Forensic Serology
Forensic Serology
Forensic Serology
Paper No. and Title PAPER No. 12: Forensic Biology and Serology
MODULE No. 2: Forensic Biology and Serology-
Module No. and Title
Definitions and Concepts
Module Tag FSC_P12_M2
1. Learning Outcomes
2. Introduction
3. Blood
3.1. Composition
4. Semen
4.1. Nature
4.2. Composition
4.3. Locating semen
4.4. Examination of Semen stains
5. Saliva
6. Urine
7. Faeces
8. Sweat
9. Nasal Secretions
10. Tears and Human Milk
11. Hair
11.1. Identification and Comparison of Hair
12. Summary
2. Introduction
Forensic biology and serology is a branch of forensic science which deals with biological
evidences and their examination. In the examination of crimes such as murder, robbery, rape
etc. the examination of biological materials play an important role in connecting the criminal
with the crime. Such biological specimens may be in the form of body fluids, stains or other
material. These materials are:
1. Blood
2. Semen
3. Saliva
4. Urine
5. Faecal matter
6. Milk
7. Hair.
The most important evidences of body fluids are blood and semen from personal
identification point of view.
3. Blood
3.1 Composition
Blood is a complex viscous red fluid with a pH of about 7.4. It is mainly composed of two
parts: Cells and plasma. The liquid part is called plasma and the solids are red cells
(erythrocytes), white cells (leukocytes) and thrombocytes (platelets). The cells are also
known as corpuscles. When the blood flows out of the body a part separates out as blood
clots consisting of blood cells, discs and fibrin. Fibrin comes out of plasma and is responsible
for clotting of blood. The liquid left is called serum. It is the plasma without fibrinogen which
turns into fibrin.
There are about five million red cells per micro litre of blood, each about ten micron in
diameter. The red cells are continuously being produced and destroyed in the body. The rate
of destruction is about ten billion cells per hour in a normal healthy adult.
b) Haemoglobin:
The colouring substance of the red blood cells is haemoglobin. The substance is of great
importance to the body functions as it is the carrier of oxygen, injected medicines, salts and
food to various body tissues. It is useful in blood examinations.
c) White cells:
The number of white cells is about four to eleven thousand cells per micro litre. They resist
the attack of diseases.
d) Platelets:
The number of platelets is about five times that of white cells. They facilitate blood clotting.
e) Serum:
Plasma and serum are complex mixtures of proteins, minerals and organic compounds
dissolved in water.
1) Cell structure
Since blood is composed of red and white cells, platelets and plasma. The cells have definite
shapes and sizes which in different species. For example, human blood cells have
characteristic non-nucleated discs with a diameter of about 0.08mm. The identification of cell
structure is possible when the blood is fresh and moist usually less than 1 day old. When the
blood has dried, it is not always possible to completely regenerate the blood cells. The study
of cells is made through microscopy.
2) Enzymatic activity
Blood contains heme group in haemoglobin. It behaves like peroxidase. It catalyses the
liberation of oxygen from oxygen rich compounds like hydrogen peroxide or sodium
perborate. The oxygen is used to carry out certain color reactions. The common color
reactions for blood detection are benzidine, phenolphthalein, leucomalachite green and
luminol reactions.
Other enzymes have been utilized in electrophoretic techniques. When they are subjected to
an electric field due to some polarity, they move towards opposite polarity. The rate of
movement varies with their structure, weight and electric charge. The pattern of separation is
characteristic of blood.
3) Spectrophotometry
Haemoglobin on treatment with acids, alkalis, reducing agents or oxidizing agents gives a
variety of products which have characteristics absorption spectra. They help to identify the
blood. The absorption bands are seen in length which are characteristic of blood.
4) Visual examination
It is useful to determine whether the given stain is of blood or not. Ordinarily, there is no
difficulty in fresh but old stains may not be identifiable. The examination permits:
Bloodstains from the height of upto 50 centimetres are round sharp edges.
