Blood

Blood is a specialized body fluid in human body system. It is also regarded as liquid vehicle by
which major organic nutrients are transported from the intestines and metabolized in the liver
where they are process and transport to other organs

Blood has many different functions, including:

 Transporting Function, oxygen, carbon iv oxide, metabolites and nutrients to the lungs
and tissues
 Osmo regulatory function, Acid-base balance, osmotic pressure
 Detoxifying functions
 Defensive or immunological function
 Regulatory or hormonal function
 Homeostatic
 Buffering /physiological buffer

Blood components and their importance

Blood has four main components: plasma, red blood cells, white blood cells, and
platelets.

Plasma

The liquid component of blood is called plasma, a mixture of water, sugar, fat, protein, and salts.
The main job of the plasma is to transport blood cells throughout our body along with nutrients,
waste products, antibodies, clotting proteins, chemical messengers such as hormones, and
proteins that help maintain the body's fluid balance.
Major components of normal human blood plasma

Sn Components Functions
1 Protein
Serum albumin, globulins
2 Lipids Fuels en route to sites for storage or oxidation, immediate
Tri acyl glycerol, fuel source for muscles and heart.
phospholipids, free fatty
acids

3 Glucose Transport form of carbohydrates

4 Amino acid Precursor in protein synthesis
5 Urea Excretion products of amino acid and nitrogen
6 Uric acid End product of purine catabolism
7 Creatine Excretory form of creatine
8 Lactate Product of glycolysis in muscle
9 Pyruvate Product of glycolysis in muscle
10 Beta-hydroxybutyrate Ketone bodies transport form of acetyl group
11 Acetoacetate Ketone bodies transport form of acetyl group
12 Bilirubin Degradation product protoporphyrin
13 In organic components
Na+, Cl-,HCO3. Phosphate

Major protein components of human blood plasma

SN Protein components FUNCTIONS

Serum albumin Osmotic regulations, transport of fatty
acid, bilirubin etc
αi-globulin Unknown
αi-lipoprotein Lipid transport
αii-globulin
α2-glycoprotein
Ceruloplasmin Cu transport
Prothrombin Blood clotting
β-globulins
β1-lipoprotein Lipid transport
Transferrin Iron transport
Plasminogen Precursor of fibrinolysin
δ-globulin antibodies
Fibrinogen Blood clotting
Red Blood Cells (Erythrocytes or RBCs)

Known for their bright red color, red cells are the most abundant cell in the blood, accounting for
about 40 to 45 percent of its volume. The shape of a red blood cell is a biconcave disk with a
flattened center - in other words, both faces of the disc have shallow bowl-like indentations (a
red blood cell looks like a donut). Production of red blood cells is controlled by erythropoietin, a
hormone produced primarily by the kidneys. Red blood cells start as immature cells in the bone
marrow and after approximately seven days of maturation are released into the bloodstream.
Unlike many other cells, red blood cells have no nucleus and can easily change shape, helping
them fit through the various blood vessels in your body. However, while the lack of a nucleus
makes a red blood cell more flexible, it also limits the life of the cell as it travels through the
smallest blood vessels, damaging the cell's membranes and depleting its energy supplies. The red
blood cell survives on average only 120 days.

Red cells contain a special protein called hemoglobin, which helps carry oxygen from the lungs
to the rest of the body and then returns carbon dioxide from the body to the lungs so it can be
exhaled. Blood appears red because of the large number of red blood cells, which get their color
from the hemoglobin. The percentage of whole blood volume that is made up of red blood cells
is called the hematocrit and is a common measure of red blood cell levels.

White Blood Cells (leukocytes)

White blood cells protect the body from infection. They are much fewer in number than red
blood cells, accounting for about 1 percent of the blood. The most common type of white blood
cell is the neutrophil, which is the "immediate response" cell and accounts for 55 to 70 percent of
the total white blood cell count. Each neutrophil lives less than a day, so your bone marrow must
constantly make new neutrophils to maintain protection against infection. Transfusion of
neutrophils is generally not effective since they do not remain in the body for very long.

