Subject FORENSIC SCIENCE
Paper No. and Title Paper No. 12: Forensic Biology and Serology
Module No. and Title Module No.14: Examination of blood
Module Tag FSC_P12_M14
FORENSIC SCIENCE PAPER No.12:Forensic Biology and Serology
MODULE No.14:Examination of Blood
TABLE OF CONTENTS
1. Learning Outcomes
2. Introduction: Blood
2.1 Function of Blood
2.2 Composition of blood
3. Examination of blood
3.1 Physical examination
3.2 Presumptive screening test
3.3 Tetraethyl Benzidine (TMB) Test
3.4 Phenolphthalein Test (Kastle-Meyer Test)
3.5 Luminol test.
3.6-Lecomalachite Green test
4. Confirmatory test of blood
4.1 Hemochromogen Crystal test(Takayama’s test)
4.2 Haemin Crystal Test(Tichmann’s test)
5. Microscopic Method
6. Spectroscopic Method
7. Some special test
7.1 Detection of species origin of blood
7.2 Precipitin Tube Method
7.3 Double Diffusion Method
7.4 Cross electrophoresis
8. Age of blood stain
9. Detection of blood group
10. Factors affecting the detection of blood group from blood stain.
11. Summary
FORENSIC SCIENCE PAPER No.12:Forensic Biology and Serology
MODULE No.14:Examination of Blood
Learning outcomes
After studying this module, you shall be able to know-
What is the blood and composition of blood
How is blood examined
What is source of blood
How will you know that examined sample is blood of human or animal
What is the age of blood stain
What is the blood group
What are the confirmatory test of blood
2. Introduction
2.1 Blood-
Blood is one of the most important physical evidence, which is frequently encountered at the
crime scene as a pool of blood, droplet, stains, etc. It can be found in almost every type of
criminal activity involving physical violence like murder, assaults, rape, etc at crime scene in
form of valuable evidence.
Blood stains are of two type-
1-Visible blood stain
2-Invisible blood stain
2.2 Component of blood-
The blood, which constitute around 1/13th of the body weight consist of medium plasma and
suspended cells like-
Source-www.netwellness.org
FORENSIC SCIENCE PAPER No.12:Forensic Biology and Serology
MODULE No.14:Examination of Blood
1. RBC (Erythrocytes) -
Red blood corpuscles containing hemoglobin are responsible for carrying oxygen from the lungs
to various parts of the body. They are formed in the bone marrow.
2. WBC (Leukocytes)-
White blood corpuscles containing antibodies that fight foreign bodies, which cause infections
and disturbs the immune system.
3. Platelets (Thrombocytes)-
Platelets are blood cells that help in blood clotting.
4. Plasma-
Plasma is the yellowish, liquid portion of the blood that contains electrolytes, nutrients, proteins
and vitamins.
2.3 Major Functions of blood are-
Transport
Maintain body temperature
Control pH (acid base balance)
Removal of toxins from the body (Excretion)
3. Examination of blood
3.1 Physical Examination-
In the natural light, the blood stains appear as brown, reddish brown stains, clot or crystals of
reddish brown color. If the stain are clear and visible then , examined under UV light ( at 230-
269nm wavelength.)
3.2 Presumptive Screening test-
The blood stain obtain from suspected area, contaminated with material should be tested for
positive blood stain. Presumptive tests produce a color reaction or release of light, in presence of
catalytic property of blood.
4
3.3 Phenolphthalein test (Kastel Meyer test)
Phenolphthalein is reduced by Zn powder in a strongly alkaline medium. If this reduced
phenolphthalein is oxidized by oxygen liberated by the action of peroxides on hydrogen per oxide
(H2O2), then a pink or purple color is obtained, if the stain of blood. The sensitivity of
phenolphthalein test is about 1:5 lakhs.
Reagent -
Stock Solution
Phenolphthalein 2.0 g
Potassium Hydroxide 20.0 g
Distilled Water 100 ml
FORENSIC SCIENCE PAPER No.12:Forensic Biology and Serology
MODULE No.14:Examination of Blood
Zinc Dust 20.0g
Working Solution:
1: Ethanol 10 ml
2: Phenolphthalein Stock 2 ml
Distilled Water, 10 ml
Ethanol 2 ml
3: 3% Hydrogen Peroxide 10 ml
Reagent Preparation:-
The reagent are formed by adding phenolphthalein (2g), potassium hydroxide (20g) distilled
water (100ml). Mix, add Zn powder, A few boiling chips and boil under reflux 2-3 hours until the
stock solution is formed. Cool and decant into a bottle containing some zinc to keep in the
reduced form. Now add ethanol (10ml), phenolphthalein stock solution (2ml), distilled water
(10ml), again ethanol (2ml), and 3% hydrogen peroxide (10ml) as working solution. Hydrogen
peroxide is used in every colour reaction or in other words, it is responsible for colour obtained in
the reaction. If the colour is obtained pink so, it confirms the presence of blood.
