differently when stained using
chemical reagents. The table below summarizes the behavior of
different types of fiber during the staining process.

Material Reagent Reaction

Silk Picric Acid Dyed
Wool Picric Acid Dyed
Silk Millon’s Reagent Brown
Cellulose Fibers Picric Acid Undyed
Stannic Chloride Black
Millon’s Reagent Undyed

b. Density – This refers to the relative distribution of molecules

throughout the fiber. Some fibers are denser compared to others and
this can be measured as weight per unit area.
c. Mechanical and Tensile Property – Fibers may be subjected to
mechanical tests that will measure their ability to withstand stress,
elongate, and eventually break. Different types of fiber exhibit differing
 mechanical and tensile properties and may be used as a standard for
identification.
d. Microscopic Examination – Microscopic examination will reveal the
type of fiber since textile fibers have different characteristics when
seen under a microscope. This is the most reliable and best means for
identifying fiber. Below is a sample table summarizing some textile
fibers and their characteristics:

Type of Appearance
Fiber
Wool With overlapping scales throughout the cuticle.
Mohair Extremely similar in appearance to that of wool but
bigger diameter and higher density; lustrous (shiny).
Cotton Flat, ribbon-like, twisted spirally on its axis, thick cell
wall, covered by a thin waxy cuticle, fiber tapers
gradually to a blunt or rounded point at one end.
Silk Smooth, cylindrical, lustrous, usually single but often
double very fine longitudinal striations with infrequent
cross-markings; more or less transparent.
Linen Straight and cylindrical, not twisted and flattened,
tapering to a sharp point, cell wall, thick, lumen
appearing as a narrow dark line in the center of the
fiber, fiber appears jointed, resembling bamboo, cross
https://www.pinterest.ph/pin/52635889370728168/ lines frequently appearing like the letter x.

https://patasicashmere.com/is-cashmere-itchy/
 22. Hoarse

23. Mouse

24. Cat

25. Chinchilla

26. Large hair

from seal

27. Female head

hair

28. Male head

hear after
treatment with
caustic soda

29. Hair of the

hand

30. Head of the

child

31. Cross section

of hair from head

32. Silk

33. Cotton

34. Flax

35. Wool

https://

www.pinterest.ph/pin/387239267939381566/

2. Chemical Test

a. Burning or Ignition Test. Fibers may be identified as to their origin

whether they are animal, vegetable, or mineral by the characteristic
 smell emitted during burning. Also, the quality of the burning and the
appearance of the burnt end, the color of the ash, and the reaction to
litmus paper of the ash solution will reveal the type of fiber.
b. Solubility Test. This test is conducted by treating the fiber with
certain chemicals. Fibers behave differently in the presence of certain
chemicals. Particular fibers may be dissolved while others will not
dissolve. This can be used as a basis to identify types of fibers.

3. Analytical Test

IR InfraRed Spectroscopy

NMRS Nuclear Magnetic Resonance Spectroscopy

X-ray X-ray Diffraction

UV-Vis UltraViolet Spectroscopy

OEM Optical Electron Microscope

Glass

Glass is normally a fused mixture of silica usually in the form of natural sand and
two or more alkaline as soda, lime, or potash. It also contains bases such as quantities of
various other elements and metals present either as incidental impurities in the basic
constituents or added them for color, degrees of hardness, heat resistance, and other
specific purposes.

Glass is often encountered as evidence in burglaries, homicides, assaults, and hit-

and-run offenses. Glass fragments easily embed in shoes and clothing of people involved
in the breakage of glass, and its recovery can help investigators piece together a crime.
One can tell whether pieces of glass recovered from the crime scene and from the person
of the suspect belong to the same class or not.

Direction of force can be determined from the pattern of glass breakage. The
determination of the direction of force is helpful information whether glass was broken
from inside or from the outside. When information regarding the direction of force is
needed, all broken glass must be recovered and submitted for analysis. Leave the
remaining glass in the window or doorframe intact and mark as to exterior and interior
surfaces.

Properties of Glass

1. Glass is somewhat flexible; it tends to bend upon the application of force.

2. Glass bends in the direction where force is applied causing the opposite side to
stretch.
3. Glass can withstand more bending rather than stretching causing the opposite
side to start breaking

The significance of knowing the characteristics of glass, either as evidence or in

the development of investigative leads, lies mainly in its physical properties. These
properties make it possible to determine that glass fragments did or did not originate
from the same source, or to determine the manner in which a piece of glass was broken.

