BLOOD AND

BLOODSTAINS
Blood and Bloodstains
• The significance of blood and blood stains as evidence in crimes of
violence is very obvious such that we need not place emphasis on
this. The test for the identification of blood is employed as an
important part of the routine investigation in many case of violent
death. The specimen usually submitted is fresh blood or fluid blood,
dried blood and clotted blood. Very often it is brought to the
laboratory in the form of dried red or brown stains on weapons,
clothing or other object.
Importance of blood and blood stains in
Forensic Investigation
• Finding and correctly interpreting blood traces can be of utmost
importance in solving the circumstances surrounding the perpetration
of the crime, the actions of the victim and the perpetrator at the
scene, their contribution to the perpetration of the crime, their
behavior after the perpetration of the crime. Unless the perpetrator is
not known in the first stages of criminal investigation, blood traces,
together with other material traces at the scene, can be a significant
controlling factor in assessing the credibility and truthfulness of the
statements of the event participants.
In criminal and judicial practice, cases have been
reported in which a thorough qualitative and
quantitative analysis of traces of the blood was crucial
for identifying the perpetrators, participants, as well as
proving their role in the perpetration of the crime. In
some cases it can also help with legal determination of
criminal offense which can lead to more accurate and
more appropriate punishment for the perpetrator. It is
very important to determine the sequence of events
during the commitment of a violent crime involving
blood.
 BLOOD
This refers to as highly complex mixture of cells,
enzymes, proteins and inorganic substances. It is the
red fluid of the blood vessels. Blood is opaque. On
treatment with either, water or other reagents
becomes transparent and assumes lake color. It is
faintly alkaline. Normal pH is 7.35 to 7.45. cts.
 Importance of the Study of Blood
1. As circumstantial or corroborative evidence against or in favor of the
perpetrator;
2. For disputed parentage;
3. 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; and
6. Determination of the approximate time the crime was committed.
Nature of Blood
1. Largest circulating tissue of the body;
2. Consists of vital substances; and
3. Fluid that circulates into the Cardiovascular System (CVS)
C. Function of Blood
1. Transport of gases (O2 and CO2), nutrients and wastes;
2. Blood regulates body temperature;
3. Blood regulates pH of the body fluids; and
4. Blood carries injected and otherwise given medicines to
the affected parts of the body
 Kinds of Blood
A. Arterial Blood – aka capillary blood, bright red in color and which is
oxygenated blood.
B. Venous Blood – dark red in color, contains increased amount of carbon
dioxide and which is non-oxygenated blood.

• Hemoglobin is responsible for red color, normal Pilipino has 200cc, 6 glasses
means loss of life, 3 glasses will cause anemia. Hemoglobin is responsible for
red color of blood which contains iron protein called globin (protein) and
hematin (organic compound of iron). 14-17 grams of hemoglobin is present
for each 100 cc of blood for adult.
Types of Hemoglobin
A. Methemoglobin (Hbm) – found in Nitrates and Nitrites
poisoning which is chocolate brown in color.
B. Sulfhemoglobin (HbS) – found in the presence of bacteria
(clostridium perfringens) in severe constipation,
enterogenous cyanosis and blood is lavender is color.
Methods of collecting Blood
A. Capillary Blood Sample – Skin/Finger/Ring Puncture, arterial blood
and small quantity of blood. Puncture sites are;
1. Ring finger (Adult and Children);
2. Ear lobes (Adults);
3. Heal or Toe (Infants and Children) – use of lancet or pricker.
B. Venous Blood Sample
Larger volumes of blood
and blood taken from the
vein.
1. Cephalic Vein;
2. Medial Cephalic Vein;
3. Basilic Vein; and
4. Jugular Vein.
C. Arterial Blood Sample (Venipuncture
Method)
Larger volumes of blood and
blood taken from the arteries.
1. Radial Artery;
2. Brachial Artery;
3. Femoral Artery; and
4. Carotid Artery.
The Chronological Test for Blood
• Benzidine Test
• Phenolphthalein Test
• Guaiacum Test
• Leucomalachite Green Test
• Luminol Test
1. Benzidine Test
Reagent:
a. Benzidine solution (a small amount of powdered Benzidine dissolved in glacial acetic acid)
b. 3% solution of Hydrogen Peroxide

Procedure:
Place a small fragment/portion of the stained material on a filter paper. Add a drop of Benzidine
solution and then a drop of hydrogen peroxide solution.

Positive Result: Intense blue color produced immediately.

Limitation of the Test: Benzidine test is not specific test for blood. Positive result may be
obtained from the substances as sputum, pus, nasal secretion, plant juices, formalin, clay and
gum. The reaction is weaker and produces faint coloration.
1. Benzidine Test
 2. Phenolphthalein
Test
Reagent:
a. Phenolphthalein solution (1 to 2 grams of phenolphthalein to 100 ml of a 25%
potassium hydroxide in water added with one gram of zinc powder heated until
colorless).
b. 3% solution of Hydrogen Peroxide

Procedure: Place a small fragment/portion of the stained material on a filter paper.

