BLOOD DETERMINATION
Forensic Identification of Blood
Categories of identification tests:
I. Presumptive or preliminary test
o Used for screening specimens that might
contain the substance or material of interest
o Both false positive and false negative results
may be obtained
o Presumptive blood tests are used to screen
evidence for the possible presence of blood
o Most are color tests and are based on the
peroxidase-like activity of hemoglobin
o Peroxidase catalyzes the following reaction
o Reduced Dye + peroxide --> Oxidized dye +
water
o The presence of hemoglobin catalyzes the
reaction, forming a colored dye product
o Positive presumptive tests proves that blood
is present but does not identify the type
o Blood Evidences
a) Wet specimen
b) Dry specimen
 Moisten clean and sterile white cloth
with distilled water
 Rub gently on the alleged blood
stain
 Air dry cloth swab
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
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o
1) KASTLE-MEYER TEST/PHENOLPHTHALEIN TEST
 The principle of the Kastle Meyer test is
based on the resultant reaction in which
phenolphthalein—a colorless compound,
catalyzed by heme with hydrogen peroxide
as the oxidant.
 The resultant reaction consists of reduced
phenolphthalein (phenolphthalin) in an
alkaline solution to produce phenolphthalein
of bright pink color.
 The pink color of phenolphthalein is carried
out only in alkaline conditions.
A color test for blood is based on the action
of the peroxidase enzyme in RBCs.
Reagents are chosen based on their reaction
to activate the peroxidase with a
corresponding color as the sign of blood.
The following is the general reaction for the
presumptive color test of blood:
 H2O2 + reduced reagent ↔ H2O + oxidized
reagent (color production)
 It is based on the blood oxidizing potential
which becomes apparent through a change
in color.
Basis of the tests:
a) Benzidine and Kastle-Meyer color tests
are based on the observation that
haemoglobin possesses peroxidase-like
activity.
b) Hemoglobin: a RBC protein that
transports oxygen in the bloodstream; it
is responsible for the red color of blood
c) Peroxidases are enzymes that accelerate
the oxidation of several classes of
organic compounds when combined
with peroxides.
1) KASTLE-MEYER TEST/PHENOLPHTHALEIN TEST
 A positive result from the KASTLE-MEYER
color test is highly indicative of blood.
 A presumed blood sample is first collected
with a swab.
 A drop of phenolphthalein reagent is added
to the sample, and after a few seconds, a
drop of hydrogen peroxide is applied to the
swab.
 Hemoglobin causes a deep pink color. If the
swab turns pink rapidly, it is said to test
presumptive positive for blood.
 Waiting for periods over 30 seconds will
result in most swabs turning pink naturally
as they oxidize on their own in the air.
 Fast and easy test used at the crime scene
to test for the presence of blood

The heme portion of the RBC reacts with
H2O2 to oxidize phenolphthalein and give a
pink product
2) LEUCOMALACHITE TEST
 The use of Leucomalachite as the
presumptive test was first developed by
Adler and Adler in 1904. Earlier, in their
study, they used LM green and violent. But
later, many researchers settle on the LM
green for blood screening.
 Leucomalachite green is a diphenylmethane dye
whose base form is colorless and produces a
blue-green color in the presence of blood.
 The principle of the LMG blood test uses the
leuco base form of malachite green (colorless)
that is made to react with blood samples to get
converted into its oxidized green form in an
acidic medium.
Procedure
 Using a moistened (with ethanol) cotton swab,
graze out some of the dried bloodstains.
 1-2 drops of LMG solution should be added.
 Wait for 10-20 seconds. Meanwhile, if the color
develops, the test is inconclusive.
 If no color change is seen, add 3% of hydrogen
peroxide to the cotton swab.
3. ADLER/ BENZIDINE TEST
 Adler test (benzidine) test is considered to be
one of the oldest tests that is used for screening
whether the given sample is blood or not.
 The test was first developed by Adler (so as on
his name) in 1904.
 The test was used for half the decade, till in
1946, it opted to be carcinogenic. And in 1976,
it was completely banned as one of the
presumptive tests for blood in the US.

The principle is based on the reaction between
the benzidine substrate as the coloring agent
that flourishes the dark blue color on reacting
with blood in presence of hydrogen peroxide.
Procedure for benzidine (Adler test):
 Take a small amount of blood sample in a petri
dish.
 Add a few drops of benzidine reagent to the
sample.
 Add 3% hydrogen peroxide
 Wait up to 20 seconds.
 Appearance of dark blue color as the indication
B. Presumptive Chemiluminescent Test for Blood Stains
 A chemiluminescent test is a light-producing
reaction. In these reactions, light is produced as
the byproduct of the electronically excited state
and their electronic transition.
 In this, chemicals react to form an excited
unstable intermediate that breaks down,
releasing energy in the form of photons of light,
and finally reaches its ground state.
 Most common presumptive test that is used for
blood detection at the crime scene is Luminol.