Glass Fractures

Glass fractures may be caused by excessive exposure to heat or caused by

impact of a blunt instrument or object or caused by projectiles.

Types of Glass fracture:

1. Radial Fractures – primary fracture resembles the spokes of a wheel

where the radiating rod originates at a common point. When glass breaks,
the lines that radiate from the hole are caused by the glass bending away
from the point of impact. The radial fractures originate on the opposite
side of the glass, because this is the surface which is the first to feel the
tension. As the front of the glass is pushed in, the opposite side is bent
backwards. When the limit of glad elasticity is reached, it breaks, with
cracking resulting along the radial lines.
2. Concentric Fractures - secondary fracture having the appearance of
circles around the point of impact connecting one radiating crack to the
other, thus forming triangular pieces of glass. While the radial fractures are
forming, triangles are created between the fractures. The newly formed
triangle glass between the radial fractures also bends away from the
direction of force. When the limit of elasticity for this triangle is reached,
the glass breaks in concentric lines. Concentric fractures originate on the
front of the glass.
3. Conchoidal Fractures - A characteristic of glass is that when it breaks,
the fracture edges appear shell-like in form - that is, having elevations or
depressions in the shape of a shell. The technical name for this condition is
“conchoidal” fracture.
Fractures Caused by Heat

Fractures caused by excessive exposure to heat can be distinguished from those

caused by impact since those due to heat do not show a regular pattern of radial and
concentric lines. Heat fractures are characteristically wave shaped.

Heat fractures also show curve patterns (stress lines) along the edges. Expansion
of the glass (stretching action) occurs first on the side exposed to the heat, and glass
splinters are usually towards that side. Reconstruction of a glass object fractured by heat
will disclose the wave-shaped fracture pattern.
Fractures Caused by a Blunt Instrument/Object

Thorough examination of glass fractures caused by the impact of a blunt

instrument/object will reveal a pattern of radial and concentric fractures. When glass
fractures are caused by the impact of a blunt instrument/object, stress lines on the edges
of both the radial and concentric fractures can be seen.

Fractures Caused by Projectiles

A small dense object such as a bullet, pebble, or steel ball may impact upon a
pane of glass with such a little force, or at such a high speed, that there is no bulging of
the glass, and therefore, no radial cracks. Penetration of high-velocity projectiles will
produce a coning or catering effect, where the opening is larger on the exit side.

Significance of determining the types of glass fractures

From the study of the types of glass fractures, one may arrive at the following
conclusions:

1. Point of Impact

Glass is at its maximum strength under compression, but weak in tension. An

impact on a pane of glass causes it to bulge. Since the side opposite the impact is
stretched more, it ruptures first. Radial cracks are rapidly propagated away from the
point of impact in short segments. Ridges will be seen as irregularities on the broken
edge of a radial crack. These ridges tend to be perpendicular to the side opposite the
impact and parallel to the side of impact. If there is high stress, minute stress cracks
called hackles or hackle marks may also be observed under the microscope at right
angles to the ridges.

2. Direction of Impact

Generally, the hole produced in the glass by a bullet is small and has sharp edges.
However, if a bullet has been fired from a very long distance and hits a window at low
speed, it will break the pane in much same manner as a stone. A shot few inches from a
glass will produce a similar result, because the pressure of the powder gas itself will
smash the glass.

A bullet will make a clear-cut hole in the side of entrance rather than on the exit
side. If a shot is fired perpendicularly, it will give a crater of uniform flaking. If the shot is
fired at an angle from the right, the left exit side of the glass will give more flaking and
vice versa. Depression will be produced on the exit side of the glass due to the rebound
of the glass. Radial fracture can be seen on the exit side and the concentric fracture on
the entrance side.

3. Entrance and Exit Hole

Point of entry is where the force is being applied and it may produce a smaller hole than
the exit. The exit is bigger than the entry for the reason that the force applied at the
glass exceeded its desired force. Point of entry has a smooth hole while the exit hole has
the characteristic of roughness.

