UNIT 8 SEROLOGY* Serology

Contents

8.0 Introduction
8.1 Bloodstains
8.2 Saliva
8.3 Urine
8.4 Semen
8.5 Patterns of Bloodstains
8.6 Summary
8.7 References
8.8 Answers/Hints to Check Your Progress

LEARNING OBJECTIVES

After studying this unit, you shall be able to learn about:

 bloodstain and bloodstain patterns;

 methods of individualisation of bloodstain;
 identification and individualisation through saliva;
 identification and individualisation through urine;
 identification and individualisation through semen; and
 bloodstain pattern analysis and its importance in forensic investigations.

8.0 INTRODUCTION
The goal of forensic science and crime scene reconstruction is to seek the
truth. There is no other agenda for the analyst. We revisit what we hope is a
not-too-distant past and make an attempt to recreate the incidents that
unfolded. The task is anything but simple and the tools employed are all of
the forensic disciplines.

Each area of forensic science provides insight and a highlight into this
past. Each has its place in evaluating the outcome of crime. “who” of
crime related information provided by the majority of the forensic
disciplines. Fingerprints, palm prints, serology, fiber evidence, hair
evidence and trace evidence allow the investigator to link people or
objects with a scene of incident. The “what” of crime has always been
the primary link to forensic pathology provides insight into some of the
events that occurred during the incident. Bloodstain spatter analysis is a

*
Contributor: Dr Rajesh Singh, Dy. Director (Biology), State Forensic Science Laboratory,
Rajasthan, Jaipur. 203
Human discipline that has reawakened to its role in modern forensic science as
Identification:
Establishing an alternative method of revealing the “what” of crime. Simply it links
Identity-II the various events of a violent incident.

In this capacity, bloodstain pattern analysis acts as a critical bridge between

classical forensics and crime scene reconstruction.

8.1 BLOOD STAIN

As a forensic scientist we should always be highly alert and attentive towards
each and every aspect of the case. Blood is important evidence present in all
types of cases like murders, sexual assaults, hit and run, etc., which are
highly related to the crime and give important information about the victim
and perpetrator.

As you know blood forms 8 % of the body weight, it’s likely to be said that
the blood forms a major part among the body fluids. So before identifying
whose blood it is, its first need is to identify whether the substance is blood or
not. If we as an investigator find a dark spot on the floor of a crime scene, the
first step is to find out if the spot is blood, mainly human blood. If the spot
turns out to be spilled ink or paint, no further testing is likely necessary.

Thus to identify the substance as blood or not, certain tests are performed.
These are chemical examinations which are based on specific chemical
reaction with the blood.

The chemical detection of blood is usually based on one of four classes of

methods which are:

Preliminary or Presumptive Test

Crystal Tests
Catalytic Tests
Instrumental Methods

8.1.1 Preliminary or Presumptive Test

Preliminary test, also known as presumptive tests are quick, simple tests on
evidence left at the crime scene to get a clue as to whether additional testing
is needed. A presumptive test either proves that further analysis is not needed
or indicates to confirm it thoroughly. Various presumptive tests for blood are
as follows:

Adlers’ or Benzidine Test: Adlers’ test is the earliest test for blood which
was developed in 1904 by Oskar and Rudolf Adler. The Adlers’ test made use
of benzidine (p-diaminodiphenyl), as it identifies the substance as blood by a
characteristic oxidation reduction reaction.

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Kastle-Meyer Test: The Kastle-Meyer reagent is made by mixing potassium Serology

hydroxide, phenolphthalein, and zinc dust. When hydrogen peroxide and the
reagent are added to blood, the hemoglobin in the blood catalyses the
conversion of phenolphthalein to its deep pink configuration.