 Luminol is 3-aminophthalhydrazide, was first
synthesized in A. Schmitz, Heidelberg, in 1902.
However, it was first observed by W. Lommel
(Germany) in 1928.
 As per forensic point of view, it was Walter
Specht (in 1937) who introduced the use of
luminol at crime scenes.
 Principle and Reaction:
o Luminol reacts with blood and hydrogen
peroxide, producing blue-white to
yellowish-green light under very low
light conditions (usually dark).
o
o
o
Luminol for large area of blood is a
presumptive test
Luminol is mixed with hydrogen
peroxide
During the reaction with hydrogen
peroxide, the luminol is pxidized and
light energy is released; therefore. The
luminol test is viewed in a darkened
area.
EXAMPLE:
o Cattle: A, B,C,F,J,L,M,S,Z
o Felines: A, B, AB
o horses: A,C, D, K, P, Q, U, T
o Dog: DEA
o Humans: A, B, O, AB
PRECIPITATION PRINCIPLE
Different Precipitation Techniques
A. In a liquid medium
B. In a gel medium
Terms:
A. “Diffusion”- usually refers to the number of
reactants moving
B. “Dimension” – usually refers to the number of
direction of the movement
II. Confirmatory test
o Are tests which are entirely specific for the
substance or material for which it is
intended
o A positive confirmatory test is interpreted as
an unequivocal demonstration that the
specimen contains the substance or material
Forensic Identification of Blood
Confirmatory Tests for Blood: usually, Ab are used to
detect the antigen
o Current immunological tests use antibodies
specific for human hemoglobin, thus combining
the confirmatory test for blood with a human
species test
 Ring test
 RID test
 Oakley and Fulthorpe
 Ouchterlony
Species Determination
Tests must be done on blood specimens to determine
the species of origin
o Species origin tests are done using
immunological methods which involve the
interaction of antigens and antibodies
o Specific antiserum can be used to test for the
presence of antigens in unknown specimens
o Note: some antigens are found in animals but
not in humans:
‘’
Therefore:
o Single Diffusion: only one reactant is
moving towards the other reactant
o Double Diffusion: both reactants are
diffusing/ moving towards each other
o Single Dimension: only one effective
direction of diffusion (usually up or down)
o Double Dimension: diffusion in multiple/ all
directions (usually radial)
A. IN A LIQUID MEDIUM
Interfacial test/ Ring test: Double diffusion,
single dimension
o introduced by Ascoli in 1902
o Principle: Ag and Ab diffuse toward each other
until they reach their ZOE point where
precipitation occurs.
o Procedure: Antiserum is deposited at the bottom
of the tube, overlaid with the Ag, and then
incubated (RT or 37oC).
o Result: (+) formation of a visible ring or layer of
precipitate at the interface of the reactants
o Antibody is added at the bottom while antigen
stay at the top because of density.
o It is expected that antigens are heavier
than antibodies hence, will move
downwards while antibodies will float
B. IN A GEL MEDIUM
o Principle: Soluble molecules (Ag and Ab) reacts
in agar gel or other semisolid media until they
reach their equivalence to form a stable
precipitate (band/ line)
o Gel media: agar, agarose, polyacrylamide gel
o Non- gel media: cellulose acetate
1. Single Diffusion, Single Dimension (Oudin)
o earliest precipitation technique(1946)
o Principle: Ag diffuses through the gel containing
immobilized Abs forming insoluble Ag-Ab
complexes
o Procedure: Abs are mixed with the liquid agar
& deposited at the bottom of the tube, allowed
to cool & solidify, then overlaid with Antigen.
Two or more unrelated antigens against their
homologous Antibodies may be tested.
o Application: permits determination of minimum
antigenic substances in a mixture such blood/
plasma / cell extract
o Note: single dimension = done in tube (because
it only moves vertically)
2. Single Diffusion, Double Dimension (Radial
Immuno-Diffusion)
o Principle: Ag binds the Abs present in the agar
o Result: Formation of a precipitin disc or ring
around the well
o Procedure:
a. Abs are mixed in a liquid gel on a flat surface
(petri dish).
b. Solidify agar and cut wells from the agar
c. Add antigen on the cut wells
d. allow for the reaction
Methods:
o
o
Mancini test/ endpoint method: Disc is measured
about 48-72 hours after placing the Ag on the
well.
Fahey/ McKelvey method: kinetic/ timed assay
method, disc is measured prior to its full
development (18 hours)
3. Double Diffusion, Single Dimension
(Oakley &Fulthorpe Method)
o described in 1953 by Oakley and Fulthorpe
o Principle: Both Ag and Ab both diffuse
towards each other
o Procedure:
a. Place Abs in a tube
b. overlay with neutral agar which is
allowed to solidify
c. overlay with an Ag
o Result: Formation of a precipitin band or line
through the gel at the point of equivalence
o Note:
- If Ab (reagent) is constant, Ag is of
various concentrations: the distance
from the Ag layer to the band is
proportional to the antigen
concentration.