Factors to be determined in glass fracture

1. Point of Impact:

Entrance Side Exit Side

1. Concentric fracture 1. Radial fracture

2. Clear cut edges 2. Rugged edges
3. Absence of depression 3. With depression
4. Absence/Little flaking 4. With flaking

2. Position of the shooter

i. Perpendicular Shot – exhibit an even distribution of chippings on the

exit side of the glass
ii. Angle from the Right – heavy flaking or chippings on the left side of
the glass
iii. Angle from the Left – heavy flaking on the right side of the glass

3. Age of Fracture
i. Age of Fracture – exhibits a regular pattern of radial/concentric
fracture.
ii. Old Fracture – presence of a short extension lines at the end of
the radial fracture.

Information Determined from Glass Evidence

a. Whether questioned fragments have originated or not from a particular source

b. What side of the glass was force applied?
c. Whether broken fragments belong to a single piece
d. Determination of class characteristics such as refractive indices, elemental
composition, densities
e. Actual identification if a reconstruction would fit the pieces of glass like jigsaw
puzzle.
f. For broken windows caused by a fired bullet, it is possible to determine the
bullet's direction by noting the pattern of breakage on the cone-shaped hole left
by the bullet. The small opening is the entrance side while the large opening is
the exit side of the bullet.
 g. For several bullet holes, the sequence by which the bullet holes were caused can
be determined by noting the radial fractures. Radial fractures caused by the
passage of a bullet will stop at any pre-existing fracture.

Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES), also known as

ICP-AES (Atomic Emission Spectroscopy), is a powerful analytical technique used for the
elemental analysis of materials, including glass. ICP-OES is particularly valuable for the
determination of trace and major elements in glass samples, making it an essential tool
in various fields, including materials science, glass manufacturing, and forensic
investigations.

Chapter V: Identification and Examination of Dangerous Drugs

"Drug testing is a form of forensic testing. Drug test results issued should be
scientifically, legally, and forensically defensible."

What is Drug Identification?

Drug Identification is a branch of Forensic Chemistry that deals with the scientific
examination of drugs and volatile substances. Drug identification is usually conducted by
a forensic chemist/chemical officer to determine the presence of dangerous drugs on
submitted specimens. The forensic chemist/chemical officer also conducts drug tests on
the body fluids of suspected drug pushers and users to determine the presence of
dangerous drug metabolites. Paraphernalia like smoking pipes, tooters, and aluminum
foils should also be submitted for examination to determine the presence of dangerous
drugs.

What are the Forms of Dangerous Drugs?

Drugs are in various forms, these include tablets, capsules, liquid, powder, brick
or decks of marijuana, crushed leaves, and uprooted plants.

Examination of the sample taken from the suspected Dangerous Drugs

Methods of Examination

There are two (2) methods of laboratory examination of suspected dangerous

drugs namely:

1. Qualitative examination - Qualitative examination for drugs involves

the identification of substances to determine their presence or absence.
This process is crucial in various fields such as forensic science,
healthcare, and law enforcement. Several methods can be employed for
qualitative drug analysis, and these methods vary depending on the nature
of the sample and the substances being tested.

Here are some common techniques used in qualitative drug examination:

a. Marquis Test: This test involves the use of specific reagents that
react with different drug classes, producing characteristic colors.
For example, the Marquis reagent can identify the presence of
opioids, amphetamines, and certain other substances.
b. Enzyme-Linked Immunosorbent Assay (ELISA): ELISA tests use
antibodies to detect and quantify the presence of drugs or their
metabolites. It's a common method for screening in clinical and
forensic settings.
 c. Thin Layer Chromatography (TLC): TLC is a chromatographic
technique that separates compounds based on their affinity for a
stationary phase. Different drugs will move at different rates and
produce distinct spots on the TLC plate.
d. Gas Chromatography-Mass Spectrometry (GC-MS): GC-MS is a
powerful technique for separating and identifying compounds. Gas
chromatography separates components based on their volatility,
and mass spectrometry provides information about the molecular
structure of the compounds.
e. High-Performance Liquid Chromatography (HPLC): HPLC is
another chromatographic technique that is widely used for drug
analysis. It separates compounds in a liquid medium and can be
coupled with various detectors for identification.
f. Nuclear Magnetic Resonance (NMR): NMR spectroscopy can
provide detailed information about the molecular structure of a
substance. It's a powerful technique but is more commonly used in
research or specialized laboratories.
g. Microscopy: Microscopic examination can be used for the visual
identification of substances. For example, the morphology of
crystals or the appearance of certain particles under a microscope
can provide clues about the nature of a sample.
h. Mass Spectrometry (MS): MS is a technique that measures the
mass-to-charge ratio of ions, providing information about the
molecular composition of a substance. It is often coupled with
chromatographic techniques for enhanced identification.
i. Fourier Transform Infrared (FTIR) Spectroscopy: FTIR
spectroscopy identifies substances based on the absorption of
infrared radiation by the molecular vibrations of the sample. It is
useful for identifying functional groups in organic compounds.

