Identification from trace

substances
By dr. Naveen
• Every criminal carries some elements with
him from the scene of crime by which he
can be linked with the crime.
Edmond Locard (1877-1966)
• Every criminal leaves some elements from
him to the scene of crime by which he can
be linked with the crime.
Zhu Yunliang
 Material
• semen stains
• hair
• saliva stains
• Bone
• Tissue
• blood stains
BLOOD AS TRACE
EVIDENCE
 Importances of Examination of
blood
• 1. identification of the victim or offender of a crime
like homicide, sex offences or where death occurs
due to rash or negligent acts on the part of persons
who are expected to act with responsibility. Apart
from identification of individuals, examination of
blood may settle other issues in criminal cases like,
to know the the cause of death;
• 2. The cause of death (e.g. detection of poison, or
some other pathology, responsible for the death) ;
•
 Importances of Examination of
blood
• 3. time of death (different chemical and
biochemical tests of blood may be helpful) ;
• 4. criminal abortion cases;
• 5. investigation of sex offence cases;
• 6. to establish relationship between the
offence, offender, the offended (victim of the
offence) and the offending agent; and, lastly,
7. The malingering cases.
 To answers the question of:
• 1 Whether the stain is due to blood or some
other material ?
• 2 If it is due to blood, then whether it is of
human origin or it belongs to some other animal?
• 3. What is the source of the blooding:
• a) Is it from arterial or venous source ?
• b) Does it belong to the victim or the accused ?
• c) Is it from an injury, or due to haemoptysis,
• haematemesis, menstruation or miscarriage ?
4. Who left the blood?(personal identification)
5. In connection with the cause of death, it should be seen
whether the blood contains any poison in lethal concentration
or there is any other abnormality which can be considered as
the cause of death, as in case of mismatched blood transfusion
or heamopoetic disorder.
6. In case of death, time passed after death.
7. Time passed after bleeding, i.e., time of assault or injury
(known from the age of the stain, whether recent or old)
8. Whether the bleeding was antemortem or postmortem.
 Blood Stains

• Blood that is in liquid pools should be

picked up on a gauze pad or other clean
sterile cotton cloth , air dry thoroughly at
room temperature. It should be
refrigerated or frozen as soon as possible
and brought to the Laboratory as quickly
as possible. Delays beyond 48 hours may
make the samples useless in serological
analysis.
∀ If close to the Laboratory, deliver
the samples immediately.

∀ If unable to deliver to the

Laboratory, or if the object must be
mailed, allow the stain to air dry
completely before packaging.
Do not heat the samples or place
it in bright sunlight to dry. Hang
clothing and similar articles in a
room where there is adequate
ventilation.
• Label all samples bits and package
each samples separately. If damp, allow
fabric to dry completely before
packaging.
• Handle fabrics as little as possible.
 Dried Blood Stains

∀ On clothing, if possible, wrap the item in

clean paper, place the article in a brown
paper bag or box and seal and label
container. Do not attempt to remove
stains from the cloth.
∀ On small solid objects, send the whole
stained object to the Laboratory, after
labeling and packaging.
∀ On large solid objects, if
impractical to deliver the whole
object to the Laboratory, scrape
the stain onto a clean piece of
paper, which can be folded and
placed in an envelope. Do not
scrape directly into evidence
envelope. Scrape blood from
objects using a freshly washed and
dried knife or similar tool. Wash
and dry the tool before each stain
is scraped off. Seal and mark the
 1. Naked eye examination
In naked eye examination, non-coagulated blood is
reasonably thick, viscid, and may be bright red or
slightly dark red in color depending on whether it is
from arterial source or from venous source,
respectively. A blood stain is reddish and soft in case
of recent bleeding. It is dark red or even blackish, if
old. Bleeding from arteries has a sprouting effect (jet
like ejection ) and is bright red when fresh. Bleeding
from a vein occurs passively, in drops. The drops
may have stellate appearance. Even when fresh, it is
rather dark.
Jet like ejection ( bleeding from arteries)
2 Chemical tests
These r Exclusionary tests
1. Benzidine Test
sensitive , one part from three
million.Benzidine is known as carcinogenic
2. Phenolphthalein Test (Kastle-meyer
test)
3. Ortho-tolidine test
 Benzidine test
cut out a small piece of stained material or tease out
fibres from the stained fabric and place it on a
porcelain tile.Add a drop of saturated solution of
benzidine in a glacial acetic acid then a drop of 10
volumes hydrogen peroxide.If the blood is
present, drak blue color is produced immediately.
a positive reaction is given by blood of almost any
age, blood that has been exposed to heat or cold,
blood stains treated with cleaning agents.
The test is very sensitive(positive with 1 in 1,000,000 dilution)
but not specific. any organic substance that liberate nascent
oxygen from the hydrogen peroxide can produce false positive
results. when a negative result is got, then there is no necessity
to go for any other test, for example, confirmatory test.
 Phenolphthalein test