The other major type of white blood cell is a lymphocyte. There are two main populations of
these cells. T lymphocytes help regulate the function of other immune cells and directly attack
various infected cells and tumors. B lymphocytes make antibodies, which are proteins that
specifically target bacteria, viruses, and other foreign materials.

Platelets (Thrombocytes)

Unlike red and white blood cells, platelets are not actually cells but rather small fragments of
cells. Platelets help the blood clotting process (or coagulation) by gathering at the site of an
injury, sticking to the lining of the injured blood vessel, and forming a platform on which blood
coagulation can occur. This results in the formation of a fibrin clot, which covers the wound and
prevents blood from leaking out. Fibrin also forms the initial scaffolding upon which new tissue
forms, thus promoting healing.
A higher than normal number of platelets can cause unnecessary clotting, which can lead to
strokes and heart attacks; however, thanks to advances made in antiplatelet therapies, there are
treatments available to help prevent these potentially fatal events. Conversely, lower than normal
counts can lead to extensive bleeding.

Plasma and Serum

The liquid component of blood is called plasma, and it is separated by spinning or centrifuging
the blood at high rotations (3000 rpm or higher). The blood cells and platelets are separated by
centrifugal forces to the bottom of a specimen tube. The upper liquid layer, the plasma, consists
of 90 percent water along with various substances required for maintaining the body’s pH,
osmotic load, and for protecting the body. The plasma also contains the coagulation factors and
antibodies.

The plasma component of blood without the coagulation factors is called the serum. Serum is
similar to interstitial fluid in which the correct composition of key ions acting as electrolytes is
essential for normal functioning of muscles and nerves. Other components in the serum include
proteins that assist with maintaining pH and osmotic balance while giving viscosity to the blood.
The serum also contains antibodies, specialized proteins that are important for defense against
viruses and bacteria. Lipids, including cholesterol, are also transported in the serum, along with
various other substances including nutrients, hormones, metabolic waste, plus external
substances, such as, drugs, viruses, and bacteria.

BLOOD GROUPS

The outer layer of Red Blood Cell membrane contains certain antigens which are chemically known as
glycoprotein and are called Agglutinogens. According to the presence of these antigens human blood can
be classified into 4 groups.

Significance of BLOOD groupings

I. Blood transfusion
II. Marriage (Rhesus compatibility )
III. Disputed paternity
IV. Detection of crime ( Medico legal case)
ABO SYSTEM

This system include 2 types of antigen A and B agglutinogens. According to the presence of A and B
antigen there are 4 groups of human blood.

(I) Group A (41%) only antigen A is present

(II) Group B (9 %) only antigen B is present
(III) Group AB (3%) both Antigens A & B are present
(IV) Group O (47 %) both Antigens A &B are absent

Plasma contains certain antibodies against the red cell agglutinogens called agglutinins which are of 2
types

Antibody A (alpha) agglutinins

Antibody B (Beta) agglutinins

BLOOD TYPES

Blood type Antigen on RBC Antibodies in Can receive blood Can donate blood
plasma from to
A A Antibodies b O&A A & AB
B B Antibodies a O&B B & AB
AB A&B Neither O,A,B & AB AB only
O Neither Antibodies a & Only O A,O, B & AB
antibodies b

O- Universal donor

AB- Universal recipient

 BLOOD GROUPS

Summary of reaction when different blood groups are mixed together during transfusion

Group RECIPIENT
O (a,b) A (b) B (a) Universal
recipient AB (o)

Universal
donor - - - -
O
(a,b)

A + +
- -
DONOR

(b)

B + +
(a) - -
AB + + +
(o) -
+ = Agglutination - = No agglutination

Agglutination. When antibodies are mixed with their corresponding antigens on the surface of large,
easily sediment particles such as animal cells, erythrocytes, the antibodies cross-link the particles,
forming visible clumps. This reaction is termed agglutination.