Bloodstains from a height of 50 to 150 centimetres have small spike like
projections along the edges.
Bloodstains fallen from a height over 150 centimetres have corrugated edges.
5. From the positions of the stains, the movement of the victim and the culprit can be
determined. If the culprit ran away bleeding, he can be tracked through blood stains.
6. The degree of fluidity, dryness and changes in color permits rough estimation of age
of the stains.
7. Gross foreign matter like hair, flesh, bones in the blood may identify the site of injury.
5) UV and IR Examination
Ultraviolet or infrared rays reveal washed or invisible bloodstains from clothes, furniture,
earth, doors etc. They also reveal stains on coloured garments or on painted surfaces. They
help to reveal minute blood traces.
6) Microscopy
7) Spectroscopy
Spectroscopic examination of blood is very useful as well as convenient. The test is usually
carried out microscopically. The blood haemoglobin is changed in two or three forms on the
slide itself and characteristic absorption are observed. Usually alkali hematin and
cyanhaemochromogen are studied for their characteristic spectra.
Ascending paper chromatography using acetic acid, methanol and water solvent system has
been employed to study the Rf values.
9) Electrophoresis
It is used for separation of various enzyme systems. It is being adopted to study the body
proteins. This technique is becoming important to distinguish between blood samples.
These tests are the first series of tests employed after visual study of stains. If a stain gives
positive color reactions in any of the two color tests, the stain is possibly a bloodstain. If it
fails to give color reactions in all probability it is not a bloodstain or the same cannot be
established.
Benzidine (0.1g) and dry sodium perborate (0.1g) are dissolved in glacial acetic acid (10ml)
and sprayed over the stain. A strong blue color indicates blood.
The reagent is prepared by dissolving leucomalachite green (0.1g), sodium perborate (0.3g)
in 65% glacial acetic acid. The reagent is applied to the stain. Intense green color indicates
blood.
Phenolphthalein (1g) is reduced and dissolved in acetic acid (100ml). Sodium perborate
(1.4g) is dissolved in the solution and applied to the blood stain. Pink coloration indicates
blood.
Luminol is a chemical which has been used to locate the bloodstains. The articles suspected
to bear stains is sprayed with luminol. It reacts with blood to give fluorescence. The
bloodstains are thus made visible. Even decomposed blood reacts with the reagent. The
reagent does not interfere with subsequent blood tests. It is prepared by dissolving sodium
perborate (0.7g) in water (100ml) and adding 3- arninophthalhydrazide (0.1g) and sodium
carbonate (5.0g) to the solution. The solution is sprayed upon the article with glass sprayers
in a dark room. Blood gives strong luminescence.
Two crystal tests are commonly employed. They are specific for blood but they are not
sensitive. They often fail if the conditions are not rigidly controlled or if the blood is
disintegrated or contaminated.
Take a dry crust or smear of blood on a slide. Put a drop of potassium iodide, bromide or
chloride (0.1gm) solution in 100ml glacial acetic acid over the blood and cover it with a
cover slip. Warm the slide gently till it gives out bubbles. Typically haemin crystals are
observed under microscope. Heating may have to be repeated a number of times.
It is also performed similarly. The reagent is prepared from one volume of glucose solution
(10%), one volume of potassium hydroxide solution (10%) and two volumes of pyridine
which are dissolved in six volumes of water.
The reaction is performed to identify the species of the origin of the bloodstains. It is very
delicate test and requires only small amounts of blood. A dilute blood solution is used for
precipitin reaction. The antisera are not diluted. The blood solution and antisera should be
free from turbidity and contamination.
4. Semen
4.1 Nature
Semen is found in liquid form, smears or stains or it may be found in vagina, anus or rectum.
Fresh semen is a gel like fluid, which liquefies on exposure to atmosphere. A normal
ejaculation is about 3.5ml, containing about thirty million sperms. The dry weight is about
seven per cent of the liquid weight. The sperm has definite morphological structure. Its
identification in a stain establishes the presence of semen.
