Module 4

Blood and Bloodstain with DNA

 Importance in the Study of
Blood and Bloodstain
 Composition of Blood
 Importance of the Study of
Blood
 Test for Blood
 Chronological Test for Blood
 Confirmatory Test for Blood
 DNA

What is SEROLOGY?

 A branch of science that deals with the study of body fluids.

  A branch of science that deals with the determination of the type and
characteristics of blood, blood testing, and the examination of blood stain, semen,
saliva, and other body fluids that may or may not be involved with DNA typing.

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 a paternity test,
determine the cause of death, or the length of time at which the victim survived an attack.

IMPORTANCE OF THE STUDY OF BLOOD:

1. As circumstance or corroborative evidence against or in favor of the perpetrator of

the crime.
2. As evidence in case of disputed percentage
3. As evidence in the determination of the cause of death and the length of time the
victim survived the attack.
4. Determination of the direction of escape of the victim or the assailant
5. Determination of the origin of the flow of blood
6. As evidence in the determination of the approximate time the crime was committed.

THE CONSTITUTION OF BLOOD

THE CONSTITUTION OF BLOOD

BLOOD

Is the circulating tissue of the body. It is referred to as a highly complex mixture of cells,
enzymes, proteins, and inorganic substances. It is the red fluid of the blood vessels. Blood is
opaque. On the treatment with either, water or other reagents becomes transparent lake color. It is
finally alkaline. Normally pH is 7.35 – 7.45.

COMPOSITION OF BLOOD

1. (45%) formed elements or the solid materials consisting chiefly of cells namely:
a. Red Blood Cells or RBC (ERYTHROCYTES) – Contains hemoglobin and carry
oxygen to various cells in the body.
b. White Blood Cells or WBC (LEUKOCYTES)- Are masses of nucleated protoplasm.
Help fight infection.
c. Blood Platelets (THROMBOCYTES)- Cells That are produced by the bone marrow
and are necessary for proper clothing of blood. Normally responsible for the reaction
of blood cloth.

2. (55%) PLASMA – The fluid or liquid portion of blood where the cells are suspended. It is
principally composed of:
a. Water ---- 90%
 b. Solid ----- 10% (largely protein in nature and consist of albumen, several globulin’s
and fibrinogen.
In the forensic aspect of blood identification, that is blood grouping; our discussion will
concentrate on the RBC and blood serum. Serum is pale yellowish liquid just like the plasma.

PLASMA

The yellowish fluid of blood in which numerous blood corpuscles are suspended. A
straw-yellow liquid formed when blood to which oxalate has been added to prevent clotting is
allowed to strand.

SERUM

A straw – yellow liquid formed when clotted blood is allowed to stand for some time and
the clot contracts.

PROBLEMS IN THE STUDY OF BLOOD:

1. where has to be searched for

2. Collection, preservation, packing and transportation of specimen suspected to contain
blood.

Blood offers little resistance to decomposition. It undergoes a rapid charge in its character with the
passage of time as process of clotting and drying commences almost immediately on exposure to
air. Sodium fluoride maybe added to blood to preserve it for a week at room temperature or
indefinitely in a refrigerator. Between 40 – 50 degrees centigrade is the ideal preserving
temperature for blood and other perishable specimens. Collection of blood stains should be done
as soon as possible; mere washing of garments/clotting removes the blood.

FLUID BLOOD:
Collect from:

1. Victims of crimes of violence

2. Parent and child in case of disputed parentage.

DRIED BLOOD OR BLOOD STAINS:

Collect from:

1. Smooth surface like walls, finished floors, table tops

2. Hard surface like axe, hammer, knives, stones, crowbars
3. Glazed surface like glass, tiles, automobiles
4. Bulky objects like blackboard, linoleum sheets, doors, window frames
5. Clothing
6. Blood absorbed by the soil

3. Does the stain contain blood or another substance?

THE EXAMINATION OF BLOOD

THE FOUR TEST FOR BLOOD STAIN

1. PRELIMINARY TEST
Determine whether the stain contains blood or another substance. Determines whether visible
stains do or do not contain blood. It is used to demonstrate the presence of blood.

