BODY FLUIDS

1 BODY FLUIDS
There may be many reasons why one would need to examine for body fluids, but
they primarily revolve around the following:

To establish corpus delicti

For example, the presence of semen in and around the scene of rape and on
the victim might be significant evidence that rape has occurred. Presence of
vaginal fluids on the suspects genital region or undergarments might also be
significant evidence to the same.
To guide the investigation towards a suspect
For example, in case of a homicide, a person seen fleeing the scene with
blood on his clothes is a more likely suspect than other people on the scene.
To obtain DNA information
The obvious point of confirming that a certain stain at a crime scene is a body
fluid is so that it can be used to compare the DNA from the stain to the
suspect or the victim, as applicable. This saves a lot of money and time that
might have been wasted in analyzing stains that are not of human origin and
can give no relevant DNA information, such as coffee stains, rust stains etc.

Therefore, when the CSI team is combing through a crime scene and suspects the
presence of body fluid, they run presumptive and confirmatory tests in order to
establish the following:

Whether the stain is of a body fluid or not?

Saves a lot of trouble and money in following false leads from coffee stains,
starch stains etc. Although there is always the chance of false positives.
Whether it is of human or other species origin?
Depending on the crime scene, one might be looking for blood from animal
(poaching, trafficking) or humans (murder, rape). This is usually determined
by immunoassays (using antibodies specific to the species), which is a much
cheaper way rather going for the whole DNA analysis.
What blood group?
On-spot testing kits available for determining blood group; helps to
consolidate an arrest while the police wait for DNA result to conclusively
prove their involvement in crime.
Whether it comes from a male or female?
Not needed in every case, can be determined only by the amelogenin allele
during DNA analysis.
Which part of the body it comes from?
Most body fluids are very obvious about which part of the body they come
from except blood. There is no way to tell which part of the body the blood
came from except for circumstantial evidence, for example, a blood stain on
the front of the shirt near the abdomen or chest is likely to happen because
of a wound in those regions.

2 SEMEN
Semen is generally found in case of sexual crimes such as rape, sexual assault etc.
and the identification of seminal fluid is essential to prove corpus delicti in such
crimes. Apart from that, semen may also be found in cases where there is no reason
to believe it could be present. A seasoned investigator will use his common sense,
good observational skills, experience and knowledge of human behavior to
determine where to look for such evidence, but there is always chance that some
circumstances might present themselves. During a grisly rape-murder, investigators
expected to find semen on the body either around the victims mouth or in the
genito-anal regions but they didnt. They were beginning to assume that the
perpetrator used a condom but on postmortem examination, the coroner did find
semen in one of the numerous stab wounds on the victims torso.

1 SEARCHING FOR SEMEN

In case of rape, unless the perpetrator has used a condom, semen stains will usually
be found on the clothing of the victim, particularly the undergarments. Semen may
also be found on bedding, mattresses, car seats, on the carpet, between floor
boards etc. in and around the immediate vicinity of where the crime occurred. The
criminal might have used any nearby fabric to clean himself or washed himself in
the washroom, meaning that there might be semen stains in the drain or toilet bowl.
A victim of sexual assault might also have considerable amount of seminal fluids in
or around their genitalia. This will be collected by the medical examiner by doing a
vaginal swab.
When running a match of semen to a suspects DNA, you dont need the suspects
semen sample. A sample of saliva from the suspect will also yield the same results.
Generally, semen stains will appear stiff and crusty on fabrics, like a starched fabric
would. On harder surfaces, it might be appear as an off-whitish stain, but that may
differ depending on the kind of surface and the chemicals upon the surface with
which the semen might react. Inspection under ultraviolet light will reveal semen
stains as a bluish white fluorescence. This is a much simpler and easier way to
detect presence of seminal fluids quickly.

2 COLLECTION AND PRESERVATION OF

SEMEN SAMPLE
When collecting semen evidence on clothes, it is important that each piece of
clothing be packed separately to avoid transfer from stains on one piece of clothing
to other. For example, in case of a gang rape, different people might have
ejaculated on different articles of clothing. It is imperative that each piece be
packaged separately to avoid mixing the DNA profiles from each stain. For the same
reason, it is also necessary that stains from one part of the clothing be not allowed