The shape and size of human spermatozoon is characteristic. But the morphology alone does
not permit individualization. Semen of a person does not contain any spermatozoon then it is
called as aspermic semen.
4.2 Composition
It is a complex mixture of organic and inorganic compounds. Important constituents of semen
from the identification point of view are proteins including enzymes, blood group factors,
choline, fructose, citric acid, uric acid and zinc. The composition varies from individual to
individual.
Enzyme, acid phosphatase found in the semen is in concentration which are significantly
higher than those found in other body fluids. Acid phosphatase offers a very delicate test for
the identification and location of semen stains though positive identification of semen are not
based upon the acid phosphatase test alone. Choline in semen is used to get crystal tests.
Fructose, citric acid and zinc are more or less absent in other body fluids and hence their
detection in semen should permit its identification but these substances have not been utilized
to any appreciable extent so far.
The suspected place or article is examined visually. A semen stain, if fresh is colourless or
pale yellow. It gives its characteristic smell. A freshly dried stain on wetting also gives the
characteristic odour. The stain becomes stiff and rough to feel on drying. The stiffness does
not wear off unless it is rubbed.
The surface or the article suspected to bear seminal stains when examined under ultraviolet
rays in darkness usually gives fluorescence. Some other substances also give fluorescence
while some other mask it. Therefore the technique though useful in most of the cases is not
infallible.
The fluorescence of semen depends upon the quantity and freshness of the semen. Therefore,
even when a stain shows weak fluorescence, it should be further explored.
The presence of acid phosphatase in semen helps to search large area and garments for semen
stains. The method is:
Large filter paper sheets are dipped in saline. Excess of the solution is removed and the
garments to be examined is pressed against the paper or vice versa. The outline of the piece
of cloth is marked on the paper to facilitate location of the semen. The filter paper sheet is
then sprayed with acid phosphatase reagent. Color spots indicate the location and size of the
seminal stains.
Chemical tests
The following chemical test are performed for detection of semen:
Barberio’s test:
A small amount of semen is taken and treated with a saturated aqueous solution of picric
acid. Spermine picrate crystals with characteristic structure, separate out.
Florence test:
A small amount of semen is treated with iodine in potassium iodide solution. Characteristic
crystals of choline iodide are formed.
The mixture of various reagents is taken and sprayed over a wet filter paper, previously
pressed against the suspected place bearing semen stains. Development of red purple color
indicates semen, the technique is extremely sensitive and several years old stains can thus be
detected. This method is however not specific. Recently modifications had been made which
claim to make it more specific.
Morphology of spermatozoon:
The identification of sperms in a stain is considered positive proof of the seminal nature of
the stain. Sperm is a unicellular organism with an oval head attached to a comparatively long
tail. If head and tail of a spermatozoon are found intact it is not difficult to identify the stain.
Intact sperm is often not available in a dried stain.
The sperms may be identified with or without staining. But staining helps:
Ordinarily double staining with haematoxylin and eosin is done. In this way the nose
is stained in pink and the rest of the head is stained purple. This differential staining
distinguishes the sperm from any other organism. Head alone can identify the stain.
Epithelial cells and leucocytes in excess mask the spermatozoon the difficulty is
overcome by staining the spermatozoon with malachite green and eosin. The
interference is considerably reduced.
Biological tests
Precipitin reaction with anti-human semen serum and specific blood group anti sera are
employed. The former determines whether it is human semen and the latter determines the
blood group of the secretor.
The techniques employed in are similar to those applied for blood grouping
The semen is a gel like mass after ejaculation. It liquefies on standing. On drying, it slowly
changes to opaque white mass with rough feel. After two or three weeks the color changes to
pale yellow and then to brown.
Sperms in fresh semen are complete with head and tail. On drying and with passage of time it
begins to disintegrate, the tail separates out from the head. The head is also affected. The
sharp division in differential staining slowly appears.