- If Ag is constant, the greater the
concentration of the Ab, the greater the
distance of band from the Ab layer to
the band of precipitate.
o The liquid agar doesn’t mix with the liquid
reagent because they do not have the same
consistency and density.
4. Double Diffusion, Double Dimension
(Ouchterlony Method)
o developed by Elek and Ouchterlony
independently in 1948
o identifies 2 antigens or 2 samples
o Principle: Ag and Ab both diffuse and bind each
other, forming a line at the point of equivalence
o Procedure:
a. Agar containing buffered saline and a
preservative is poured on a flat glass surface
(peti dish, glass plate, microscope slide),
solidify
b. cut wells
c. Place Antigens and Abs on separate wells,
kept for several days/ weeks.
d. Allow to react to form a precipitin line at the
point of equivalence.
Note: The number of precipitin lines indicates the
minimum number of distinct antigenic substances
present in the antigenic solution.
o if both Ag and Ab are of the same MW, the line
is straight; otherwise the line tends to be
concave toward the reagent of higher MW.
o Types of Reactions: (observed when related
antigens in adjacent wells react with Ab against
various Ag determinants)
i.
Type I (identity) - fusion of bands of precipitate
ii.
Type II (nonidentity) – the precipitate lines
intersect/ cross because the samples contain no
antigenic determinant in common.
iii.
Type III (partial identity) – obtained when two
antigens compared possess common
determinants but also display antigenic
differences. It is distinguished by spur
formation.
AGGLUTINATION PRINCIPLE
Principles of Agglutination
o Agglutination is a visible manifestation of
antigen-antibody interaction; however not all
antigen-antibody interactions lead to
agglutination. Rather, agglutination occurs only
when the properties and relative concentrations
of Ag and Ab allow sufficient lattice formation.
o Carrier particles may be needed to indicate
visibly that an antigen-antibody reaction has
taken place.
o Artificial carriers: latex particles and
colloidal charcoal
o biologic carriers: RBC, bacteria
o Agglutination is a two-step process that
results in a stable lattice formation:
1. DIRECT IMMUNE AGGLUTINATION
o Aggregation of particles with the native Antigens
or the antigens that are naturally present on the
surface of particle such as RBC or bacteria.
o Applications:
a. Hemagglutination: detect antigens on RBCs by
using known antisera (Ex: ABO, Rh forward
typing)
Adhesion-Elution/Absorption Method
o Adhesion: known antibodies are made to react
or adhere to the unknown specimen
o After washing the excess or unreacted
antibodies, the specimen undergoes elution
o Elution: the process which removes the reacted
antibodies
o Known RBCs are introduced into the system to
determine which antibodies have remained.
Adhesion Stage: formation of antigen-antibody
complex where unreacted antibodies will be washed off
You can’t immediately drop the NSS or antibodies to the
cloth, only to the tube/slide, because the lattice
formation is affected by many factors. For example, to
enhance lattice formation, you have to agitate and mix.
Also, the ratio of blood to antibodies must be equal
which can’t be achieved in the cloth.
2. Detect Antibodies to RBCs by using known Antigens
(Ex: ABO reverse typing)
Elution Stage: the antigen-antibody complex will break
sensitization under 40C for 30 minutes
o
o
o
It is applied to pre-screen evidence in an
effort to identify discrete areas for ACP
testing
Many body fluids fluoresce when excited
with light at 450-nm wavelength
Semen stains have tendency to fluoresce
more intensely than most other body fluids
b. Acid phosphatase test
o Identification of an enzyme known as acid
phosphatase
o Considered as a presumptive identification
of seminal fluid because other body fluids
might also give a positive reaction
o Purple color
IDENTIFICATION OF SEMEN
o The identification of semen is important in many
cases of alleged sexual assault
o Seminal fluid is a protein-rich body fluid
origination primarily from the prostate and
seminal vesicles
o Spermatozoa, commonly referred to as “sperm”,
are the male gametes or sex cells produced in
the testis. Not all men produce spermatozoa
I.
Presumptive testing
a. Alternate light source/UV light
o Not all semen stains are visible to the naked
eye
II.
Confirmatory testing
a. Microscopic identification of spermatozoa
 Positive swabs or a small cutting from a positive
stain can be smeared onto a microscope slide
and then stained for visualization
 Nuclear fast red (red stain) and
picroindigocarmine (green stain) and are
sometimes referred to as the Christmas tree
stain
b. Protein confirmation of semen
o Prostate specific antigen (PSA)/p30
o
o
o
o
o
Value of the protein in forensic science is
that it allows semen to be identified in stains
and swabs
Male who is vasectomized or a male with a
congenital or other defect of the male
reproductive system, spermatozoa may not
be present in the semen
Electrophoretic or diffusion methods such as
crossover electrophoresis and ouctherlony
double diffusion or ELISA
Commercial kits
This can be used alone to confirm semen, or
it can be used in conjunction with the
microscopic method
FORENSIC DNA ANALYSIS