8.1.2 Crystal Tests

The crystal tests, which are now rarely used, are all based on the formation of
haemoglobin derivative crystals such as haematin, haemin and
haemochromogen. Two common confirmatory tests on bloodstains are the
Teichmann and Takayama tests which are as follows:

Teichmann Test: The Teichmann test is much older, having been developed
or invented in 1853 by Polish anatomist Ludwig Karl Teichmann. In the
Teichmann test, the reagent is a mixture of glacial acetic acid and sodium
chloride. The reagent causes hemoglobin molecules to cleave, producing
brownish crystals of pure hemin that have a violet, almost black, sheen.
(Hemin is the form of heme that contains the Fe3+ ion.)

Takayama Test: Probably the best known of the crystal tests was developed
by Japanese criminologist Masaeo Takayama in 1912. An alkaline solution
of pyridine is added to the stain and, if blood is present, salmon-pink color
crystals of a complex between pyridine and haem form as the slide is
warmed.

8.1.3 Catalytic Tests

These methods depend on the fact that the haem group of haemoglobin
possesses a peroxidase-like activity which catalyses the breakdown of
hydrogen peroxide. The oxidizing species formed in this reaction can then
react with a variety of substrates to produce a visible colour change. Among
substrates in common use are benzidine and various substituted benzidines,
ortho-tolidine, leucomalachite green, leucocrystal violet and phenolphthalein.
The reaction with 3-aminophthalhydrazide (luminol) to form a luminescent
rather than a coloured product is also a catalytic test. The catalytic tests are
extremely sensitive (blood can be detected to dilutions of about 1 in 100,000
times), but are subject to a number of interferences and are therefore not
totally specific for blood. Substances which can interfere include enzymes
such as catalase and peroxidases (which can occur in both plant and animal
materials), oxidising chemicals and metals – in particular copper and iron.
But it doesn’t mean it is useless in forensic field. It helps to identify and
emerge the hidden evidence.

8.1.4 Instrumental Tests

The blood obtained from the crime scene is usually not in wet condition, it
gets dried up with the passage of time. This dryness of blood results in
rupture of red blood cells (RBCs) and it produces difficulties in analysis.
However, to overcome the problem, methods are available for use on dried
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Human blood samples.
Identification:
Establishing
Identity-II The two most popular tests are:

Absorption-inhibition test
Absorption-elution test

Both tests take advantage of the tendency of antibodies to attach themselves

to and agglutinate with antigens.

8.1.5 Individualisation of Blood Stains

The potential for the individualisation of blood is based on typing of proteins
and enzymes. Blood proteins and enzymes have the quality polymorphism or
of being iso-enzymes, which means they exist in several forms and variants.
Generally people are familiar with at least one common polymorphism in
blood. There are several methods available for individualisation of
bloodstain; we will discuss here the important and economical methods.

8.1.5.1 Serum Protein Polymorphisms

A number of different proteins and enzymes are synthesised in the cells of a
single organism. They each have their own distinctive properties and
functions and together they define and control complex pattern of metabolic
and developmental processes which characterise the species and the
individual.

Proteins are composed of one or more polypeptide chains which are made up
of the long strings of amino acids linked by polypeptide bonds in a specific
linear order. 20 different amino acids may be present and typical polypeptide
chains have sequence 100-500 amino acids long, so the number of possible
structures is enormous. The sequence of base pair in a given gene determines
the sequence of the amino acids in a corresponding polypeptide chain. Thus
the structure and proteins of all individual are controlled by the base pair
sequence of his genes.

The haptoglobin groups are polymorphic in all human populations; two

alleles Hp1 and Hp2 accounting for three common phenotypes Hp 1-1, Hp 2-1,
Hp 2-2.

8.1.5.2 Haptoglobin (Hp) System

Haptoblobin is a glycoprotein that combines with free haemoglobin from the
lysed red cells, thus preventing its excretion by the kidneys. The haptoglobin
groups are polymorphic in all human population. The haptoglobin (Hp)
molecules consist of four chains (αβ)2 linked with disulphide bonds. The
HPα- chain is polymorphic with three common phenotypes Hp1-1 (HP1),
Hp-2-1 (HP2, 1) and Hp 2-2 (HP2) that can be readily detected by
conventional starch gel electrophoresis.