It is important to note that the choice of method depends on

factors such as the nature of the sample, the required sensitivity, and
the available equipment. Often, a combination of techniques is used for
comprehensive drug analysis. It's important to note that the
interpretation of results requires expertise to avoid false positives or
negatives. Additionally, the legality and ethical considerations
surrounding drug analysis should always be taken into account.

2. Quantitative examination - Quantitative examination for drugs involves

determining the amount or concentration of a specific substance within a
given sample. This type of analysis is crucial in various fields, including
clinical chemistry, pharmaceuticals, forensic science, and environmental
monitoring. Several techniques are commonly used for quantitative drug
analysis, and the choice of method depends on factors such as the nature
of the sample, the required sensitivity, and the specific drug or substance
being measured.
Here are some common techniques for quantitative drug analysis.
1. High-Performance Liquid Chromatography (HPLC): HPLC is widely
used for quantitative analysis of drugs and pharmaceuticals. It
separates components in a liquid medium and can be coupled with
detectors such as UV, fluorescence, or mass spectrometry for
precise quantification.
2. Gas Chromatography (GC): GC is suitable for volatile and semi-
volatile compounds. It separates components based on their
 vaporization properties and can be coupled with detectors like
flame ionization detectors (FID) for quantitative measurements.
3. Liquid Chromatography-Mass Spectrometry (LC-MS): LC-MS
combines liquid chromatography with mass spectrometry, providing
both separation and identification capabilities. It is highly sensitive
and is commonly used in pharmaceutical and forensic laboratories
for quantitative drug analysis.
4. Atomic Absorption Spectroscopy (AAS): AAS measures the
absorption of light by atoms in the vapor phase, allowing for the
quantification of certain elements, including some drug
components. It is particularly useful for metals and metalloids
analysis.
5. Inductively Coupled Plasma Mass Spectrometry (ICP-MS):
ICP-MS is a powerful technique for the quantitative analysis of
elements. It uses an inductively coupled plasma as the ionization
source and mass spectrometry for detection.
6. Ultra Violet -Visible Spectroscopy: UV-Visible spectroscopy
measures the absorbance of light in the ultraviolet and visible
regions. It is commonly used for quantitative analysis of compounds
that absorb UV or visible light.
7. Enzyme-Linked Immunosorbent Assay (ELISA): ELISA,
although often used for qualitative analysis, can be adapted for
quantitative measurements. It relies on the reaction of an enzyme
with its substrate, and the resulting color change can be correlated
with the concentration of the substance being measured.
8. Nuclear Magnetic Resonance (NMR) Spectroscopy: NMR can
be used for quantitative analysis, especially in research settings. It
provides information about the molecular structure and can be
calibrated for quantitative measurements.
9. Capillary Electrophoresis (CE): CE separates ions based on their
charge and size. It can be used for quantitative analysis, especially
for charged compounds, and is known for its high separation
efficiency.

Steps common to qualitative and quantitative methods

The following are the steps common to qualitative and quantitative methods of
analysis:

1. Selection of method to be used

2. Physical test
3. Sampling
4. Sample preparation
5. Chemical test
6. Confirmatory examination
7. Calculation and interpretation of dates
8. Drawing of conclusion and writing report

Two phases in the examination of the suspected Dangerous Drugs

1. Screening test/Preliminary test (also known as the color test) - This test is
non-specific and preliminary in nature. It is employed to reduce the family or
group of drugs to a small and manageable number.
Screening test includes a series of color tests producing characteristic
colors for each family or group of drugs. This is done by adding a specific reagent
to an unknown sample in a spot plate. This test is quite simple to perform even by
 investigators in the field. As a matter of fact, field tests using these techniques
are being taught in Narcotics Investigation Courses. Test reagents and basic
apparatus are commercially available.