When antibodies bind to antigens in the blood which results in clumping. This agglutination could lead
to a variety of problems including clogged blood vessels and the stoppage of blood circulation.

In summary:

O: Universal donor because It can donate blood to all O, A, B & AB because agglutination do not occur.

A B: Universal recipient because it can receive blood from all O, A, B & AB because agglutination do not
occur.

A: can donate blood to A and AB. B can donate blood to B and AB. AB can donate to only AB
 Determination of ABO system

Mix one drop of blood with 1 ml of isotonic saline in a test tube.

-Put 2 separate drops of the diluted blood on a glass slide.

-Add one drop of anti-A serum (blue coloured) to one blood drop and one drop of anti-B (yellow
coloured) to the other drop.

-Mix the blood with anti-A and anti-B sera gently using the blunt end of two separate pins.

After 2 minutes examine for agglutination:

a- If agglutination occurs with anti-A serum only, the blood group is type A.

b- If agglutination occurs with anti-B serum only, the blood group is type B.

c- If agglutination occurs with both anti-A and anti-B sera, the blood group is type AB.

d- If no agglutination occurs with either anti-A or anti-B, the blood group is type O.
 RHESUS FACTOR (Rh system)

This is the system of antigens or agglutinogens normally present in the red cell of Rhesus
Monkey (hence the name rhesus). There are 6 varieties known as dominant C, D and E antigen
and recessive c, d and e antigen. However, antigen D has the strongest antigenic effect.
Therefore if antigen D is present it is known as rhesus positive and if antigen D is absent it is
known as rhesus D negative.

Rhesus factor and pregnancy. If the pregnant mother is rhesus negative but the baby she is
carrying is rhesus positive. The child must have inherited her Rh D positive from her positive
daddy. When antigen from Rh D positive baby enters the mother’s blood stream, immune system
may react to the D antigen on the baby’s cell and it will be treated as foreign invader as such the
mother will produce antibodies against it. This is known as Sensitizing event. This event is not
harmful at first pregnancy but can cause problem when the mother is pregnant again with another
rhesus positive baby. The woman during the first pregnancy can quickly multiply and cross the
placenta to attack the blood cells of the baby. Routine injection of anti-D immunoglobulin (anti-
D) to guard against harmful effect of antibodies.

Rhesus disease causes anemia which under sever condition can lead to life-threatening problems
such as heart failure, fluid retention and swelling.

IDENTIFICATION OF BLOOD AND OTHER BODY FLUIDS

Various body fluids may have to be identified in the investigation of a crime

and they are usually presented as dry stains. The most important of these
stains is blood.

Microscopic Identification
BLOOD STAINS
Chemical Identification
Two identification of blood stains is the primary importance whenever blood
has or may have been shed. The questions asked in most cases are what
sort of fluid is it? If blood is it human, if human to what group does it belong
to or Is it the same as control ?

The first step in the investigation is to distinguish blood stains from others
due to rust, boot polish, fruit juice, jam, chemicals, paints etc. At the same
time blood must be looked for in places where it has not been seen, but
where the circumstances of the case suggest that it may be found e.g in the
crevices of a folding knife which has been rinsed or shoes which have been
roughly wiped clean etc. Where actual stains, smears or splashes are seen
appearances and distribution are of their great evidential value. Spots and
splashes are produced by drops of liquid blood.

If these drops hit a surface perpendicular they produce a star-shaped flash,

while if they hit it obliquely (slanting) they produce a characteristic “failed”
splash, with the tail pointing away from the direction of impact. A number of
such splashes distributed over an area will serve to locate the position of
shedding, this may be important in for e.g the investigation of an indoor
murder case. Hence, drops of the dried blood can tell by their appearances,
from which direction they came.