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Technique: Serology

Electrophoresis is performed using 10%-13% hydrolysed starch gel and

following buffers: -

1) Tank buffers (Borate, pH 8.1)

a) Boric acid = 18.55 gm
b) Sodium hydroxide= 2.0 gm
c) Dissolved in 1 litre distilled water
2) Gel buffer (Tris/ citrate pH 8.6)
a) Tris = 9.20 gm
b) Citric acid = 1.05 gm
c) Dissolved in 1 litre distilled water

3) Sample application: Mix one drop serum or plasma with little

haemoglobin to make it pink (about 0.4 ml serum or plasma with 5µl
haemoglobin) and load the mixture on Whatman No. 3 filter paper insert.
Place inserts in gel 6 cm from cathode.

4) Electrophoresis: 6 V/cm for 3 to 4 hr.

5) Staining :

i) Benzidine staining method:

Prepare fresh Benzidine staining solution as follows:

a) Benzene =0.2 gm
b) Distilled water = 100 ml
c) Warm to dissolve benzidine, then add glacial acetic acid = 0.5 ml
d) Hydrogen peroxide (30%) = 0.2 ml

Drop the solution by pipette over sliced gel and note the results for
haptoglobin (Hp) phenotypes.

ii) Dianisidine staining method:

a) Staining buffer (1.5 M Acetate buffer, ph 4.6)

Sodium acetate (anhydrous) 12.3 gm. dissolve in 50 ml distilled water

and adjust pH to 4.6 with glacial acetic acid. Adjust final volume to 100
ml.

b) Stain:

O- Dianisidine = 35 mg
Ethanol = 35ml
Staining buffer = 35 ml
207
Human Distilled water = 80 ml
Identification:
Establishing Hydrogen peroxide (add just after use) = 1 ml
Identity-II
Pour the stain on the sliced gel placed in a box. Cover the box and rock it in a
few times. Leave until the brown bands are intense. Note the results for
haptoglobin. The gels may be stored up to few weeks after washing in 2 %
acetic acid and placing in 2.5% teepol.

6) Reaction Mechanism:

Haptoglobin has the property of binding the haemoglobin and forming a very
stable complex. The separation of haptoglobin types is determined by the size
of the molecules of the haemoglobin- haptoglobin complex, which is in turn
dependent on the type of the haptoglobin present.

The haeme group of the haemoglobin exhibits a peroxidase like activity

which is capable of catalysing the breakdown of hydrogen peroxidase and
thus liberating the nascent oxygen. The nascent oxygen thus liberated
oxidises the leucobases, such as the benzidine sulphate into a rich blue-
coloured benzidine salt. The proposed mechanism of the reaction is given
blow:

Figure: 1 Reaction of Benzidine

The other half of the gel may be stained with amino black 10 B solutions for
transferrin as follows:

a) Amino acid 10 B = 0.1 gm

b) Acetic acid = 10 ml
c) Methanol = 90 ml

After 1-2 min staining wash gel with methanol (90%) and acetic acid (10%)
solution overnight. Note the results for transferrin (Tf) phenotypes.

8.1.5.3 Red Cell Enzyme Polymorphism

A variety of different enzymes and proteins are synthesised in the human
body, and the primary amino acid sequence of each of their distinctive
polypeptide chains is coded in the DNA of a separate gene locus. Thus there
must be a large number of so-called “structural” gene loci in the genetic
make-up of every individual. In addition, as a result of mutational events
there may occur at any given gene locus a series of different alleles each of
which determines a structurally distinct form of the particular polypeptide
chain. Therefore the extent to which individual members of a population
differ from one another in the structural characteristics of the enzymes and
208
proteins they synthesise will depend on the numbers of different alleles which Serology

are present at these different loci, and on the relative frequencies with which
they occur.