Add a drop of Phenolphthalein solution and then a drop hydrogen peroxide solution.

Positive Result: Rose color develops/deep pink/permanganate color.

Limitation of the Test: The test will also give positive result to copper salts, potatoes
and horseradish.
2. Phenolphthalein Test
 3. Guaiacum Test
A fairly delicate test showing the presence of fresh blood in a solution of 1:50,000
dilutions. It may not react to very old stain.
Reagent:
a. Fresh tincture of guaiac resin (few lumps of this to 95% alcohol, then filter)
b. 3% Hydrogen Peroxide solution or few drops of turpentine

Procedure: Place a small pieced of a stained fabric on a porcelain dish. Soak with
fresh tincture of guaiac. Add a few drops of Hydrogen Peroxide.

Positive Result: Beautiful blue color that appears immediately.

Limitation of the Test: The test also reacts with saliva, pus, bile, milk, rust, iron,
salts, cheese, glutten, potatoes, perspirations and other oxidizing substances.
3. Guaiacum Test
 4. Leucomalachite Green Test
This test is not as sensitive as the benzidine test.
Reagent:
a. Leucomalachite green solution (1 gram leucomalachite green dissolved in 48 ml
glacial acetic acid and diluted to 250 ml water).
b. 3% Hydrogen Peroxide

Procedure: Place a small pieced of a stained fabric on a filter paper. Add a drop of
Leucomalachite green solution and after a few seconds add a drop of hydrogen
peroxide.
4. Leucomalachite Green Test
 5.Luminol Test
– An important presumptive identification for blood. The reaction of luminal with blood results in the
production of light rather than color. By spraying luminal reagent onto a suspected 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. Luminol test is extremely sensitive test. It is capable of detecting
bloodstains diluted up to 10,000 times. Luminol is known to destroy many important blood factors necessary
for the forensic characterization of blood, so its use should be limited to seeking out blood invisible to the
naked eye.

Positive Result: Luminescence or emission of light.

The principle involved in the four preliminary color test for blood. The peroxidase present in hemoglobin acts
as career 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 – This refers to an enzyme that accelerates the oxidation of several classes of organic compounds
by peroxide.
5.Luminol Test
The Confirmatory Test for Blood
The actual proof that a stain is blood consists of establishing the
presence of the characteristic of blood pigment hemoglobin or one of
its derivatives. Hemoglobin is the red coloring matter of the red blood
cells of the blood.
 1. Microscopic Test for Blood
Method of Microscopic Examination:
a. Take two small fragments of the dried blood.
b. Place each fragment on separate slides with a drop of 0.9% salt solution.
c. The slides are put in a covered dist to prevent evaporation and the preparation allowed to
stand for 1-2 hours.
d. One of the slides is examined as wet preparation.
e. The other preparation is spread evenly over the slide, allowed to dry and stained by:
1. Fix preparation in absolute methyl alcohol for 3 minutes. Stain in a 0.5% aqueous solution of eosin
for 1-3 minutes. Loffer’s methylene blue is added for 1-3 minutes. Eosin stains the red blood cells,
white methylene blue stains the nulei.
2. .Fix smear with methyl or ethyl alcohol for 3 minutes. Pour off alcohol and flood smear with
Geimsa’s stain. Stain for 15 minutes, cover to prevent evaporation, wash in water and dry.
3. Wright’s Stain – The smear is flooded with the stain and allowed to stand for a minute. Distilled
water is added until a metallic scum forms on the surface. Let stand for 3 minutes, wash with water
Visible Result:
• a.Mammalian red blood cells – circular, biconcave discs with nucleus.
Appear as characteristics non – nucleated discs. Exception is camel
and closely related animal as llama whore red blood cells are oval but
also without nucleus.
• b.Birds, fish and reptile red blood cells – larges, oval and nucleated.
• c.Amphibian red blood cells – larger than mammals, oval and
nucleated.
• d.Lamprey eel red blood cells – circular and nucleated.
 2.Microchemical Test and MicrocrystallineTest for Blood
The identification of blood can be made more specific if microchemical or microcrystalline test is
applied or performed. Takayama test and Teicmann test are the most popular ones.

The Teicmann Test – The test depends on the addition of the specific chemicals to the blood so
that characteristics crystals with hemoglobin will be formed.
Reagent: Sodium Chloride, Glacial Acetic Acid
Procedure: Place a minute fragment of the stain on a glass slide. Add a small crystal of sodium
chloride and 2 to 3 drops of acetic acid. Place cover slip and heat gently over small flame to
evaporate the acid. Cool and examine under the high power objective.
Positive Result: Dark brown rhombic crystal of haemin or haematin chloride arranged singly or in
cluster.
Limitation of the Test: The test will also give positive results for indigo – dyed fabrics. If the stain
is old or washed or is changed by chemical reagents, the crystals are not formed. The addition of
too much salt or presence or moisture in the acid or over – heating of the slide may result in
failure.
Microchemical Test MicrocrystallineTest
 2.1.Acetone – Haemin Test
The test depends on the addition of specific chemicals to the blood so that
characteristic crystal with hemoglobin will be formed.