2. CONFIRMATORY TEST
Determines whether bloodstain really contains blood. Test that positively identifies blood.

3. PRECIPITIN TEST
Determines whether blood is a human or non-human origin, and if non-human, the specific
animal family from which it originated.

4. BLOOD GROUPING TEST

Determines the blood group of human

THE PRELIMINARY TEST FOR BLOOD (PRELIMINARY COLOR TEST)

FOLLOWING ARE PRELIMINARY TEST FOR BLOOD:

1. Benzidine Test or Benzidine Color Test

2. Phenolphthalein Test (also known as Kastle – Meyer Test)
3. Guaiacum Test (Van Deen Test, Day’s or Schonbein’s Test)
4. Leucomalachite Green Test
5. Luminol Test

BENZIDINE TEST

An extremely sensitive test that can be applied to minute stain. For many years the most
commonly used preliminary test for blood. The Benzidine test never fails to detect blood even
when very old, decomposed stain with all sorts of contamination is examined. The positive result
is only indicative that the blood maybe present.

REAGENT: Benzidine solution (small amount of powdered benzidine dissolved in

glacial acetic acid) and 3% solution of hydrogen peroxide.

POSITIVE RESULTS: Intense blue color produced immediately

Limitation: Benzidine test is not a specific test for blood. Positive results may be obtained
from substances as sputum, pus, nasal secretion, plant juices, formalin, clay, gun. The reaction is
weaker and produce faint coloration.

PHENOLPHTHALEIN TEST

An alternative test to benzidine test. It can detect blood in a dilution of 1:80,000,000

parts. A positive result with this test is highly indicative of blood. The negative result is, therefore,
valuable and is conclusive as to the absence of blood.

REAGENTS: Phenolphthalein solution (1 – 2 grams’ phenolphthalein to 100 ml of a

25% KOH in water added with one gram zinc powder heated until colorless) and 3% solution of
hydrogen peroxide.

POSITIVE RESULT: Rose color develops or deep pink color or permanganate color.

LIMITATION: Test is also given by copper salts, potatoes and horseradish.

GUAIACUM TEST

A fairly delicate test showing the presence of fresh blood in a solution of 1:50,000
dilution. It may not react to very old stains.

REAGENTS: Fresh tincture of guaiac resin (Few lumps of this to 95% alcohol, then
filter) and 3% of hydrogen peroxide or few drops of turpentine.

POSITIVE RESULTS: Beautiful blue color that appears immediately.

Limitation: The test also reacts with salvia, pus, bile, milk, rust, iron salts, cheese,
gluten, potatoes, perspiration and other oxidizing substances.

LEUCOMALACHITE GREEN TEST:

Not as sensitive as the benzidine test

REAGENT: Leucomalachite Green solution (1 gram leucomalachite green dissolved in

48 ml. glacial acetic acid and diluted to 250ml. water) and 3% hydrogen peroxide.

POSITIVE RESULTS: Malachite green or bluish green

PRINCIPLE INVOLVED IN THE FOUR PRELIMINARY TEST FOR BLOOD

(BENZIDINE TEST, PHENOLPHTHALEIN TEST, GUAIACUM TEST AND
LEUCOMALACHITE GREEN TEST)

The peroxidase present in hemoglobin acts as carrier of oxygen from the hydrogen
peroxide to the active ingredients of the reagents (benzidine, guaiac, phenolphthalein and
leucomalachite) and produces the characteristic colored compounds by OXIDATION.

PEROXIDASE

Enzyme that accelerates the oxidation of several classes of organic compounds by peroxide.

HEMOGLOBIN

The red coloring matter of the red blood cells of the blood.

LUMINOUS TEST

An important presumptive identification test for blood. The reaction of luminol with
blood result in the production of light rather than color. By spraying luminol reagent onto a
suspect item, large areas can be quickly screened for the presence of bloodstains. The sprayed
object must be located in a darkened area while being viewed for the emission of light.
(LUMINESCENCE). Luminol test is extremely sensitive test. It is capable of detecting
bloodstains diluted up to 10,000X. Luminol is known to destroy many important blood factors
necessary for the forensic characterization of blood, so its use should be limited only to seeking
out blood invisible to the naked eye.