Studies on a variety of different enzymes and proteins have led to the

discovery of a large number of structurally variant forms which are
genetically controlled. The inherited variants of different enzymes and
proteins which we find among human populations today must be attributed to
specific gene mutations which occurred in single individuals among our
ancestors in earlier generations. Thus it appears that at almost all loci coding
for enzymes and proteins a large number of different mutant alleles which
may be generated by separate mutations; actually exist among living
members of our species.

8.1.5.4 Enzyme Polymorphism

The concept of enzyme polymorphism is based on the occurrence of variant
forms of an enzyme controlled by two or more alleles in frequencies
exceeding 1 percent. Enzyme variants may originate from duplications and
deletions of the genetic material, but most mutations leading to amino acid
substitution may cause alterations in the electric charge, kinetics properties,
and rate of synthesis or stability of the enzyme. Makert and Moller (1959)
coined the term isozyme or isoenzyme to describe the existence of different
molecular forms of an enzyme which catalysed the same biochemical
reaction, but different in their electrophoretic properties.

The technique most commonly used to detect enzyme polymorphism is

electrophoresis in starch gels, although agarose and polyacrylamide are also
suitable gel media. The amount of material (usually blood) required is small
and the technique is convenient for screening a large number of samples in a
relatively short time period. Some important techniques are:

a) Adenylate Kinase (AK) System

Adenylate kinase, also called ATP: AMP phosphor transferase occurs in red
cells as well as in muscles and other tissues. It catalyses the following
reversible reaction.

Adenosine diphosphate Adenosine triphosphate +

Adenosine monophosphate (2 ADP ATP + AMP)
Three different groups of isozymes of adenylate kinase – AK1, AK2 (Khoo
and Russell, 1972) and AK3 (Wilson et al., 1976) determined by three
separate gene loci AK1, AK2 andAK3 have been identified. Genetically
determined polymorphism of AK1, isozymes in red cells was first
demonstrated by Filders and Harris (1966) and three distinct phenotypes AK
1, AK 2-1, and AK 2 (AK1 1; AK1 2,1 and AK1 2) could be found by starch
gel electrophoresis. They showed with the help of family and mother/child
studies these to be genetically determined by two alleles AK1 (AK*1) and
AK2 (AK*2). These results were confirmed by Harris 1969; Harris et al. and
many more.
209
Human Technique:
Identification:
Establishing
Identity-II Electrophoresis is performed using 10-12% hydrolysed starch gels and the
following buffers:
1) Tank Buffer (pH 7) :
Citric acid 86.16 g
Dissolve in about ½ 1 distilled water and leave in refrigerator for about 2 hrs.
Adjust pH to 7 with strong NaOH solution. Make up the total volume to 1
liter with distilled water.
2) Gel Buffer (pH 7) :
L-Histidinemonohydrochloride 1.05 gm.
Dissolve in about 50 ml distilled water and adjust pH to 7 with NaOH
solution. Make up the total volume to 100 ml with distilled water. Dilute 1:10
before use.
3) Sample Application :
Apply haemolysates on Whatman No. 3 filter paper inserts at center of the
gel.
4) Electrophoresis : 5 V/cm for 16 to 18 hrs. at 40˚C (till haemoglobin runs
towards cathode about 4.5 cm from the origin). After electrophoresis, cut the
gel at 4 cm from the origin towards anodic end and 8 cm towards cathodic
end.
5) Staining :
(i) Staining Buffer (0.1 M, pH 8.0):
Tris- 1.2 gm
Dissolve in about 50 ml distilled water and adjust pH to 8.0 with 1 N HCl.
Make up the total volume to 100 ml with distilled water.
(ii) Agar Gel :
Dissolve 200 mg agar in 22 ml staining buffer by heating till it is clear and
then cool it to about 550˚C.
Staining Mixture
Glucose 40 mg
Magnesium chloride 82 mg
Adenosine-5 – diphosphate 10 mg
ADP (disodium-salt)
NADP 7 mg
MTT 2.5 mg
PMS 2.5 mg
Glucose-6-phosphate dehydrogenase (G6PD) 10 µl
Hexokinase 2 µl

