BLOOD AND BLOOD STAINS

What is the importance of studying blood?

• As circumstance or corroborative evidence against or in favor of the perpetrator

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

What is BLOOD?

➢ Blood has been called 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

✓ (45%) formed elements or the solid materials consisting chiefly of cells namely:
▪ Red Blood Cells or RBC (ERYTHROCYTES) around 4 – 5 million of red cell
per cc. of blood.
▪ White Blood Cells or WBC (LEUKOCYTES)
▪ Blood Platelets (THROMBOCYTES)
✓ (55%) PLASMA – The fluid or liquid portion of blood where the cells are
suspended. It is principally composed of:
▪ Water ---- 90%
▪ 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 is 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 is 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

Blood is difficult to be searched, the collection, preservation, packing and

transportation of specimen suspected to contain blood is another. 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.

Blood Collection

FLUID BLOOD are usually collected from victims of crimes of violence, parent and
child in case of disputed parentage.

DRIED BLOOD OR BLOOD STAINS are collected from smooth surface like walls,
finished floors, table tops, hard surface like axe, hammer, knives, stones, crowbars,
glazed surface like glass, tiles, automobiles, bulky objects like blackboard, linoleum
sheets, doors, window frames, clothing, and blood absorbed by the soil

Blood Examination

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 humans.

THE PRELIMINARY TEST FOR BLOOD (COLOR TEST)

▪ Benzidine Test or Benzidine Color Test

▪ Phenolphthalein Test (also known as Kastle – Meyer Test)
▪ Guaiacum Test (Van Deen Test, Day’s or Schonbein’s Test)
▪ Leucomalachite Green Test
▪ Luminous Test
Benzidine Test

This is 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.
PROCEDURE: Place a small fragment/portion of the stained material on a filter paper.
Add a drop of benzidine solution and then drop of hydrogen peroxide solution.
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 produces faint coloration.

Phenolphtalein Test

This is 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.
PROCEDURE: Place a small fragment/portion of the stained materia on a filter paper.
Add a drop of phenolphthalein solution and then a drop of hydrogen peroxide solution.
POSITIVE RESULT: Rose color develops or deep pink color or permanganate coor.
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.
PROCEDURE: Place a small piece of the stained fabric on porcelain dish. Soak with
fresh tincture of guaiac. Add a few drops of hydrogen peroxide.
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

This is a 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.
PROCEDURE: A small piece of the stained fabric on a filter paper. Add a drop of
leucomalachite green solution and after a few seconds add drop of 3% hydrogen
peroxide.
POSITIVE RESULTS: Malachite green or bluish green

Take Note – The principle involved in blood testing is that 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.

Hemoglobin is the red coloring matter of the red blood cells of the blood.

Luminous Test

It is an important presumptive identification test for blood. The reaction of luminol with
blood results 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 (3) confirmatory tests for blood are:

Microscopic Test - Useful for the demonstration 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.

Take Note: The Mammalian red blood cells are 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 – also known as Microcrystalline test which include Teichmann

Hemin Reaction/Teichman Test/Haemin Crystal Test, Haemochromogen crystal Test
or Takayama Test, Acetone-Haemin Test. One of the two popular microchemical test
is the Takayama Test, a delicate test for the presence of hemoglobin.
PROCEDURE: Place a small piece of suspected material on a glass slide. Add 2 – 3
drops of Takayama reagent. Cover with glass slip.
POSITIVE RESULTS: Large rhombic crystals of a salmon pink color arranged in
clusters, sheaves and other forms that appears 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.

Spectroscope Test – is 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.

THE PRECIPITIN TEST

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

Reagent: Precipitin/antiserum

PROCEDURE: Scrape off blood stain if on hard material. Powder the scrapings and
exact with saline solution. If the stain is cloth, paper or similar material, cut a small
portion and then place in a test tube and add extract with saline solution. Allow mixture
to stand overnight. 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: 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: The precipitin reacts not only with blood proteins but also with other
body proteins as those as saliva, semen, mucus and other body fluids.

THE BLOOD GROUPING AND BLOOD TESTING

The Four Blood Groups

Group “O”
Group “A”
Group “B”
Group “AB”
Inheritance of Blood Groups

Knowledge of genetics will make it easier to understand the principle involved in the
inheritance of blood groups. The inheritance of blood group is predetermined by the
presence and absence of two facts or GENES called Gene A and Gene B.

GENES - any of the complex chemical units in the chromosomes by which hereditary
characters are transmitted, responsible for the transmission of hereditary
characteristics. They occur 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.

PHENOTYPES – the term used to denote the expression of the inherited characteristic
as found in the individual. Actually the blood groups

GENOTYPES - Are paired genes.

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.
▪ Determination of whether a man accused of fathering a child out of wedlock
could or could not be its parent.
▪ Determination of whether a child born of a married woman could or could not
have been fathered by her legal spouse.
▪ 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.
▪ 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.