Survival of sperms:
The sperms remain motile only for a few hours under normal conditions. The motility seldom
continues after twenty four hours. But if the semen is frozen, the sperms may remain alive for
long periods. It shows motility when the sample is brought to normal temperature.
In humid and warm climate, the sperms get destroyed in a short while. In dry and cool
climate the dead sperms may be identified even after months.
In vaginal swabs of a living person, sperms may be detected upto about five to ten days,
though the number of sperms detected goes on decreasing with passage of time.
In dead persons, detection of sperms depends upon the condition under which the dead body
remained. It may be detected in unputrefied bodies even after a few months. But if the body
has putrefied the sperms are also destroyed.
Saliva stains may be found at the scene, on handkerchief, on discarded cigarette stubs, spittal,
on cups, tumblers, bottles on postage stamps or envelopes or even tooth picks or they may be
found on a piece of cloth used as gag.
Saliva contains an enzyme (ptyalin) which when added to starch, hydrolyses it. Saliva extract,
therefore, when added to starch inhibits its color reaction with iodine.
Saliva of secretors contains blood group substances and can be grouped. The saliva on
cigarette stubs is often in criminal investigation. DNA profiling of the saliva stains has
enhanced the evidential value of the saliva stains and has brought it at par with the other body
fluids like that of blood and semen.
Food material mixed in saliva may interfere with blood grouping. Saliva does not give
specific precipitin test.
6. Urine
Urine is identified from the comparatively large amounts of urea in it. The stain located with
ultraviolet rays. It is then extracted with water and tested for urea. Urine stains can now be
individualized through DNA profiling.
7. Faeces
Faeces are involved in cases of sodomy and bestiality. The stains are found on the
undergarments, on the flies of the trousers and on flaps of the shirt of the active agent.
Faeces are identified microscopically. Bacterial studies of the faeces may also lead towards
individualization but the same has not been exploited as a routine at present.
Sweat stains are involved only in the case when some garments are left at the scene of crime
or where the culprit has perspired profusely and left sweat stains. The composition of sweat is
similar to urine. However, if the two substances are warmed, the odour is characteristically
different. Paper and gas chromatography techniques hold bright future for identification of
sweat stains. Sweat stains of secretors can be blood grouped. DNA profiling of the stains is
possible.
9. Nasal Secretions
Nasal secretions is similar in chemical composition to saliva. They can be blood grouped, if
they belong to secretors. They are liable to DNA profiling.
Tears and human milk stains likewise permit their identification. Blood grouping of the same
is possible if they have come from the secretors. DNA profile can be developed for
individualization.
11. Hair
Hair is one of the common and important physical evidence encountered in a crime scene.
Individualization of human hair i.e. whether it is from head or body. Forceful removal of hair
may have blood or skin with the root. In such cases DNA typing can be done. There have
been advancement in the DNA typing technology. Earlier only hair strand with root attached
could be used for typing but now with advanced technology hair without root can also be
used if there are enough number of hair strands.
Examination of the hair evidence found at crime scene is the first step which should be
performed. Hair should be examined to establish the origin of the hair i.e whether it is human
hair or animal hair. Comparison of hair with victims and suspects should be done in case of
human hair.
Medullary index
Scale structure
Medullary shape
As forensic evidence, hair is used to compare and determine whether the hair recovered at
crime is of same origin as that of suspect or not. Scalp or pubic hair are most commonly
found at crime scene.
Color, length and diameter of the hair are the most important characteristics that should be
matched while comparing hair samples. Presence or absence of medulla, its distribution,
shape and color intensity of pigment granules found in the cortex are also important
characteristics for comparison of hair. Hair grows at an average of 1 centimetre per month.
Following features are observed and compared for identification through hair:
Color of hair
Area of origin
Presence of micro residual material
Unusual appearance of hair
Pigments
Color of Hair
The general color of human hair varies from black, blackish brown or reddish brown. In
absence of pigmentation they turn grey and then eventually white. Dyed or bleached hair can
be predicted by examining under microscope. Time passed since colouring can also be
estimated if growth of hair since hair was coloured or bleached is present. The cortex is
coloured throughout when dyed or coloured. Yellowish tint is observed on bleaching hair due
to removal of pigments from hair. Granules are present in naturally coloured hair. The texture
of naturally coloured hair is similar to a picture coloured by a crayon whereas smooth
uniform color is displayed in case of artificially dyes or coloured hair.