to come in contact with other parts of the clothing. To ensure this, as far as possible,
folding the article of clothing should be avoided. If that is not possible, one may also
cut out the suspected stained part and package it separately.
The stain must be dried before packing. However, exposure to sun or heat can be
detrimental to DNA evidence so ideally you should leave it under the shade, with
probably the fan on, for quick drying. Once the seminal stain has tried, the stains
become brittle and there are chances that the crust might break up into smaller
pieces and be lost. Friction can also cause damage of the spermatozoa. To avoid
this, the garment should not be folded in the area of the stain. Additionally, the
clothing (or pieces of the fabric) should be laid gently between two sheets of
cardboard (or similar material) and the sheets tied together with a string to avoid
movement.
If the stain is upon a hard surface that cannot be moved to the lab, a sample of the
fluid is collected by swabbing the surface with a cotton tip. If the sample has dried,
the sample is swabbed with a tip that has been wetted with saline solution (salt
solution). The swab is then allowed to dry naturally before it is put inside a test tube
and sealed.
NOTE: We do not simply use water, since moisture may lead to growth of microorganisms. A highly concentrated solution prevents the growth of these organisms.
NaCl (sodium chloride) is found to be an ideal salt because it does not interfere with
the DNA analysis process.
When collecting seminal fluid samples from the body, the medical examiner
generally swabs the area in a manner similar to the above. Any pubic hair
containing semen traces is picked and put in a test tube after drying naturally.

3 EXAMINATION FOR SEMEN

Semen is produced by the male reproductive organs and consists primarily of
sperms (spermatozoa) along with glandular secretions which make up the seminal
fluids. 1 mL of semen is likely to contain 100 million spermatozoa on an average, a
normal healthy male ejaculates approximately 3.5 mL of semen.

A normal sperm measures approximately 0.05 mm in length and most of the nuclear
DNA is contained in the head. However, as the semen lies exposed or dries up,
there are chances that the heads might break off from the spermatozoa. Also, in
cases of people who have undergone vasectomy, their ejaculation fluid is not likely
to contain any sperm. For this reason, when examining probable semen stains, we
check for both seminal fluids and sperms.
PRESUMPTIVE TEST
The Florence test uses a potassium tri-iodide reagent to give rise to dark brown
crystals of choline periodide. It tests for choline in the stain. The Barberio test uses
picric acid to react with spermine in seminal fluids to give rise to spermine picrate
crystals. Neither of these are very dependable as they are neither specific nor
sensitive.
The best way to identify semen is by testing for acid phosphatase, an enzyme. It is
also found in vaginal fluids but in semen, it is present in the seminal fluids in far
greater quantities than in any other body fluid. Acid phosphatase can be tested by
extracting a part of the suspected stain in a small amount of sterile water and
treating it with an acidic solution of alpha-naphthyl phosphate followed by
Bentamine Fast Blue B dye. A positive semen sample will turn an intense purple
colour in the presence of seminal acid phosphatase. It is possible that certain
vegetable juices, contraceptive creams, vaginal secretions might also a give
positive reaction but no at the same speed as seminal fluids. This test can
essentially be considered a confirmatory test if your suspect has undergone
vasectomy operation and shows no sperm in his seminal fluids.

CONFIRMATORY TEST
The conclusive test for semen is identifying the presence of spermatozoa in the
stain. A drop of the extract of the stain (small part of the stained fabric dissolved in
small amount of sterile water) is put on a microscope slide, stained and observed
under a microscope.

Sperms as seen under high power microscope

4 SALIVA
The identification of saliva stains may be important depending on the kind of
investigation of criminal incidents. From evidence such as a handkerchief or
cigarette butt left at crime scene to bite marks, saliva might provide a lot of
pertinent information. Generally we use saliva to obtain DNA information, but if the
person is a secretor, it may be possible to identify their blood type and other
protein variants. Besides, saliva is the body fluid that police generally collect for
control samples.

5 SEARCHING FOR SALIVA

Since saliva appears similar to water and leaves no distinctive texture or colour on
fabrics after it has dried up, it is often to determine where it might be located at a
crime scene. Investigators mostly use circumstantial indications to determine where

saliva might be present. For example, in the case of a masked robbery or burglary,
chances are that saliva might be found on the lower half of the mask; on any
cigarette butt left on scene, on glasses, mugs, cups, bottles, etc. that either the
victim or suspect might have drunk from; on bite marks on skin (post or antemortem).
There might be chance that dried saliva stains might show up under light of any
wavelength, but such cases are extremely rare and the process can be time
consuming, scanning all of the crime scene with all possible wavelengths of light.

6 COLLECTION
AND
SALIVA SAMPLE

PRESERVATION

OF

As with all other body fluids, the article with saliva stain should be dried naturally
under shade before packaging. Heat or UV from sun can damage the DNA in the
stain. Care must to taken that the article of clothing is not folded on the stain and
packaged such that no other part of the article comes in contact with the stained
part. If necessary, use tissue paper in between each fold to keep them separate
from contact.
When collecting control samples, the subjects are asked to either spit directly into a
sterile test tube or the inside of their mouth is swabbed and the swab is placed
inside a test tube and sealed. This is also the same method by which saliva is
collected from bite marks on any body part.