THE CONFIRMATORY TEST FOR BLOOD

The actual proof that stain is blood consists of establishing the presence of the characteristic of
the red blood cells of the blood.
THE THREE CONFIRMATORY TEST FOR BLOOD (OR THE THREE TEST TO
DETERMINE IF STAIN IS REALLY BLOOD)

1. Microscopic Test
2. Microchemical Test
3. Spectroscope Test

MICROSCOPIC TEST

Useful for the demonstration and mensuration of blood corpuscles for making the
distinction between mammalian, avian, piscine, and reptilian blood and for the investigation of
menstrual, lochial and nasal charges. In short, it differentiates mammalian, avian, piscine and
reptilian blood.

MAMMALIAN RED BLOOD CELLS

Circular, biconcave disc without nucleus birds, fish and reptiles red blood cells larger,
oval and nucleated Amphibians-animal living on land breeding in water. Red blood cells are larger
than mammals, oval and nucleated.

MICROCHEMICAL TEST OR MOCROCRYSTALLINE TEST:

a. Teichmann Hemin Reaction/Teichman Test/Haemin Crystal Test

b. Haemochromogen crystal Test or Takayama Test
c. Acetone-Haemin Test

HAEMOCHROMOGEN CRYSTAL TEST OR TAKAYAMA TEST

One of the two popular microchemical test. A delicate test for the presence of
hemoglobin.

POSITIVE RESULTS: Large rhombic crystals of a salmon pink color arranged in

clusters, sheaves and other forms that appear within to 6 minutes when viewed under the low
power objectives. To hasten result heat maybe applied.

REAGENT: Takeyama reagent (3 cc. of 10% NaOH, 33 cc. pyridine, 3 cc. of saturated
glucose solution and diluted with 7 ml. of water.

SPECTROSCOPIC EXAMINATION

The almost delicate and reliable test for the determination of the presence of blood in both
old recent stains. This is performed by means of an optical instrument known as
SPECTROSCOPE.

I. PRECIPITIN TEST

PRECIPITIN TEST

Is the standard test used to determine whether the stain/blood is of human or animal
origin

Reagent: Precipitin/antiserum

POSITIVE RESULT: A white cloudy line or ring or band at the contact points of the
fluid that appears immediately or within one or two minutes.
 LIMITATION OF PRECIPITIN TEST; The precipitin reacts not only with blood
proteins but also with other body proteins as those as saliva, semen, mucus and other body fluids.

II. BLOOD GROUPING AND BLOOD TESTING

BLOOD GROUPING TEST OF FRESH BLOOD (Direct Technique Method) USING THE
A-B-O SYSTEM

THE FOUR BLOOD GROUPS:

1. Group “O”
2. Group “A”
3. Group “B”
4. Group “AB”

AGGLUTINOGEN OR ANTIGEN

These are characteristic chemical structures or “principles” that the found on the surface
of each red blood cells which stimulates the production of agglutinins or antibodies. There are two
different agglutinogens or antigens classified as AGGLUTINOGEN A OR ANTIGEN A AND
AGGLUTINOGEN B OR ANTIGEN B.

ANTIBODY OR AGGLUTININ

These are properties or “principles” contained in the serum which cause agglutination or
clumping together of the red blood cells. They are antitoxic substances within the body which
reacts when confronted with a specific antigen to protect the system. There are two different
agglutinins classified as Anti-A and Anti-B. Agglutinins are demonstrable in about 50% of newly
born infants.

We have the four groups because of the presence of absence of two antigens A and B in
the RBC and two agglutinins Anti-A and Anti-B in the serum.