A stain can be proved to be blood in several ways

1. Microscopy could be used. The microscopic recognition of the

characteristically shaped (i.e, star shaped red crystals) red cells are
conclusive evidence that the stain is blood. But it is impossible for
dried stains to show under a microscope.
Other tests have to be used in cases of dried stains. These tests are of
two types.
They are:
a. Those utilizing the peroxidase activity which is fundamental to
the blood role in tissue oxygenation, and which is performed by
the haemoglobin molecule. This test is highly sensitive and can
be used in the routine localization of blood stains but are not
completely specific.
b. Those depending on the direct identification of blood pigments
i.e haemoglobin and its derivatives. This test is more specific,
less sensitive and can be applied only to detected, visible items.
The test to be performed on suspected blood stains are divided
into:
1. Preliminary test
2. Confirmatory test
3. Species identification
4. Grouping
PRELIMINARY TEST

This utilizes the peroxidase activity of haemoglobin (Hb) molecule. The

peroxidase test depends on the fact that these enzymes will catalyse the
oxidation of Hb molecules and some other oxidations. The reagents used
contain.

1. A suitable Hydrogen peroxide e.g H202.

2. A colourless reduced compound which gives an intensely coloured
oxidized product.
3. Acid/Alkaline to adjust the pH to that suitable for the reaction.

In an acidic medium, the most suitable reducing agent sued is 0-tolidine

(substituted for benzidine because it is carcinogenic).

Peroxidases aid in the transfer of excess 0 2 in the reaction. The

peroxidase in the blood are extremely stable and can be detected in
stains which are very old or even heated. A negative result from a
peroxidase test is complete proof of the absence of blood. But some
chemicals like oxidants or catalysts for the reaction and some plant juices
which also contain peroxidase will give positive (+ve) results.

PROCEDURE FOR THE PEROXIDASE OR BENZIDINE TEST

Transfer the stains into a white piece of paper by rubbing a filter paper on
the suspected stain or the area where blood is being sought and add few
drops 0-tolidine (The 0-tolidine is normally made in acid). Add few drops
of H202 in the presence of Hb, the H202 gives H20 and 02. The 02 will now
combine with 0-tolidine to give a blue complex.

Hb
2H202 2H20 + 02
Peroxidase

02 + 02 –toluidine Blue 0-toludine

(reduced) (Oxidized)
This reaction suggest it is blood if positive, but does not confirm it is blood.

if plant peroxidase are present a green vegetable smearing will generally be

observed on the area being tested. The reaction in this case will be much
less intense and usually disappears when the stain is a few days old and the
peroxides of plants are easily destroyed by heat unlike those of blood.

Confirmatory test (Takayama test)

In this presence of Hb is confirmed and the reagent used contain hydrazine

sulphate and pyridine. If the article being examined gives a positive
peroxidase reaction but shows no visible blood staining, the identification
cannot be carried any further.

Procedure

Scrap a small part of the stain into a slide and grind to powder.

Add a few drops of a mixture of hydrazine sulphate and pyridine (50:50)

Cover with a cover slip and warm briefly, then allow to cool.

Observe under the microscope

The result shows beautiful characteristics red star shaped crystals (or salmon
pack crystals).

This confirms the presence of blood.

The chemistry of the reaction is crush that the hydrazine sulphate reduces
the Hb to heamochromogen and the pyridine now combine with the
heamochromogen to form pyridine heamochromogen crystals which are the
beautiful red star shapes.

Hydrazine
Hb heamochromogen
Sulphate

Pyridine
Heamochromogen + Pyridine heamochromogen
Crystals

A stain having been identified conformatorily as blood and assumed to be of

human, the age i.e how old the stain had been there and from which sex are
questions that can be asked.

SPECTROPHOTOMETRIC METHOD

The age of a blood stain can be detected using spectrophotometric

technique although experience of the analyst is an added advantage.
Normal, Hb shows two absorption bands in its spectrum (a graph of
Absorbance vs wavelength) in the visible region at 540 and 578n,
respectively with an intermediate position of minimum absorption at 560nm.
It has been shown that the height and precise wavelength of the peak at
578nm changes as the stain ages and experienced analysts have found a
way of measuring this change.

It is also important to decide whether or not a stain is of menstrual blood.

