BLOOD STAINS

EXAMINATION

BY
DR AISHWARYA
EXAMINATION OF BLOOD STAINS

 Examination of blood/bloodstains consist of following

steps:
a. Is it bloodstain?
b. If blood, whether human or animal? (What is species?)
c. If human, then-
- Age of stain,
- Sex,
- Source,
- Antemortem/postmortem,
- Blood group
 Distribution of pattern of bloodstains
 BLOOD STAIN

 Bloodstains may resemble rust stains or

pan stains.
 Identification of blood in stain is based
on presence of:
- Blood cells — RBCs, WBCs, platelets
- Hemoglobin and its derivatives
- Serum proteins
 It is done by screening and
confirmatory tests
 SCREENING TESTS

 Screening tests are based upon the principle of presence of enzyme

peroxidase in RBC’s.
 Following are chemical screening tests.
1. Benzidine test
2. Phenolphthalein test (Kastle-Meyer test)
3. Leucomalachite green test
4. Ortho tolidine test (Kohn & O’Kelly test)
5. Luminal test
Advantages-
 Screening tests are sensitive tests
 Less time is required and are economical
 If screening tests are negative, then no need to proceed for confirmatory
tests.
Disadvantages:
 These are only screening tests and are not confirmatory
 Tests can be positive with any organic substance, which contains
peroxidase enzyme example sputum, pus, body fluid, green vegetable,
apple, potato, onion, pan etc. So false positive results may be obtained.
 Benzidine is known carcinogen agent.
 Benzidine Test

Principle:
 Benzidine substrate acts as
the coloring agent that
flourishes the dark blue color
on reacting with blood in
presence of hydrogen
peroxide.
Phenolphthalein test (Kastle-Meyer
test)

Principle:
 Phenolphthalein is colourless
when it's reduced (has
electrons), and pink when it's
oxidized with hemoglobin (no
electrons)
 Leucomalachite green test

Principle:
 Leuco base form of malachite
green (colorless) react with
blood samples & converts into
its oxidized green form in an
acidic medium.
Ortho tolidine test (Kohn & O’Kelly
test)

 A test for the presence of hemoglobin in feces or urine.

 The O-toluidine can be in solution, in a tablet or impregnated onto
absorbent paper.
 A positive test is the development of a green to blue color.
 Luminal test

Principle:
 Luminol reacts with blood and
hydrogen peroxide, producing blue-
white to yellowish-green light under
very low light conditions (usually
dark).
 CONFIRMATORY TESTS

 These are:
1. Crystal tests, e.g. Techiman test, Takayama test
2. Microscopic examination
3. Spectroscopic examination
4. Electrophoresis
5. Chromatography
 Crystal Tests

 Teichmann test (Hemin crystal test): stain

extract and few crystals of sodium chloride are
taken and heated with glacial acetic acid. If stain
happened to be blood, dark brown rhomboid
shaped crystals will be formed as visible under
microscope.
 Takayama test (Hemochromogen crystal
test): when bloodstain extract is heated with
Takayama reagent (Pyridin + NaOH + glucose &
distilled water), pink feathery crystals will be
visible under microscope.
 Microscopic Examination

 Microscopic examination can be done by wet film or stained

smears with Leishman’s stain.
 Presence of intact red blood cells confirmed presence of blood
 Human- RBCs are circular, biconcave, non-nucleated cells with
a diameter of 7.2 μ.
 Mammalians- RBCs are circular, biconcave and nonnucleated
cells except that of camels.
 In camel- RBCs are oval, biconvex and non-nucleated.
 In birds, fish, amphibians and reptiles- RBCs are oval, biconvex
and nucleated cells.
 Spectroscopic Test

 The spectroscopic test is one of the confirmatory

test for blood, its sensitivity 1:5,000
 If the sample contains fresh oxygenated blood
(oxyhemoglobin), 2 dark absorption bands will be
seen
 If stain is old or has been exposed to any extent,
methemoglobin will have been formed, a darker
pigment formed when blood is decomposing
 Carboxyhemoglobin has a spectrum similar to
oxyhemoglobin, which remains unchanged after
addition of ammonium sulphide which reduces
oxyhemoglobin.
 DETECTION OF SPICES ORIGIN

 A wide variety of tests are available for the determination of species

origin of an identified bloodstain, and most use immunoprecipitation to
effect a result.
Electrophoretic Methods
 2 methods are usually used for identifying bloodstains:
 Separation and identification of hemoglobin by electrophoresis
 Separation and identification of serum proteins by
immunoelectrophoresis
Precipitin Methods
 If host animal (e.g. a rabbit) is inoculated with
a human serum protein, the immune system
of the rabbit will normally recognize the
protein as foreign and produce antibodies
(globulins) against it.
 Harvesting the antibodies provides an
antiserum to the protein (antigen) and when a
sample of the antiserum and the antigen are
brought in contact, a precipitin reaction
normally occurs.
 AGE OF BLOOD STAIN

 Age of a bloodstain can be known by:

a. Change in color, i.e. gross examination: fresh stains appear red and
sticky. As age advances, it turns brown due to oxidation of hemoglobin
to methemoglobin.
b. Color changes measured on colorimeter or spectrophotometer
c. Immunoelectrophoresis can be used to determine the age of
bloodstains. There is gradual disappearance of beta-globulins and
gamma-globulins with increase in the age of blood stains
 SEX DETERMINATION

Sex from bloodstains can be identified by

 Leishman stained blood films — for presence of Davidson body
 Demonstration of Y chromosome on fluorescence microscopy
 DNA analysis
 SOURCES OF BLOOD

 Arterial blood
 Copious
 Bright red in colour
 Spurting
 Venous blood
 Dark red
 Oozes out gradually
ANTE MORTEM / POST MORTEM
BLOOD

 Ante mortem
 Due to presence of fibrin blood effused during life
can be peeled off in scales upon drying
 Post mortem
 Blood flowed after death tend to break into a
powder upon drying
 PATTERN OF BLOOD STAINS

1. Drops on a horizontal surface.

2. Splashes, from blood flying through the air
and hitting a surface at an angle.
3. Pools around the body, which can show if
it's been dragged.
4. Spurts from a major artery or vein.
5. Smears left by movement of a bleeding
person.
6. Trails, either in form of smears when a
bleeding body is dragged, or in droplets
when it is carried.
Categories of Blood Stain

1. Passive (dripping)
2. Transfer (smearing)
3. Projected
 Occur in shootings, trauma from blunt weapons, hacking, or
slashing attacks.
 Blood Spatter
Passive Spatter Projectile Spatter Contact Spatter

Passive Spatter: Projectile Spatter: Smearing:

Blood that drips because Blood that drops as a This includes marks
of gravity and as just an result of an external which have been left as
after effect of the impulse is called imprints of something
violence is called the projectile spatter. This drenched in blood
passive spatter. includes blood coming in contact with a
This can range from projecting from various target area.
isolated drops of blood to wounds as a direct
stagnated pools. result of the violence.