Dissolve in 5 ml staining buffer, mix with agar gel, and pour it immediately
over the sliced starch gel. When overlay is set incubate the gel in darkness at
370˚C for 30 min. Read the result for AK.
210
6) Reaction Mechanism : Serology

AK converts ADP into ATP and AMP, and since glucose and hexokinase are
present in the reaction mixture, glucose-6-phosphate is formed with the
regeneration of ADP. In the presence of G6PD and coenzyme NADP,
glucose-6-phosphate thus formed is oxidised to 6-phosphogluconate and
during this reaction NADP is reduced to NADPH. NADPH in the presence of
PMS, in turn reduces yellow MTT to purple formazan which is thus
deposited at the site of AK activity. Therefore AK isozymes are seen as
purple band against a yellow background.

Figure: 2 AK isozymes-sequence of biochemical reactions involved.

b) Adenosine Deaminase (ADA) System

Adenosine deaminase shows an electrophoretic polymorphism genetically

determined by the occurrence of two common alleles ADA1 (ADA*1) and
ADA2 (ADA*2) at an autosomal locus; correspondingly there are 3
phenotypes: ADA 1, ADA 2 and ADA 2-1 The ADA1 and ADA2 alleles
have been observed with frequencies of about 95 per cent and 5 per cent,
respectively in most European populations, ADA2 is relatively less frequent
(approximately 2 per cent) in Africans (Blacks), but quite frequent
(approximately 12 per cent) in Indians (Bhasin and Fuhrmann, 1972a). The
211
Human presence of high ADA2 allele frequency in India suggests that perhaps this
Identification:
Establishing allele is of local origin proliferating in the region on account of some
Identity-II selective advantage.

The rare ADA alleles encountered in Indian populations include ADA3

among Jats of Punjab (Singh et al., 1974); ADA4 among Muslims of Delhi
(Papiha et al., 1976) and Ghanchi and Anavil of Gujarat (Papiha et al., 1981);
ADA6 among Vadabalijas of Orissa (Reddy et al., 1989)

Technique:

Electrophoresis is performed using 10-12% hydrolysed starch gels and the

following buffers:

1) Tank buffer (pH 5.0) :

Citric acid 21gm

Dissolve in about ½ l distilled water and adjust pH to 5 with NaOH. Make up

to 1l.

2) Gel Buffer (pH 5.0) :

Use 1:20 dilution of tank buffer.

3) Sample Application: Haemolysates are loaded on Whatman No. 17 filter

paper inserts which are placed in the middle of the gel, about 10 cm from
cathode end.

4) Electrophoresis: 5-7 V/cm for 16 to 18 h at 4˚C (till haemoglobin runs

towards cathode about 3-4 cm from origin).

5) Staining :

(i) Staining Buffer (0.06M Tris, pH 8.0) :

Tris 727 mg

Dissolve in 50 ml distilled water, and adjust pH to 8 with dilute HCl. Adjust

total volume to 100 ml with distilled water.

(ii) Agar Gel

Dissolve 200 mg agar in 22 ml staining buffer by heating till it is clear and

then let it cool to about 55˚C
Staining Mixture:
Adenosine 10 mg
Sodium arsenate 40 mg
MTT 2.5 mg
PMS 2.5 mg
Xanthine Oxidase (XOD) 10 µl
212 Nucleoside Phosphorylase (NP) 10 µl
Dissolve stain in 5 ml staining buffer, mix with agar gel and immediately Serology

pour it over the sliced starch gel. Keep it in dark and when overlay is set
incubate in darkness at 37˚C for 45-60 min. interprets the results for ADA.