Immune electrophoresis is used in this detection. Menstrual blood does not
clot because it contains a factor which inhibits the clotting reaction i.e the
conversion of soluble fibrinogen into insoluble fibrin. Although there is no
universally applicable method of identification, good results have been
obtained by the use of an anti-fibrinogen antiserum in immune-
electrophoresis followed by a calculation of the ratio. Fibrinogen/total protein
for the stain in question.

Analysis of detecting the sex of blood stains are much more complicated and
nto yet completely solved though recent progress seem to promose good
results especially with DNA.

SPECIES IDENTIFICATION

Once a stain has been identified, it is important to know if it is human blood.

Other species like dog, cow, sheep, horse, pit etc. have sometimes to be
identified if expected. The test used is the precipitin test which is an
application of the antigen body reaction.

Then a small quantity of the blood of one species is injected into the body of
another, the injected animal reacts self-protectively by producing an
antibody to the proteins of the foreign blood. This antibody produces by the
organism is specific for the injected blood.

When the injected animal is bled and the serum separated it becomes the
reagent for the type of blood that was injected into the animal and it is called
anti-serum of that blood. This serum is the serum that contains a large
number of antibodies in its haemoglobulin faction to a specific antigen. This
is produced by the injection of an antigen (immunogen) to an animal to
immunize it. The level of the antigen in the antiserum take about three
weeks to be at its peak after immunization. This is the technique of
immunology. The anti serum obtained from the injected animal can be used
in species identification.

In practice, specimens of all the bloods which may have to be identified are
injected into a series of experimental animals, each receiving blood from a
different species. After a suitable time, the animals are bled and their blood
sera separated each batch of serum then becomes the reagent for the type
of blood that was injected into the animal from which that serum came.

Such anti-sera are available commercially in performing the test, a filtered

isotonic saline extract of the stain is allowed to come into contact with the
anti-serum, a reaction between the two is then shown by the formation of a
cloudy precipitate at the line or plane of contract.

PROCEDURE

Extract stain and filter (use centrifuge). Put a few drops of filtered
appropriate antiserum into a tube and layer the extract into an anti serum
with a Pasteur pipette. Allow to stand for about 10mins or more and
observed the inter face for a white precipitate. Result of the white precipitate
shows a positive result.

Control could set up using an unstained area of the material bearing the
stain, in case anything extracted from the material itself is giving a false
positive.

Suppose a blood stain jacket thought to be human is being examined, two

saline extracts will be made, one of the stain and the other of an equal are of
clean fabric from another par of the jacket. A lock of tubes is set out for
varieties in a gel preparation) in which the two extracts are tested with
several anti-sera as shown below. The anti-sera include both the species
being tested for and several other suspected species.
Below is a specimen result

Serum Anti-human Anti-dog Anti-cow Anti-pig

Jacket stain X 0 0 0
Jacket Unstained 0 0 0 0
area (control)

X = PRECIPITATION

0 = No precipitation

This reaction is specific e.g sheep and goats will give the same result.
Human, monkey and chimpanzee will give the same result etc.

Blood Stain Grouping

Having known that a stain is blood and of human, the blood group is
important. With the fact that only ABO antibodies occur naturally the
grouping of dried stains are considered in the ABO grouping system.

In blood stains, the antigens does not leave the blood despite the fact that
the stain is dried but the antibodies are unstable and soon become
undetectable on drying, hence it is often necessary to compare blood stains
formed on an exhibit with the blood of a victim or accused.

A straight forward grouping as in intact fluid blood (fresh) group is not

possible since the blood cells in a stain have been analysed. the ultimate aim
in all types of grouping is to identify the type of antibody or antigen present
in the blood.

Methods of Grouping
Stains grouping by identification of Antibodies: This is called Lattes
method. Antibodies are difficult to detect on stains unless the stains are
fresh and dense enough for crusts of dried blood to be detectable.

Method: Emmerse 3 small crusts of the stains in a few drops of washed

diluted saline suspension of A, B and O cells respectively. These ABO cells
are well washed free of antibodies of the bloods from which they came).
Shake gently and observe for agglutination, after about 10min.