Procedure-
A small cutting, swab or extract of the suspected bloodstain is placed on filter paper or spot test
paper Two or three drops of ethanol are placed on the stain. Two drops of working
phenolphthalein solution are added to the stain.. After waiting to insure that no color develops at
this stage, two or three drops of 3%Hydrogen peroxide are added. An intense pink color indicates
the positive test for peroxides activity and indicates the presence of hemoglobin.
3.4 Tetra methyl Benzidine (TMB) Test-
Reagent -
Acetate Buffer
Sodium acetate 5.0g
Glacial Acetic Acid 13 ml
Distilled Water 57.0.0 ml
Working Solution
TMB solution 1.5g
Acetate Buffer 20.0 ml
Reagent Preparation-
Take 1.5gm of benzidine and 13ml of glacial acetic acid and57ml of distilled water .After
shaking benzidine solution is ready for test.
Procedure-
Place cutting or swabbing of the stain on filter paper or spot test paper. A drop of TMB Solution
is placed on the stain, followed by a drop of 3% Hydrogen Peroxide and mix with glass rod.
FORENSIC SCIENCE PAPER No.12:Forensic Biology and Serology
MODULE No.14:Examination of Blood
Appearance of immediate blue-green color is a positive test for
peroxides activity i.e indicative of presence of hemoglobin.
3.5 Luminol test-
Luminol reagent-
Sodium perborate 0.7g
3-Aminophthal hydrazide 0.1g
Sodium bicarbonate 5.0g
Reagent Preparation-
The reagent are formed by taking 0.7g of sodium perborate and 0.1g of 3-aminophthalhydrate is
mixed with 0.5g of sodium bicarbonate.
Procedure:
Take the suspected blood stain and add few amount of Luminol reagent appearance of
fluorescent color indicate positive test of blood.
3.6 Leucomalachite Green test-
Reagent-
Leucomalachite green 0.1g
Sodium perborate 3.2g
Glacial acetic acid 65%(Solution) 66ml
Distilled water 33ml
Reagent Preparation-
The reagent of Leucomalachite green test is formed by the combination of Leucomalachite
green(0.1g), sodium perborate (3.2g), and Glacial acetic acid (66ml) in 33ml distilled water.
If the color appear green, it confirms the presence of blood.
FORENSIC SCIENCE PAPER No.12:Forensic Biology and Serology
MODULE No.14:Examination of Blood
Procedure-
Add Leucomalachite green solution on the stain. If indicate green colour appears it indicate
the the presence of blood.
4. Confirmatory test of blood:
4.1 Takayama test - It is also known as Haemochromogen test.
Takayama reagent –
Saturated solution of glucose 3ml
Pyridine solution 3ml
10%NaoH (sodium hydroxide solution) 3ml
Glacial Acetic Acid 7ml
Reagent preparation-
Taken the 3ml of saturated solution of glucose,3ml of pyridine solutiona and 3ml of NaOH
solution along with 7ml of Glacial acetic acid.
Procedure:
Place the material to be tested on a microscopic slide and cover with a cover slip. Add a drop of
Takayama Reagent and allow to flow under the cover slip. Warm slide gently on a hot plate at
65°C for 10-20 seconds. Allow to cool and observe under microscope at 100X. The appearance
of pink needle shaped crystals of pyridine Haemochromogen (Pyridineferroprotoporphyrin) is
positive reaction for haeme and confirms the presence of hemoglobin.
4.2 Teichmann’s test-
Teichmann’s reagent-
KCl, KBr and KI 0.1 g each
Glacial Acetic Acid 100ml.
Reagent preparation-
For the Teichmann’s test, the reagent are formed by the combination KCl, KBr and KI at about
0.1g each in 100ml of Glacial acetic acid. The reagent react with hemoglobin and give brownish
rhombic crystal, Confirms the presence of blood.
Procedure-
Place material to be tested on a microscopic slide and cover with a cover slip. Let the reagent
flow under the cover slip. Warm the slide gently on a hot plate at 65°C for 10-20 seconds. Allow
FORENSIC SCIENCE PAPER No.12:Forensic Biology and Serology
MODULE No.14:Examination of Blood
to cool and observe under microscope at 100X. The appearance
of brown rhombohedron shaped crystals of ferroprotoporphyrin
chloride is a positive reaction for haeme.
5. Microscopic test-
Microscopic Method of examination is having paramount importance relating to identification of
blood stain-
1. Determination of the species of origin by nucleated RBC’s along with the cell structure.
2. Sex determination by examining chromatin bodies in Leucocytes.
3. Detection of blood related pathological condition.
6. Spectroscopic Method-
In this method identification of haemoglobin and its derivatives is done by characteristics
absorption band,when viewd through microscope.
Test-
A small portion of the suspected stain (as small as 2mm) is put in0.5% Potassium cyanide
solution for 15min rest and then fiterate.The filtered thus obtaind is take in 1cm. cell and passed
a U-V light in a spectrometer, the absorption observed at from 300 - 600 micron. The
absorption maximum at 422 milli micron obtained that the presence of cyclohaemoglobin.