7 EXAMINATION FOR SALIVA

Saliva is secreted by the salivary glands and is mostly made of water (99.5%). The
other 0.5% contains electrolytes, mucus, glycoproteins, enzymes, antibacterial
compounds (IgA, lysozyme) etc. The enzymes found in saliva are functional for
digestion of various kinds of food molecules. One such enzyme found in abundance
in the saliva is amylase which helps in digestion of starch. This is the enzyme we
test for to confirm that a particular stain is saliva.
PRESUMPTIVE TEST
The Phadebas test can be done in two ways:
A part of the suspected fabric can be cut out and put in a small amount of Phadebas
solution. The solution will turn blue in the presence of saliva. Another way to do the
same test is to overlay a Phadebas paper which is white in colour over the object
and lightly spritz water all over it. Any area with saliva will turn the corresponding
area on the paper blue.
CONFIRMATORY TEST
This is only possible if there is a considerable amount of saliva sample to test from.
The extract is mixed with starch solution, then add a few drops of iodine. Presence
of saliva will break down the starch and thus iodine will not be able to react with it

to give a negative colour. However, in the absence of saliva, we will get a deep blue
colouration when iodine is added.

8 BLOOD
Blood is one of the most important and frequently encountered body fluid evidence
in criminal investigation. It is mostly found in crimes of physical violence such as
murders, assaults, rape, etc. Knowing the nature and possibilities of recording this
kind of evidence is imperative to understanding its value and ensuring that it is not
underestimated or overstated.

9 SEARCHING FOR BLOOD

When it comes to looking for blood, where you look for it depends on the nature of
the crime. The main place to search for is obviously at the scene of crime and any
other secondary sites, on the clothing and body of the victim and suspect, the
weapon of offence (if any), vehicle and route etc. Clothing, bedding, upholstery,
carpets, furniture may have blood spatter or smears from any kind of activity from
the victim or suspect (wiping of weapon, bullet, handprints etc.). In some cases,
there might attempts made to wash or wipe the blood off the floor or other stained
area or the weapon of offence or clothes and body parts. They might have also tried
to wash off the blood in the basin, in which case stains might be found trapped in
the drain pipe. Even with all attempts to keep blood off clothes, some spatter might
be found on the seams or underneath the sleeves of the clothes. In outdoor scenes,
one need to go through layers of soil, through piles of stones or under them, grass
blades, dried leaves and twigs to find any amount of significant blood. The victim
might carry blood and scrapes of skin under their nails or in their hair. If a vehicle
has been used, there are chances that some blood might have been deposited on
the floor of the vehicle, in the crevices of the upholstery or elsewhere within the car.
During rape, there could have transfer of blood from the victim to the genital region
of the perpetrator and their clothing, particularly the underwear.
Usually, a simple visual examination of the crime scene can reveal a lot of blood
stains. However, it must be kept in mind that the colour of the bloodstains on any
surface may vary according to their age, amount of blood present and the nature of
the surface. On certain surfaces, it might be difficult to detect bloodstains as well
(such as dark clothes). Blood stains that are recently made are usually reddish in
colour but the more time they are exposed for, the more brown they appear. Blood
on walls might react with paint, and similarly putrefaction and reactions with other
chemicals on other surfaces to give different colours ( black, green, blue, greyish)
that might not appear like blood. Bloodstains on metal surfaces might form dark
crusts that appear very much like rust. On glass, blood appears similarly glossy to
paint.
An easier way to detect blood traces that are not visible to the naked eye is to use a
luminol spray and UV light to scan the surfaces. Although this is not very specific to
blood and a few oxidizing agents might also show up on the scan, still it is a more

sensitive and dependable method of detection, especially for tiny stains and those
that have been attempted to wash off.