RESULTS
O cells A cells B cells Result
- + + Group 0
- + - Group B
- - + Group A
- - - Group AB

The O cells are used to exclude false results due to agglutination by

substances other than the anti-A and anti B agglutinis (antibodies)

The disadvantages of this method is that its neither A nor B are agglutinated
it will be difficult to know whether the blood is AB or whether the test is not
working hence known stain controls are used.

NOTE:

Group 0 will agglutinate A and B cells

Group A will agglutinate B cells only

Group B will agglutinate A cells only

Group AB will agglutinate none

Grouping by identification of antigens

There are three method of grouping by identification of (agglutinogens)
antigens. These include Absorption/elution techniques, absorption-inhibition
and mixed agglutination technique.

(1) Absorption/Elution Techniques: In this method, antigen and

antibodies are allowed to react. The excess antibodies are eluted and
subsequently identified.

Method: The stain is transferred or mopped on a clean thread/fibre. The

thread is out into three parts (if the sain is on a hard surface, then swob
the stain with a clean thread and allow to dry).

The thread is glued on a cellulose acetate (plastic) sheet. The thread

contains the antigen of the blood.

, Anti-B Anti-H

+ + +
A cells B-cells 0 cells

To the 1st thread add 2 drops of anti-serum, to the 2 nd, 2 drops of anti-b
serum and finally 2 drops of anti-h to the 3rd thread.

Cover the cellulose sheet and put in the fridge over night or 3 hours
minimum at 400C Adsorption will take place. After the time of adsorption,
excels serum is washed off with cold saline and the acetate sheet dipped into
a beaker containing cold saline. The beaker is left again in the fridge for 30
minutes. Adsorption will be completed by this time. remove from the fridge
and blot with tissue paper. Add 2 drops of saline to each of the thread.
Elution: Place in an oven for 30 minutes after the above at 40 0C. At this
temperature whatever had been adsorbed comes out into the saline but if no
adsorption had taken place only the thread will float.

Absorption/Inhibition: This technique does not involve elution. After the

reaction, the thread is taken off and the antigen left in it, identified. if the
blood group is A and A cells are added, agglutination is less or none
inhibited. It is not a very sensitive method. The method is obsolete
(outdated).

Mixed agglutination: In the mixed agglutination, it could be carried out like

the absorption/inhibition and also by adding blood cells around the thread, so
that agglutination will occur if the particular antibodies are added to the
thread. Although sensitive, it is tedious and complicated.

OTHER BODY FLUIDS (NOT BLOOD)

Other types of stains, which are not blood, may need to be identified. These
may include semen, saliva, urine and faces, etc.

Semen Identification

The identification of semen is important in every sexual case. in most

assaults, the stain may be found on the clothing of one or both parties. When
full penetration and intercourse have occurred, i.e in rape, incest, etc, it will
be found on vaginal swabs if taken in time while in cases of buggery
(bestiality, unnatural vice etc) it could be found in anal swabs.
Preliminary Test: This test also called the acid phosphatase or ‘AP’ test
depends on the fact that human semen contains a uniquely high
concentration of an enzyme-acid phosphatase derived from the prostrate
gland which catalyses in acid conditions, the hydrolysis of phosphoric esters.

Procedure:

Spread the garment and transfer for stain onto a filter paper by pressing a
while filter paper damped with distilled water on the areas being examined.
This will extract some of the enzymes on the filter paper. This is treated with
a reagent buffered to a slightly acid pH and containing the phosphoric ester
of a reactive phenol and a suitable coupler (or colour developer or dyestuff
intermediate)

The phosphoric ester most commonly used is sodium – naphytyl phosphate

(an ester of phenol – naptol). While the coupler or dyestuff intermediate used
are brentamine Fast Blue (or Black) B salt (carcinogenic) which gives an
intense purple (or black) or 4 methyl umbelliferyl phosphate could be used
as a coupler and result detected by examination of the paper under filtered
UV radiation since the unbellife- one liberated fluorescence.