Pigments
Various pigments are present in hair. One of the pigment is melanin which is responsible for
color of the hair. Presence of large amount of eumelanin (one of the types of melanin) results
in dark black hair. In case of red hair, pigment called pheomelanin is present. According to
the types of pigments present and its distribution, the comparison between two specimens can
be done. The pigment distribution and internal structure can be helpful in the identification
between species.
Identification of Race
Through the gross characteristics of hair, a person’s identification race can be predicted. A
cross section of it can show the structure, diameter, the thickness of cuticle, pigmentation,
hardness of hair etc. which can be helpful in identification. For example, In Negroid race, the
pigmentation is more and their distribution is also disturbed. Hair are hard, small, curly and
stiff in nature. In Mongoloid race, hair are usually straight and have more pigments, their
cuticle is thick and cross section is circular in shape.
Various factors affect the reliability of hair association which includes experience training,
adequacy of equipment and suitability of known hair standards. Although hair evidence is a
valuable tool in human identification, it is difficult to establish a statistical probability for a
particular association due in part to the lack of reliable quantitative assessments of the
microscopic characteristics present in hairs.
Various microscopes used for hair examination are:
a) Stereomicroscope
For initial examination of mounted or unmounted hairs stereomicroscopes are used with
magnification range of upto 100X.
c) Comparison Microscope
While comparing microscopic characteristics of hairs, high quality transmitted light
microscope is necessary. High quality objectives are important. The objectives and eyepieces
selected should permit observations in the range of approximately 40X to 400X. A high-
intensity tungsten light source, suitable for photomicrography and equipped with a daylight
correction filter which is present for providing adequate lighting. Both sides of a comparison
microscope should be balanced for light intensity and color. A comparison microscope may
be equipped with one of several types of stages.
Density
It is one of the most important physical properties of hairs. It is best studied and compared by
density gradient tube method. The question and sample hair are placed in the columns of two
similarly prepared gradient tubes and allowed to settle. They settle at the levels where the
densities of the hairs correspond to the liquid mixtures. If the hairs settle at the same height in
two columns, a common source is indicated.
Refractive Index
Refractive index of hair is determined by Beckline method. There are minor variations in the
refractive indices of hairs of the same individual, the variations are statistically ignored.
Refractive index of hair also gives rough idea about the age and sex of the source of origin.
The phenomenon of deviation of light passing through some transparent materials, differently
at the different angles is called birefringence. The deviation are characteristic of the
materials. The hairs also show the phenomenon but due to inconsistency in the deviations of
different hairs of the same person, it has not been used to any appreciable extent to identify or
individualize hairs.
Forensic biology and serology is a branch of forensic science which deals with
biological evidences and their examination
Blood is a complex viscous red fluid with a pH of about 7.4.
The colouring substance of the red blood cells is haemoglobin.
Benzidine (0.1g) and dry sodium perborate (0.1g) are dissolved in glacial acetic acid
(10ml) and sprayed over the stain. A strong blue color indicates blood.
Luminol is a chemical which has been used to locate the bloodstains
Semen is found in liquid form, smears or stains or it may be found in vagina, anus or
rectum.
Semen is a complex mixture of organic and inorganic compounds.
A semen stain, if fresh is colourless or pale yellow. It gives its characteristic smell.
A small amount of semen is treated with iodine in potassium iodide solution.
Characteristic crystal of choline iodide are formed.
Saliva of secretors contains blood group substances and can be grouped
Urine stains can now be individualized through DNA profiling.
Faeces are identified microscopically.
Density is one of the most important physical properties of hairs.
Refractive index of hair is determined by Beckline method.