6) Reaction Mechanism:

Adenosine is converted to inosine by ADA at the sites of enzyme activity and

this in turn is converted to hypoxanthine in the presence of nucleoside
phosphorylase and arsenate. Hypoxanthine is then oxidized by the action of
xanthine oxidase, and during this reaction MTT in the presence of
phenazinemethosulphate (PMS) is reduced to form purple insoluble
formazan. The ADA isozymes are therefore seen as purple bands against a
yellow background. The sequence of biochemical reactions involved is given
below:

Adenosine

Adenosine deaminase

Inosine

Sodium Nucleoside
arsenate phosphorylas
e
Hypoxanthine
MT
(Yellow)
Xanthine
PM
Formazan (purple)

Xanthine

Figure: 3 ADA isozymes show the changes of color band in biochemical reaction.

Check Your Progress 1

1) How will you identify whether the fluid is blood or not?

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213
Human 2) Explain common confirmatory tests on bloodstains.
Identification:
Establishing
Identity-II ……………………………………………………………………………
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3) Discuss briefly individualisation of blood stains.

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8.2 SALIVA
Saliva is a viscid, watery fluid, secreted into the mouth by the salivary glands
that functions in the tasting, chewing, and swallowing of food, moistens the
mouth, and starts the digestion of starches.

8.2.1 Composition
Salivary fluid is an exocrine secretion consisting of approximately 99%
water, containing a variety of electrolytes (sodium, potassium, calcium,
chloride, magnesium, bicarbonate, phosphate) and proteins, represented by
enzymes, immunoglobulin’s and other antimicrobial factors, mucosal
glycoproteins, traces of albumin and some polypeptides and oligopeptides of
importance to oral health.

There are also glucose and nitrogenous products, such as urea and ammonia.
The components interact and are responsible for the various functions
attributed to saliva.

Total or whole saliva refers to the complex mixture of fluids from the
salivary glands, the gingival fold, oral mucosa transudate, in addition to
mucous of the nasal cavity and pharynx, non-adherent oral bacterial, food
remainders, desquamated epithelial and blood cells, as well as traces of
medications or chemical products. At rest, without exogenous or
pharmacological stimulation, there is a small, continuous salivary flow (SF).
Whereas, stimulated saliva is produced in the face of some mechanical,
gustatory, olfactory, or pharmacological stimulus, contributing to around
80% to 90% of daily salivary production. A healthy person’s mean daily
saliva production ranges from 1 to 1.5L.

214
8.2.2 Collection of Salivary Stains Serology

A. Questioned Samples

 Dry Stains:

a) Swabbing: This method is best to apply on non-absorbent surfaces.

Lightly moisten sterile swab with distilled or sterile water is rubbed over
stain while rotating. Swab is then allowed to air dry. Combination of first
moistened swabbing followed by second dry swabbing is recommended.
Both swabs are examinee in laboratory.

b) Cutting: Stained portion should be cut from item.

c) Scraping: Stained portion is scrubbed into clean piece of paper using

clean blade.

d) Lifting: Works for non-absorbent surface; use fingerprint lifting tape

that does not interfere with DNA testing to lift stain; lifted stain should
be covered with a piece of lifter’s cover.

 Wet Stains

a) Swab: Sample should be absorbed onto sterile cotton swabs. Stain

should be concentrated on tip and allowed to air dry.

b) FTA paper: Sterile disposable pipetteshould be used to collect liquid

saliva. It should be spotted on FTA paper and then allowed to air dry.

B. Reference Saliva (Buccal Samples)

a) Swab: Swabs should be placed inside of cheek using two swabs. Swabs
should be kept on rotating during collection. Swabs are then allowed to
air dry.

b) Filter paper: Subject’s saliva sample should be placed on marked area

of filter paper and allowed to air dry.

8.2.3 Examination of Saliva

After collection of saliva sample we perform following test for examination
of saliva sample

a) Starch- Iodine Test:

Reagents Preparation:

1) 0.5% soluble starch solution (50 mg soluble starch / 10 ml H2O).

2) Lugol’s iodine solution.