Reagent: Acetone, Dilute Acetic Acid or Oxalic Acid

Procedure: Place dried stain on glass slide and cover with cover slip with a needle
interposed to prevent direct contact of the cover slip with the slide. Add a drop of
acetone then a drop of acetic acid.

Positive Result: Small dark, Diachronic Acicular Crystals of Acetone Haemin.

2.2.Haemochromogen Crystal Test of the
Takayama Test
A delicate test for the presence of hemoglobin. The test
depends on the addition of specific chemicals to the blood so
that characteristic crystals of hemoglobin derivatives will be
formed.
 Spectroscope Test for Blood
The most delicate and reliable test for the determination of the presence of blood in both old
and recent stains. This test is performed by means of an optical instrument known as
Spectroscope, an optical instrument for forming and examining spectra.

Procedure: Dissolved bloodstain in water or saline solution. Place in small chamber (glass) with
parallel sides so arranged that the rays of light will pass directly through it. The chamber is placed
in the spectroscope and the instrument is so adjusted that the spectrum is clearly visible.

Positive Result: Upon absorbing some of the rays from the spectrum, it produced characteristic
dark colored bands, which vary with the type of blood pigment.

Principle involved in the Spectroscopic Test is the absorption properties of translucent colored
fluids can be observed on the solar spectrum.
Precipitin Test for Blood
• Reagent: Precipitin/Antiserum
• Procedure: Scrape off bloodstain if on hard material. Powder the
scraping and extract with saline solution. If the stain is cloth, paper or
similar material, cut a small portion and the place in a test tube and
add extract with saline solution. Allow mixture to stand overnight and
centrifuge to clean the solution. Dilute with saline solution. Layer an
extract of the bloodstain on top of the human antiserum/precipitin in
a capillary tube.
Positive Result:
1.Development of a white cloudy line at the contact point of the fluids that appears immediately or within
one or two minutes.
2.Human blood, or for that matter, any protein of human origin in the extract will react specifically with anti –
bodies present in the serum as shown by the formation of cloudy ring or band at the interface of the two
liquids. Principle Involved in the Precipitin Test: When a rabbit is injected a human blood serum or whole
human blood, the precipitin that develops in its serum will react with the protein of human blood serum,
other human body fluids and other human proteins or any other protein as human origin.

Limitation of the Test: The precipitin reacts not only with blood proteins but also with other body proteins as
those on saliva, semen, mucus and other body fluids. For this reason the test does not identify specifically
human body but only a protein material from the specific animal type. In order that a conclusion of human
blood is arrived the precipitin test must be corroborated by supplementary chemical, microscopic or
spectroscopic tests. The specificity and delicacy of the precipitin reaction is great, but the reaction may be
inhibited or even destroy by a number of factors. Chemicals like acid, alkalis, alcohols, cresols, formaldehyde,
corrosive sublimate or other germicide may alter blood to such as extent that the reaction cannot be formed.
Heat had the same effect. Fluid blood loses its power may stand 150. Rust and postmortem decomposition
may react with it poorly. Old stains may be identified after long period of time.
Precipitin Test: Negative and Positive
 Definitions of Terms
1. Gene – This refers to any of the complex chemical units in the chromosomes by
which heredity characters are transmitted that occur in pair that is a factor
occurring singly in a garmete. There are two genes or factors called gene A and
gene B, These are found in the chromosomes. Since chromosomes go on pair,
each of which carries or fails to carry one of these genes and an individual’s
genetic constitution may be represented by AA, AB, BB, BO, AO, OO which are
called genotypes, where O represents the absence in the chromosomes of
either the A or B gene that is responsible for the transmission of hereditary
characteristics.
2. Chromosomes – This refers to any of the microscopic rod – shaped bodies
bearing genes responsible for the transmission of hereditary characteristics are
observed to occur in pairs.
3. Phenotypes – This refers to term used to denote the expression of the
inherited characteristics as found in the individual that is actually the blood
4.Genotype – This refers to paired genes. It is either homozygous or
heterozygous.
5.Homozygous genotype or pure genotype – This refers to paired genes that are
similar.
6.Heterozygous genotype or hybrid – This refers to paired genes that are
dissimilar or not alike.
7.Gamete – This refers to sexual cells; reproductive cell that unites with one
another to from cell that develops into a new individuals.
8.Sperm cell or microgamete – This refers to male sexual cell.
9.Egg cell or macrogamete – This refers to female sexual cell.
10.Zygote – This refers to pair of genes occurring in a gamete produced during
fertilization. The cell formed by the union of an ovum and sperm.
11.Alleles – This refers to pairs or contrasting genes, which determines the
expression of the inherited characteristics of an individual.