BLOOD GROUP ANTIGEN/ ANTIBODIES/

AGGLUTINOGEN AGGLUTINIES PRESENT
PRESENT IN THE RBC IN THE SERUM
A
A ANTI-B
B
B ANTI-A
AB
A&B NO A & NO B or NONE

NO A & NO B or NONE ANTI-A & ANTI-B

(+) Means agglutination or clumping of RBC

(−) Means absence of agglutination or no clumping of RBC

INHERITANCE OF BLOOD GROUPS

Knowledge of the gas of genetics will make it easier to understand the principle involved
in the inheritance of blood groups. The inheritance of blood groups are predetermined by the
presence and absence of two facts or GENES called Gene A and Gene B. Before we understand
the inheritance of blood groups following are definition of items:
 1. GENES- Any of the complex chemical units in the chromosomes by which
hereditary characters are transmitted. Responsible for the transmission of hereditary
characteristics. They occurs in pair. There are two genes or factors called gene A and
gene B. these are found in the chromosomes. Since chromosomes go in pair, each of
which carries or fails to carry one of these genes. An individual’s called genotypes,
where O represents the absence in the chromosomes of either the A or B gene.

2. Chromosomes- any of the microscopic rod-shape bodies bearing genes responsible

for the transmission of hereditary characteristics.

3. PHENOTYPES- Term used to denote the expression of the inherited characteristic

as found in the individual. Actually the blood groups

4. GENOTYPES- Are paired genes. It’s either homozygous or heterozygous.

5. PHENOTYPE- Term used to denote the expression of the inherited characteristics as

found in the individual. Actually the blood group.

IMPORTANT APPLICATION OF BLOOD GROUP DATA

Questions of illegitimacy and relationships in may cause maybe solved by means of the blood
groups as determined by the agglutinogens A, B, M, and N.

1. Determination of whether a man accused of fathering a child out of wedlock could or

could not be its parent.
2. Determination of whether a child born of a married woman could or could not have
been fathered by her legal spouse.
3. Determination of whether a child could or could not belong to a given set of parents
in the case of accidental interchange of infants in a hospital.
4. Determination of whether a child who has been lost and later recovered after a long
interval could or could not belong to a given set of parents.

DNA
[Deoxyribonucleic Acid]

- A chemical substance found in all cells of living organism whose composition have been passed
from parents to offspring. It is called as the genetic or hereditary material. A person`s DNA is
the SAME in every cell.

Chemically, it is an acid and is composed of three sub-units, namely:

1. the phosphate group
2. a deoxyribose sugar, and
3. one of the four bases –
Adenine (A),
Thymine (T),
Cytosine (C), &
Guanine (G)

DISCOVERY
In 1953, DNA - the so called building block or genetic blueprint of
life was first characterized by scientist Francis H. CricK and James Watson as the material that
make up the genetic code of all organisms.
Crick and Watson were completely unaware of the potential impact their discovery would have on
forensic science in particular and the criminal justice system as a whole. Resting on the
established natural truth that no two individuals have the same DNA blueprint, with the exception
of identical twins. DNA typing or "profiling" as it is now called was CONCEIVED.

In 1984 DNA analysis was first recognized as having application to Forensic Science by the
British Molecular Biologist Alec Jeffreys and thereafter directly used in a rape and murder case to
exonerate an innocent suspect and to convict the guilty accused.

HOW DNA ANALYSIS IS DONE

DNA TYPING is done by first carefully extracting the DNA from the evidentiary
samples. The DNA is then analyze to give a particular pattern. The patterns are compared with
that of a known individual to determine a match.

SOURCES OF BIOLOGICAL EVIDENCE

 Blood
 Semen
 Saliva
 Urine
 Hair
 Teeth
 Bone
 Tissue

GENERAL CHARACTERISTICS OF GENOMIC DNA

• Each person has a unique DNA profile (except identical twins because they have the same
DNA profile)
• Each person`s DNA is the same in every cell (DNA from skin cells will match from blood
cells)
• An individual`s DNA profile remains the same throughout life
• Half of your DNA comes from your mother and half from your father

DNA ANALYSIS

 DNA contains “detectable” patterns unique to each individual.

 DNA is a robust molecule and is stable.
 DNA can be isolated from a wide range of biological samples.
 Source of is the same in all sources (blood, semen, sputum, skin etc.).
 Precise patterns can now be detected even with very small samples (a blood spot,
single hair follicle, lip-prints on a glass, physical fingerprints, saliva, skin on a
cigarette butt, etc.)