7.Detection of species origin of blood-
The biological evidence has been identified necessarily to determine and confirm whether it is of
human origin or not. If it is non-human origin, then to which species it belongs to the species
specific proteins in the bloodstains or other body tissues may be identified with the help of
species specific antibodies-
The species specific proteins from the bloodstain or tissue are extracted in normal saline (8.5 g of
sodium chloride in one liter of distilled water) or 5% ammonia solution.
The following method are applied to detect the species of origin-
7.1 Precipitin tube method-
Take six precipitin tubes (number can vary on the number of anti sera used) and place them
vertically in a precipitin tube stand and label. Put a drop of the bloodstain/tissue extract in the
tubes. Carefully add one drop of antiserum for species origin (anti-Human serum, anti- Fowl
serum, anti-Dog serum, anti-Cow Serum, anti-Goat serum, etc.) along the walls of tube. Leave
undisturbed for 30 minutes at room temperature. Carefully examine the white ring at the interface
of two solutions. If ppt is formed, it belongs to that specific anti-serum.
7.2 Double diffusion method-
In this method both of the reactants, antigen and antibody diffuse towards each other in agar gel
plate, and when an antigen combines with its specific antibody at optimum proportions, precipitin
are formed. Fill the central well with tissue extract and peripheral wells with different anti sera
for species origin like (anti-Human serum, anti-Fowl serum, anti-Dog serum, anti-Cow Serum,
FORENSIC SCIENCE PAPER No.12:Forensic Biology and Serology
MODULE No.14:Examination of Blood
anti-Goat serum, etc.). Cover the Petri dish and keep gel in a
moist chamber for overnight. Examine the gel for the presence
of precipitin band form
7.3 Crossover Electrophoresis –
In a buffered gel, the stain extract (antigen) is placed in the cathodic well and the anti serum in
the anodic one. When electric current is applied the globulin antibodies migrate catholically
because of electro endosmosis. The other serum proteins migrate anodically.
A precipitin reaction occur midway between the paired well , When an antigen
combines with its specific antibody.
Fill the cathodic side well of the pair with stain extract and anodic well with species
specific antiserum (anti-Human serum, anti-Fowl serum, anti-Dog serum, anti-Cow
Serum, anti-Goat serum, etc.).
Place the slide on the electrophoresis chamber. The stain extract should be nearest to the
cathod and the antiserum nearest to the anode and Connect the gel to tank buffer
chambers by two pieces of filter papers on each side. Run electrophoresis for 20 minutes
at 150 volts. Record the conditions.
After that electrophoresis, switch off the power supply and remove the slide and then
observe the slide with the aid of a lamp. A fine white line of precipitate between a pair of
wells is a positive reaction.
8. Age of blood stain-
Age of blood stain can be calculated by colour and nature of the stain –
In Fresh blood stain observed bright red in colour ,moist and sticky. Within 24 hours, The stain
turns reddish brown in colour. After 24 hours, The stain turns dark brown and finally black.
9. Detection of blood group-
After determining the origin of blood. It is also examined by prosecution to mention blood group.
The modern serological techniques have divided blood in two three constituent classes for the
discrimination of human blood,A,B and O.
9.1 The blood grouping and typing antigen-
Although several type of antigen have been discovered, in so for as dried blood stain are
concerned, only ABO,MN and Rh antigenic system have been found of practical utility in the
crime work.
9.2 The Polymorphic Enzymes-
These enzyme are located in red blood cells,which are responsible to catalyze biochemical
reactions in body. The enzyme which have same function to perform .but differ in physic-
chemical properties are called as isoenzymes. Many such isoenzyme have been identified in dried
blood stains using electrophoresis techniques. Few example of these isoenzyme are
phosphoglucomutase (PGM), Adenylate kinase (AK), Erythrocyte acid phosphatase (EAP) and
esterase D(EAD).
PGM system which has three common varients -
(a) PGM-1
FORENSIC SCIENCE PAPER No.12:Forensic Biology and Serology
MODULE No.14:Examination of Blood
(b) PGM2-1
(c) PGM-2 have been found very stable.
9.3 Rh system-
This system is however not found useful in the analysis of dried blood stain .It is more often used
in disputed paternity cases in which whole blood analysis is done.
10. Factor affecting the detection of blood group from bloodstain -
Following factors are responsible detection of blood group from stains
1. Putrefaction
2.Thermal condition.
11. Summary .
The blood is important evidence found at crime scene ,that leads to further investigation
of various crimes
Blood examination has important role in solving suicidal ,accidental and
homicidal cases.
Various test are performed to identify and confirms blood stain of human or animal
origin.
Identification of paternity and maternity cases by blood grouping.
Species and origin of blood detection given indication of various animals.
Determination of age of blood stains.
FORENSIC SCIENCE PAPER No.12:Forensic Biology and Serology
MODULE No.14:Examination of Blood