10 COLLECTION AND PRESERVATION OF

BLOOD SAMPLE
When collecting blood from the victim, we must be sure that there are chances that
the blood stains could be the offenders, which means that that the assailant must
also have been injured. When collecting blood stains from the body post-mortem,
we swab it as done before and wait for it to dry before sealing it inside a tube.
Occasionally, for toxicology or other purposes, it might be necessary to take blood
in a syringe and store in an ice-cold vial (with or without anticoagulants). Samples of
dried blood might also be found under the nails, in which case it should be scraped
off with a clean knife onto a clean piece of paper.
When collecting a blood sample from a pool of liquid blood at the scene, you can
either use a dropper to collect it and put it in a vial on ice. Alternatively, you can
also use a piece of sterile cotton or filter paper dipped in saline water to absorb the
blood and dry it before storing.
When collecting blood stained clothes from the victim, one might have to cut them
off their body. However, one must make sure that they dont cut through stained
areas or through holes caused by bullets or cuts made by weapons. Another major
concern is to avoid contaminating the clothes during seizure and storage. The first
rule to collecting is to ensure that the garment is thoroughly dry. Then wrap it in
clean paper before folding it, making sure that no stained area comes in contact
with the other. If the whole item is too big to pack, cut out the area of the stain and
pack it similarly. Avoid packing articles from the same person in one evidence bag.
The best practice is to package every item separately. Also, avoid packing blood
evidence in polythene bags or airtight containers. It is difficult for moisture to
escape from such environment and there are chances that this might lead to growth
of bacteria and destroy the biological evidence on it. A paper bag allows for
breathing, thus keeping the evidence relatively dry.
If the bloodstain is obtained from an immovable surface like a wall or furniture,
which cannot be transported, the blood stained area should be removed by cutting
or scraping the stain, when dry, onto a clean piece of paper with a clean knife.

11 EXAMINATION FOR BLOOD

An average human adult has about 5 litres of blood in their body which constitutes
to 1/13th of the body weight. Essentially blood has two main components - the
cellular part containing red blood cells (erythrocytes), white blood cells (leucocytes)
and platelets (thromobocytes), and plasma which is fluid and contains water,
proteins, minerals, organic compounds, clotting factors etc. These are what a tube
of blood will separate into if left out in the open without anticoagulants and this is

what happens when you cut yourself and bleed. The clot collects the solid cellular
part and separates it from the plasma.
Exposed blood, after clotting, with undergo further complex changes while drying.
The cell structure might weaken, depending on the kind of environment, and the
proteins may lose some of their function. Growth of bacteria and fungi, heat and
light may bring about other changes, not only in the appearance of the stain but
also in the chemical buildup.
PRESUMPTIVE TEST
Benzidene test: Although this test is not very specific for only blood, it is highly
sensitive and can detect blood even if it is present in the 1 out of 300,000
molecules. The test is based on the peroxidase enzyme like property of the
haemoglobin in blood. Peroxidase enzyme initiates the oxidation of bezindene and
give a blue coloured compound. However, vegetable peroxidase, chemical oxidants
or any other similar contaminants may also give similar reactions, which is why it
needs to be confirmed by another test. Leucomalachite green and phenolphthalein
also give a similar reaction. In presence of blood, LMG turns green and
phenolphthalein (in presence of alkali) turns pink from colourless.
Phenolphthalein test is also known as the Kastle-Meyer test.

CONFIRMATORY TEST
Luminol can be a good confirmatory test, but if you want to make it more specific,
you can do one of the following in the laboratory.
Hamein crystal test: A small crystal of sodium chloride and 2-3 drops of glacial
acetic acid are placed on a fragment of the stain on a glass slide. A cover slip is
then placed on it and the acid evaporated by heating it gently over a flame. Dark
brown rhombic crystals of haemin chloride form from blood stains.
Spectroscopic examination: the stain is dissolved with some saline in a test tube
and a hand held spectroscopic examination done. Blood shows a characteristic
pattern of spectral bands.
Microscopic examination: For relatively fresher stains present in considerable
amount, a part of it can be dissolved and observed under the microscope for blood
cells. However, this is seldom preferred since most of the times, the blood stain
samples are too damaged to examine under a microscope.

12 OTHER BODY FLUIDS

FAECAL MATTER
Although faecal matter is not something usually found at a crime scene, there might
be present because of some abnormal mental aberrations of the criminal or as a
result of nervousness and/or natural urge. Alternatively, this kind of evidence is
mostly found in case of anal assaults. Faecal stains are very visible because of their

grainy, yellowish-brown stain accompanied by a characteristic smell. Microscopic

examination of faecal matter may reveal the presence of particular parasites and
stomach flora/fauna as well as undigested food matter.

SWEAT
Perspiration stains on fabrics are usually found at the indicative positions e.g.
armpits, collar etc. and may sometimes form off-white stains or discolour the fabric.
The best test for perspiration is to remove a small sample of the cloth and hold it
lightly over a heat source so that it fumes. The odour is characteristically distinct
and typical of sweat.

URINE
Depending on the volume of urine deposited and the time since deposition, the
stain might be wet or dry. The composition and colour of urine might differ but
generally it is pale yellow in colour and has a characteristic smell of ammonia/urea.
Urine stains appear fluorescent yellow under UV light.

13 OTHER IDENTIFYING FACTORS FROM BODY

FLUIDS
See additional notes