Reaction: If seminal stain is present the acid phosophatase enzymes will

carry out the following reaction.

The ‘AP’ test is the only test for semen which can be used to localized stains
as well as identify them but a positive AP result only strongly suggest the
presence of semen. it is not a confirmatory test because the test can be
positive for some plant juices (e.g from gallardia flowers and cauliflower)
which contain acid phosphatease although these juices are not likely to occur
in places where semen is being sought. But of most importance is that both
vaginal secretions of young girls and certain used in contraceptive jellies
give positive AP reaction.
Confirmatory Test: The normal and absolutely conclusive test (although not
always possible) is the microscopic identification of spermatozoa, which only
occur in the male ejaculate. The human spermatozoon has an ovoid head
(about 4 micros long) and a thin flagellate tail (40-50 microns long). A good
microscope is therefore necessary for its identification.

Procedure: Extract or scrap part of stain on a glass slide and stain with
haemaeoxyling and eosin dye and view under the microscope. the
spermatozoa will be seen under the microscope. The head is usually seen
most. Due to the staining with the dye, the head/acrosone will be red and the
nucleus blue.

Staining with the dye is essential because unless the stain is a fresh one, the
sperms will probably have lost their tails by the time they reach the
microscope slide. Also because isolated unstained head are easily confused
with other things like stray yeast cells. If these yeast cells or others are
numerous enough to make the identification of the spermatozoa heads
difficult under the microscope, fluorescence microscopy can be employed
after staining with acridine orange when the reproductive sperm cells will
fluoresces with a different colour from the non-reproductive cells like yeast.

There are cases where the sperms are scarce and hard to find. They may be
concentrated by shaking them off the stained area into water with the ultra-
sonic vibration, then collecting at the bottom of the water by centrifugation
before carrying out the test.

A seminal stain does not necessarily both give AP test and contain
spermatozoa. this is because it is possible to wach stained garment in
detergent to remove all the enzyme, but leave some of the sperms. Also the
sperms are not always detectable in known seminal stains. this is because
some men, although are potent and capable of ejaculation, contains few or
no sperms (i.e Oligospermia and azzospermia respectively).

Once semen has been identified its position and condition become facts of
importance. There is yet no known way of measuring age of a seminal stain
but its colour and texture enable an experienced analyst to make a fair
estimate of whether it is recent i.e days or old i.e weeks or months.

Saliva identification

The teeth may be used in a struggle leaving salvia stain, On clotting, hence it
might become necessary to identify saliva. Saliva stains usually contain
squamous epithelial cells because these cells occur in the living of the
month. But it is much more easy and positive to identify saliva by the
presence of ptyalin – the amylase which it contains.

Amylase is a starch hydrolyzing enzyme starch gives a blue black colour with
iodine. The test or stain portion is extracted by washing with little distilled
water (or use moistened filter washing with little distilled water (or use
moistened filter paper to transfer stain. This will transfer the enzyme add few
drops of starch solution and few drops of iodine is added and incubated at
370C (for 2-5 mins). A control using an unstained portion of the garment is
carried out along with the stained portion.

If the test portion does not give blue black colouration but the control give a
blue black coloration, it indicates the presence of amylase which has
hydrolysed the starch. Blue black colour shows absence of amylase and the
stain therefore does not contain amylase.
 URINE STAINS

It is not often necessary to identify urine stains but if needed qualitative

analysis of ions is carried out. Urine contains 2-25% urea and about 1% NaCl,
and its dry stains show a characteristics whitish fluorescence.

The chloride ions are identified by a simple spot test while the urea is
identified by qualitative rests after extraction of the stain with acetone.

FAECES STAIN

The identification of faeces stain is not common but if needed, microscopic

investigation is carried out. Vegetable residues and occasionally muscle
fibers from meat can be identified.

Chemically, blue pigments or their degradation produces can be tested for

intestinal worms or recognizable fragments of them or worm eggs can also
eh used for identification.