Color Test - Upon addition of specific reagents to a sample of dangerous drugs, a

specific color reaction is produced such as:

Drug Test Reagent Result

Cannabis Duquenois-Levin - The Duquenois-Levine 1. vanillin, Violet/Purple
test, also known as the Duquenois test, is 2. acetaldehyde
a chemical color test used to 3. hydrochloric acid.
presumptively identify the presence of
Fast Blue B salt purple red
marijuana (cannabis) and related
compounds in plant material or other
substances. It is not a definitive test for
the identification of specific drugs but
rather a preliminary screening test that
can indicate the possible presence of
cannabinoids.
Cocaine Cobalt Thiocyanate test or CT test - 1. cobalt(II) chloride Blue
The Cobalt Thiocyanate (CT) test, also 2. potassium
known as the Scott test, is a chemical test thiocyanate
used for the preliminary detection of
cocaine in a substance or on surfaces. It is
a color test that relies on the formation of
a distinct color complex when cocaine
reacts with a cobalt thiocyanate reagent.
The test is relatively simple and quick,
making it useful for law enforcement and
forensic purposes. However, like other
color tests, it is not a definitive method for
identifying substances and is primarily
used as a presumptive test.
Wagner test - The "Wagner Test" for cobalt thiocyanate bright blue
cocaine, also known as the Wagner color/Brown
Reagent or Wagner's Reagent, is a
chemical color test used to detect the
presence of cocaine in a substance. It's a
presumptive test, meaning it provides an
initial indication of the possible presence
of cocaine, but it is not considered a
definitive or confirmatory test.
Confirmatory testing, such as gas
chromatography-mass spectrometry (GC-
MS), is typically required for precise
identification and quantification of
cocaine.
Opium Marquis Test - The Marquis Test is a 1. Formaldehyde Violet Ferric
chemical color test used to identify and 2. sulfuric acid Sulfate
detect the presence of specific classes of Brownish purple
drugs, primarily in illicit substances. This
test is named after its developer, Eduard
Marquis, and is commonly used for
preliminary drug screening. It can help
identify a range of substances, including
amphetamines, ecstasy (MDMA), and
other drugs. However, like other color
tests, the Marquis Test is not definitive
 and should be followed by more accurate
confirmatory testing methods.
Morphine Marquis Test Violet to
reddish-purple
Mescaline Marquis Test Orange
Methamphe Marquis test Orange to
tamine brown
Hydrochlori
de (Shabu)
Simon test - The Simon's Reagent Test is 1. sodium blue color.
a color-based chemical test used to nitroprusside (or
identify the presence of specific sodium
substances, including methamphetamine
nitroferricyanide)
and its hydrochloride salt form
(methamphetamine hydrochloride or meth 2. acetaldehyde
HCl). This test can help determine whether
a sample contains methamphetamine, but
it does not provide information about the
purity or quantity of the substance. As
with other color tests, it is considered a
presumptive test, and confirmatory testing
is necessary for accurate identification.
Ecstacy Simon test Blue

Note:
It must be noted that Positive results of these tests are not conclusive, as
there are substances that may give same positive color reaction/s upon
addition of the specific reagents. Hence, confirmatory tests must be
performed by the forensic chemist chemical officer on case to establish the
presence and identification of dangerous drug. It must also be noted that
only those specimens that yielded presumptive positive results are subject
to confirmatory test in order to confirm if the positive result of the
screening test is really positive.