• Also called Kastle-Mmeyer test

to a solution extracted from the stain with
distilled water, add 10 to 20 drops of
phenolphthalein reagent[ phenolphthalein 2 gm ,
sodium hydroxide 20 gm, distilled water 100 ml]
and then a drop or 2 drops of 10 volumes
hydrogen peroxide.If blood is present a pink or
purple color develops immediately.
 Ortho-tolidine test
• To a solution extracted from the stain add 2
to 3 ml of 4 percent ortho-tolidine
followed by a few drops of hydrogen
peroxide.Blue color appears if blood is
present.
 3. Microscopical and
Microchemical tests

• These are conformatory test

1. Takayama Crystal Test
2. Teichmann Crystal Test
3. Spectroscopic Test
 Takayama crystal test
• Place a small piece of suspected material on a
glass slide add 2 to 3 drops of takayama reagent[
sod.hydroxide, pyridine,glucose] and cover with a
coverslip.Pink, feathery crystals of
haemochromogen or reduced alkaline haematin
arranged in clusters, sheaves, etc. appear in one to
six minutes.Slight warming of the slide hastens the
reaction.The result is negative if crystals are not
formed in half an hour. The test give good result
even with old blood.
 Teichmann’s test
– A small crystal of sodium chloride and two to
three drops of glacial acetic acid are placed on a
small piece of the suspected stain on a glass
slide.A coverslip is applied and the acid is
evaporated by heating over a small flame.It is
examined under the microscope after
cooling.Faint yellowish-red to brownish-black
rhombic crystals of heamin or hematin chloride
arranged single or in clusters are seen if blood
is present.
Bubbles of gas are given by haematin crystals
on addition of a drop of hydrogen
peroxide.The reaction is negative if the
stain is old, is washed or treated by
chemicals,presence of too much
salt,moisture in acid and over heating.
 Spectroscopic examination
• It is the most delicate and reliable test for
detecting the presence of blood in both recent and
old stains.Less than 0.1 mg of blood is sufficient.
• The blood is dissolved in water, normal sline or
dilute ammonia and is placed in a small glass test
tube which is then kept between the spectroscope
and the source of the light.
• The extract of the blood must be dilute and
if turbid it should be filtered. The solution
of the blood has the property of absorbing
some of the rays from the spectrum
producing characteristic dark absorption
bands which vary with the type of the blood
pigment present. [see page 378]
4.The Blood is Human’s or not?—
Species Identification