Procedure:

1) Place 3 tubes in a rack and add the following:

215
Human a) In the first tube, place a 5 mm x 5 mm piece of sample to be tested.
Identification:
Establishing
Identity-II b) In the second tube, place a similar sized unstained control piece.

c) In the third tube, place a 5 mm x 5 mm piece of a known saliva stain.

2) Add 3 drops of soluble starch solution to each tube.

3) Mix, cork and incubate the tubes for 1 hour at 37⁰C.

4) Add 2 drops of Lugol’s iodine and note the colour formed.

5) A dark blue starch-iodine complex should be observed in the second and

fourth tubes. The absence of the dark blue colour indicates that the starch
has been hydrolyzed and is no longer available for complexing.
Therefore, the lack of blue colour is a positive result for amylase activity,
indicative of the presence of saliva. This is not a confirmatory test for
saliva.

Notes:

 To dissolve the starch, heat the solution to a boil. Allow solution to cool
to room temperature. Always use soluble starch. Do not use hydrolyzed
starch.

 Starch solution must be prepared fresh daily. Never use starch solution
older than 24 hours.

Check Your Progress 2

4) What is the composition of Saliva?

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5) How to collect the Salivary Stains from crime scene?

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6) Discuss starch- iodine test.

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8.3 URINE Serology

The fluid and dissolved waste substance excreted by the kidneys constitute
urine. Urine is a liquid byproduct of the body secreted by the kidneys through
process called urination and excreted through urethra.

8.3.1 Physical Properties

Urine is a transparent, yellowish fluid; shade however, depends on its
concentration. Its color is due to pigment Urochrome derived from the
breakdown of hemoglobin from the worn out RBCs. It is hypertonic to blood
plasma. Its specific gravity ranges between 1.003 & 1.04 and pH ranges from
5 to 8.

Urine has a characteristic unpleasant odour. If allowed to stand, urea is

degraded by bacteria to ammonia which imparts a strong smell to urine.

8.3.2 Chemical Composition

Urine consists of water, organic and inorganic substances.Water alone forms
about 95% of it and other substances form only 5%. The organic substances
are mainly nitrogenous compounds but small amounts of non-nitrogenous
compounds are also present.

Nitrogenous organic compounds include urea, uric acid, creatinine and

hippuric acid. Out of all these, urea is the principle component of human
urine. Non-nitrogenous organic compounds include Vitamin C, oxalic acid,
phenolic substances and traces of glucose.
The inorganic substances include ammonia & mineral salts such as chlorides,
sulphates and phosphates of sodium, potassium, calcium & magnesium.
Sodium chloride is the principal mineral salt of the urine.
Urine also contains some other substances like pigments & drugs coupled
with some epithelial cells and leucocytes.

8.3.3 Collection of Urine Samples

Urine found at the scene of crime can be removed by dropper or gauge and
placed in a sterile test tube or other container. If the sample is present on
cloth, entire article should be submitted.

8.3.4 Examination of Urine Stains

1) Physical Examination- A suspected urine stain may fluoresce pale
yellow or pale blue, when viewed under long and short wave Ultraviolet
light. Safety glasses, which absorb UV radiation, must be worn when
viewing material for fluorescence.

2) Odour test-The characteristic odour of urine may be detected by placing

a small sample of the stain in a test tube and heating it gently over flame.
Avoid scorching the test material. 217
Human 3) Urea Nitrate Crystal test- An aqueous extract of stain is made and a
Identification:
Establishing thin film of it is made on a microscopic slide. Add one drop of
Identity-II concentrated. Nitric Acid (HNO3) and cover. In presence of urea,
hexagonal stacked crystals of urea nitrate are formed.