3. Confirmatory Test - A confirmatory test is the method employed to confirm the

results of the screening/preliminary test. This test involves the application of an
analytical procedure to identify the presence of a specific drug or metabolite. This is
independent of the screening test and which uses techniques and chemical principles
different from that of the initial test in order to ensure reliability and accuracy. There
are several methods used in the confirmatory test. Some of these methods are:
a. Chromatography - is the process of separating the mixture and comparing
the migration of each component with a standard. Some chromatographic
techniques include:
i. Gas chromatography - is a powerful analytical technique commonly
used for the identification and quantification of drugs and their
metabolites in various samples, including urine, blood, hair, and drug
formulations. GC is widely employed in forensic science, clinical
toxicology, and drug testing laboratories due to its high sensitivity,
specificity, and ability to separate and analyze complex mixtures.
Inside the GC instrument, the sample is carried by a carrier gas
(usually helium or nitrogen) through a column packed with a stationary
phase. The column is heated, and as the sample components travel
through it, they separate based on their chemical properties, such as
volatility and affinity for the stationary phase. The most volatile
compounds move through the column more quickly than less volatile
ones.
 As the separated compounds exit the column, they pass
through a detector. Various detectors can be used in GC, with the most
common being the Flame Ionization Detector (FID) and the Mass
Spectrometer (MS). The detector responds to the presence of specific
compounds by producing electrical signals, which are then recorded as
peaks on a chromatogram. The resulting chromatogram provides
information about the retention times of the compounds, which can be
compared to reference standards for identification. The area under
each peak corresponds to the quantity of a specific compound in the
sample, allowing for quantification.
ii. Thin Layer chromatography - is a chromatographic technique
commonly used for the qualitative analysis of drugs and other
compounds in forensic, pharmaceutical, and research settings. TLC is a
quick and relatively inexpensive method for separating and identifying
different compounds present in a mixture. It is particularly useful for
the preliminary screening of drugs and can provide information about
the number of components in a sample.
The TLC plate is placed in a developing chamber containing a
solvent or mobile phase. The plate is immersed in the solvent but not
submerged. As the solvent travels up the plate by capillary action, it
carries the sample with it. The components of the sample separate
based on their affinities for the stationary phase and the mobile phase.
After the solvent has traveled a specific distance up the plate
(known as the solvent front), the plate is removed from the chamber
and allowed to dry. The separated compounds on the TLC plate are
typically not visible to the naked eye, so they need to be visualized.
This can be done using various techniques:
1. UV light: Some compounds may fluoresce under ultraviolet
(UV) light.
2. Chemical reagents: Specific reagents can be sprayed or
applied to the plate to react with compounds, producing visible
spots or colors.
3. Iodine vapor: Iodine vapor can be used to visualize spots on
the TLC plate.
iii. High-Pressure Liquid chromatography - is a widely used analytical
technique for the separation, identification, and quantification of drugs
and various other compounds in a wide range of applications, including
pharmaceuticals, clinical chemistry, forensics, and research. HPLC is a
powerful method because it can handle a variety of sample types and
provide high sensitivity and precision. HPLC is commonly used in
pharmaceutical laboratories to analyze drug formulations, in clinical
labs for drug screening and therapeutic drug monitoring, and in
forensic labs for drug identification. It allows for both qualitative and
quantitative analysis, making it a versatile tool in drug analysis and
research.
b. Spectroscopy - a confirmatory method whereby light is used to identify the
sample specimen. Spectroscopy is a powerful analytical technique used in
drug analysis to identify, characterize, and quantify drugs and pharmaceutical
compounds. Spectroscopy involves the measurement and analysis of the
interaction between matter and electromagnetic radiation (light) at various
wavelengths. There are several types of spectroscopic techniques commonly
used in drug analysis:
i. Fourier Transform Infrared Spectroscopy (FTIR) is a widely used
spectroscopic technique in drug analysis. It is valuable for identifying
and characterizing drugs and pharmaceutical compounds based on
their unique infrared absorption patterns. FTIR can provide information
 about the functional groups, chemical bonds, and molecular structures
present in a drug substance.
ii. Ultraviolet-Visible (UV-Vis) Spectroscopy - UV-Vis spectroscopy
involves measuring the absorption of ultraviolet and visible light by
molecules in a sample. It is widely used for pharmaceutical analysis to
determine the concentration of a drug in a solution by measuring the
absorbance of light at specific wavelengths. UV-Vis spectroscopy is also
used to study the electronic transitions of compounds, helping to
identify functional groups and assess the purity of drug substances.
UV-Vis is used for screening dangerous drugs in urine specimens.
iii. Infrared (IR) Spectroscopy - IR spectroscopy measures the
absorption and vibration of infrared light by molecules. It is valuable for
identifying functional groups in drug molecules, as different functional
groups absorb infrared radiation at characteristic frequencies. IR
spectroscopy is used to confirm the identity of drugs and assess their
purity and structural integrity.
iv. Nuclear Magnetic Resonance (NMR) Spectroscopy - NMR
spectroscopy provides detailed structural information about molecules
by analyzing the nuclear magnetic properties of certain nuclei (e.g.,
hydrogen and carbon) in a sample. It is essential for elucidating the
complete structure of complex drug compounds, including their
stereochemistry. NMR can also be used to determine the purity and
concentration of drug samples.
v. Mass Spectrometry - Mass spectrometry involves ionizing molecules
and measuring their mass-to-charge ratio. MS is crucial for the precise
identification and quantification of drugs and their metabolites. It is
commonly used in pharmaceutical research, clinical toxicology, and
forensic drug analysis.
vi. Fluorescence Spectroscopy - Fluorescence spectroscopy measures
the emission of fluorescent light from a sample following excitation by
light of a specific wavelength. It is employed in drug discovery and
pharmacology to study drug-receptor interactions, ligand binding, and
enzyme assays. Fluorescence can also be used to quantify the
concentration of fluorescently labeled drugs or biomolecules.