• Immunization-the provocation of
immune

• Method : 1. Ring Precipitation

2. Double agar diffusion test
 Ring precipitation
• Principle :
Blood serum contains proteins in colloidal suspension
and when human serum is injected into an animal the
animal becomes immunised against these proteins and
antibodies develop in its blood.If human serum is then
brought into contact with this animal serum the antibodies
in the animal serum react with the proteins in the human
serum and a visible precipitate forms.The antibodies
causing this reaction are known as precipitin and the
animal serum is known as antihuman precipitin test.
• A rabbit or a fowl is injected with human blood
every third day for 3 to 5 injections.After this the
animal is killed and the antiserum is collected.A
suitable antiserum should react immediately or
within a minute on the 1:1000 dilution
• A white ring with well defined borders appears in
case of positive reaction.The ring is mostly
situated in the antiserum
• In case of negative reaction no ring appears
• A positive reaction should begin in 10
minutes and should be read in half an hour.
• For medicolegal purpose all the doubtful
reactions are read as negative.
 Double aggar diffusion test
• A piece of stained material or extract is
placed in a central well cut in agar gel,
while each of six well sourrounding this
conains a drop of antiserum specific for the
globulin of a particular species of
animal.The advantage of this method is that
extract can be tested against different
antisera simultaneously.
 5.Personal identification
• Who do the blood stain belong to?
• By DNA typing, we can come to a
conclusion that the blood stain belong to
someone with a very high
probability(>99.99%).
The examination of seminal fluid
as trace elements
 Civil importances
• Compensation on the ground of acquired sterility
• Disputed paternity
• Artificial insemination Legitimacy
• Compensation on the ground of failure of vasectomy
cases, leading to pregnancy of the wife
• Divorce cases: non-ejaculation amounts to impotence
 Criminal importances
• In relation to sex offence cases
a) concerning commission of sex offence
b) identification of the offender
 Physical appearance
• When dried on cloth, the area appears
slightly glistening, starchy hard in feeling,
irregular in shape and distribution, and is
whitish on dark clothes, and has
fluorescence property when examined under
ultraviolet light in a dark room
 2. ACID PHOSPHATASE test
• Acid phosphatase is present in almost all body
fluids. But its concentration in seminal fluid is
maximum (about 20 to 400 times more than normal
of about 20 units present in other body fluids).
• this is a sensitive but not specific test. this test is a
screening test, because many other body fluids also
give positive reaction, However if the test is negative
then presence of seminal fluid in the stain is
excluded.
 Confirmatory test
• Microscopic examination
the dried seminal stain extract can be
stained with some dye(methylene blue or
eosin). Tail and the posterior third of the
head of spermatozoa takes eosin or reddish
stain and the anterior two third of the head
of spermatozoa takes very light or faint,
basic or blue stain.
 Personal identification
• DNA typing
 Confirmation Personal
identification
Saliva stain 1. buccal squamous cells Nuclear DNA
2. amylase maker.
Vaginal fluid Vaginal epithelial cells Nuclear DNA
stain maker
Faecal stain Vegetable cells and Nuclear DNA
muscle fibres maker
Urine stain Urea and epithelial cell Nuclear DNA
(from urethra or bladder) maker
 Parentage testing
• When the mother of a child names a person
as to be the father of the child and the man
denies this, it remains to be proved whether
the putative father is the actual father of the
child. Apart from morphological features,
examination of genetic makers come for
consideration to settle the dispute.
 The basis of parentage testing
• 1. A gene will be present in a child, only if at
least one of the parents has it; and that,
• 2. If a locus in one parent is homozygous then its
gene must appear in the child.
• 3. A child must have a gene from each parent.

• A child must, and can only, have a gene from each

parent.
 1 2 3 4 5 6 7 8 9
10 11 12
L AF M 13
C 14
AF2 M2 C2 AF3 M3 C3
1 1 1
L AF4 M4 C4
 locus AF M C

AF: alleged father; M: mother; C: child

If there are some loci that disobey the law, we
can exclude the man as the father of the child.
The only chance of exception of the theory is
the chance of mutation which may occur in
0.1-0.2% new-borns. So at least 3 inconsistent
loci are necessary for the exclusion
conclusion.
Blood group Red Cells Antigens Antibodies

A A Antigens Anti-B antibodies

B B Antigens Anti-A antibodies

AB AB Antigens None

O None Anti A and Anti B antibodies

 Heredity of the blood group
Parents' Blood Types Possible Children Impossible Children

A&A A, O B, AB

A&B A, B, AB, O none

A & AB A, B, AB O

A&O A, O B, AB

B&B B, O A, AB

B & AB A, B, AB O

B&O B, O A, AB

AB &AB A, B, AB O

AB & O A, B AB, O

O&O O A, B, AB
 Inheritance of MN types
Group of children
parents Possible Impossible
_______________________________________
M*M M N,MN
M*MN M,MN N
M*N MN M,N
N*N N M, MN
N*MN N ,MN M
MN*MN M,N,MN None
 Paternity Test Case
GM M C AF PI
ABO B B O 1.3632
LDLR BB AB AB 2.3474
GYPA BB BB AB 1.1382
HBGG BB BB BB 1.8109
D7S8 BB AB AA 1.6428
GC AB AB BB 1.5437
DQA1 3，3 1.2,3 1.2,1.3 2.6838
CSF1PO 12,12 10,12 10,12 2.0559
TPOX 9,11 11,11 8,11 1.6567
THO1 9,9 6,9 6,6 8.8810
D16S539 8,10 9,10 9,11 1.6119
D7S820 10,12 11,12 11,12 1.6835
D13S317 9,10 9,10 8,9 1.8031
PI(总) 6644.1011
 PI

• The paternity index (PI) compares the

likelihood[probability] that a genetic marker
(allele) that the alleged father (AF) passed
to the child to the probability that a
randomly selected unrelated man of similar
ethnic background could pass the allele to
the child.