LINE-UP of CASES where DNA ANALYSIS can be of help:

 Sexual assault cases like RAPE

 Murder
 Homicide
 Robbery
 Hit and run
 Extortion
 Paternity cases
 Identification of remains in mass disaster
 Fight against human trafficking especially for kids
 Prevention of illegal adoption of kidnapping
  Stolen or trafficked children

IDENTIFYING DNA EVIDENCE

GUIDELINES FOR COLLECTING AND SUBMITTING DNA EVIDENCE

DOCUMENTING, COLLECTING, PACKAGING and PRESERVING DNA EVIDENCE from

CRIME SCENE

If DNA evidence is not properly documented, collected, packaged and preserved, it will not
meet the legal and scientific requirements for admissibility in a court of law.

1. If DNA evidence is not properly documented, its origin can be questioned.

2. If it is not properly collected, biological activity can be lost.
3. If it is not properly packaged, contamination can occur.
4. If it is not properly preserved, decomposition and deterioration can occur.

REQUESTING EVIDENCE EXAMINATIONS

All requests for evidence examinations should be in writing addressed to the DIRECTOR,
thru the Deputy Director for Forensic Investigation Service , Attention: DNA Analysis
Laboratory, Forensic Chemistry Division, NBI, Taft Avenue, Manila and contain the
following information:

 The requesting and submitting contact person’s name, agency, address and telephone
number.
 Description of the nature and the basic facts concerning the case (brief history of the
case).
 The name(s) of and descriptive data about the individual(s) involved (subject, suspect,
victim) and the agency-assigned case identification number.
  A list of the evidence being submitted herewith(enclosed) or under separate cover.
 State what types of examinations are requested.
 State where the laboratory report should be sent.
 Chain of Custody of Evidence

. PACKAGING AND TRANSPORTING EVIDENCE

• Take precautions to preserve the evidence.
• Place porous evidence in individual protective covering such as paper envelopes.
Stabilize the evidence to avoid movement or friction during transport.
• Wrap and seal each item of evidence separately to avoid contamination.
• Place the evidence in a clean, dry and previously unused inner container.
• Seal the inner container with tamper-evident tape.
• Affix EVIDENCE and appropriate BIOHAZARD labels to the inner container.
• Affix the evidence examination request and all case information between the inner and
outer container.
• Place sealed inner contained in a clean, dry and previously unused outer container with
clean packaging material.
• Completely seal the outer container so that opening of the container would be evident.
• Address the outer container as follows:
FORENSIC CHEMISTRY DIVISION
Attention: DNA Analysis Laboratory
National Bureau of Investigation
Taft Avenue, Manila 1000

DNA TYPING/PROFILING
It is an analytical procedure of identifying the most likely owner of a piece of evidence /biological
sample. To determine the genotype of the donor/source of the DNA material, the NBI make use of
the fully automated hi-tech equipment called ABI-PRISM 310 GENETIC ANALYZER and 3130
GENETIC ANALYZER, using different typing system.

ACCEPTABILITY IN COURT

The proposed Rule on DNA Evidence take effect on October 15, 2007 following its publication in
a newspaper of general circulation on October 2, 2007.

EN BANC A. M. NO. 06-11-5, SC

Rule on DNA Evidence Resolution
Cases resolved cause of DNA Analysis:
1. Edgardo A. Tijing &n Bienvenida R. tijing v. Court of Appeals and Angelita Diamante,
G.R. 125901, March 08, 2001, (J. Quisumbing)
2. People v. Vallejo (G.R. No. 144656, May 09, 2002)
3. Carmelo Cabatania v. Court of Appeals & Carmelo Regodas, G. R. No. 124814, october
21, 2004
4. People of the Philippines v. Joel Yatar alias “Kawit” G.R. No. 150224, May 19, 2005, Per
Curiam
5. People of the Philippines v. Rufino Umanito G.R. No 172607 April 16, 2009, (J.
Quisumbing)

SUMMARY

In this module, we learn that blood consist of different elements. We also tackled
serology and there importance in criminal investigation. Blood examination involves in different
methods in the solution of crime. It contains also DNA topic that consist its definition and
admissibility in court.