Examination of the Urine Specimen

The rate of excretion from the body depends on the drug's solubility in fat. Water-
soluble drugs (such as cocaine) are excreted quickly, while fat-soluble drugs (such as
marijuana) may take several weeks or months before excretion. Drug tests must be
conducted to apprehend individual/s who is/are suspected to be a user/s; and to those
who are charged with the offense of "Illegal Use of Dangerous Drugs".
Validity Test for Urine Specimen
A validity test for urine, often referred to as a "urine validity or integrity test," is
a part of the drug testing process designed to determine whether a urine sample has
been tampered with or diluted in any way. Drug testing laboratories and employers use
these tests to ensure the reliability of urine drug test results by confirming that the
collected specimen is genuine and has not been altered to produce a false negative
result.
Reasons for Conducting Validity Tests
 In cases of unobserved urine collection
 When there is suspicion that the urine specimen has been tampered
Instances when to allow Unobserved Urine Specimen Collection
 When the donor is physically unable to go to the laboratory
 When the donor is involved in a crime scene
  When the donor is involved in post-accident trauma
 When the donor is critically ill
Different Types of Tampered Urine Specimen
The following are the different types of tampered urine specimen:
1. Adulterated a specimen containing either a substance that is not a normal
constituent for that type of specimen or containing an endogenous substance at
a concentration that is not a normal physiological concentration
2. Diluted refers to a specimen with less than normal physiological constituents
3. Substituted a specimen which has been derived through switching or
replacement of the original sample.
Ways to Adulterate Urine Samples
The following are the different ways to adulterate urine samples:
(a) Addition of salt
(b) Addition of juice
(c) Addition of detergent
(d) Addition of bleach and other
(e) Adulterants
(f) Addition of illicit drugs
Ways to Substitute a Urine Sample
The following are the ways to substitute a urine sample:
1. Urine from friends or other persons not using drugs may be used as a substitute
specimen.
2. Replace the sample with other substances similar to urine in appearance.
Ways to Dilute a Urine Specimen
The following are the ways to dilute a urine specimen:
1. Internal Dilution (e.g. Intake of plenty of water before collection or drinking of
herbal tea, etc.)
2. External Dilution (e.g. Addition of water to previously collected urine)
Parameters for Validity Tests
The following are the parameters for validity tests
Physical characteristics such as color, odor, etc
(a) Initial Validity Tests:
 Volume
 Temperature
 PH
 Specific gravity
 Nitrites
 Creatinine
 Oxidizing agents
(b) Confirmatory Validity Tests
 Physical characteristics such as color, odor, etc..
 Volume
 Temperature
 PH
 Specific gravity
 Nitrites
  Creatinine
 Oxidizing agents
Other Methods for Confirmatory Validity Tests
Physical characteristics-visually determined
 Volume-same as physical characteristics
 Temperature - using thermometer
 pH - pH Meter calibrated with appropriate buffers
 Specific gravity - use a refractometer
When do we consider a urine specimen as invalid?

A urine specimen is considered invalid under the following circumstances:

- Adulterated, substituted or diluted

- Improperly collected, handled and stored
- Improperly documented