4) Gee’s method for detection of Urea- A portion of the suspected stain is

extracted with acetone and the acetone extract is concentrated by
evaporation. The concentrated extract is filtered and evaporated to
dryness. A few drops of acetone is added to the residue and stirred with a
narrow glass rod. A drop of the solution thus prepared is taken on a
microscopic slide and again allowed to evaporate. At this stage
separation of urea crystals (long colorless 4 or 6 sided rhombic prisms)
may be observed. A glass rod dipped in concentrated nitric acid is lightly
run across the surface of the crystal line area. Formation of characteristic
urea nitrate crystals (colorless rhombic or 6 sides tiles) may be observed
under microscope indicating the presence of urea in the suspected stain.

5) Detection of Creatinine- A concentrate of the stain is placed on a strip

of chromatographic paper and it is treated with 2N sodium hydroxide
solution and after it one drop of 5% picric acid is added to it. The
development of red-orange color indicates the presence of creatinine in
the stain.

6) Indican test- This test though not always positive, may be undertaken
for the identification of urine in addition to the above tests. 1ml of
resorcinol reagent (1gm resorcinol in 20ml of ethyl alcohol) is added to a
small quantity of aqueous extract of the stain followed by the addition of
1ml of cupric bromide. The mixture is extracted with amylacetate. Red
coloration of the extract indicates the presence of indican (a potassium
salt).

8.3.5 Determination of Species

Determination of origin of urine stain by precipitin method has not been
found satisfactory. However, Popielski, Brzecka and Szczecin (1963)
described a method of concentrating urinary proteins and thereafter
subjecting that concentrated urine proteins to precipitin test by agar gel
double diffusion method for the determination of species.

8.3.6 ABO Blood Grouping

ABO Blood grouping of urine stains can yield useful results when
absorption-elution test is employed.

8.3.7 Individualisation from Urine Samples

The use of biological samples and bodily fluids as sources of DNA for
identification has been thoroughly investigated and documented. Urine
however, has not been heavily studied as a potential source of DNA for
218 forensic identification purposes.
Urine is not considered an ideal source of DNA due to low concentration of Serology

nucleated cells present in human urine. The nucleated cells found in urine are
typically white blood cells and epithelial cells.

Check Your Progress 3

7) What is the Chemical composition of urine?

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8) Describe the Test methods for urine samples.

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9) How will you determine the Species or origin?

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8.4 SEMEN
Seminal fluid is a complex minute of glandular secretion. Seminal fluid
accounts for approximately 60% of the ejaculate.

Seminal fluid contains the “flavin” that causes semen to fluorescence under
the ultra-violet light.
Prostatic fluid accounts for approximately 30% the ejaculation. This portion
of the semen contains high concentration of acid phosphatase (AP) and
prostate specific antigen (PSA). Both are very useful to the forensic analyst
for detection of semen in case of rape, unnatural offence, hanging, etc. It is an
alkaline fluid with pH=7.4.
A vasectomy is the surgical removal of a bilateral segment of the ducts
deferens. Aspermia is the condition that causes males to produce no
spermatozoa. However, seminal fluid continues to secrete. 219
Human 8.4.1 Biological Characters
Identification:
Establishing
Identity-II A typical ejaculation releases 2-5 ml of semen that contains seminal fluid and
sperm cells i.e. spermatozoa. A normal sperm count ranges from 107 – 108
spermatozoa per millilitres of semen.

Each testis is composed of numerous coiled seminiferous tubules in which

the spermatozoa are found from spermatogonia. The process of generating
spermatogonia is referred to as spermatogenesis.

The spermatozoa are then transported and stored at the tubular network of the
epdidymis where they undergo functional maturation. Then through
epdidymis it transfers to ejaculatory ducts. Then spermatozoa then follow the
ejaculatory duct into the prostatic urethra where they are joined with
secretion of prostate.

8.4.2 Structure of Spermatozoa

Human spermatozoa have three distinct regions:

1) Head
2) Middle piece
3) Tail

Figure 4: Structure of Spermatozoa

Source: https://brainly.in/question/38704167

The head is shaped like flattened ellipse and contains a nucleus with densely
packed chromosomes. At the tip of the head is the acrosomal cap, a
membranous compartment containing enzyme essential for fertilization. The
neck is also observed in some type of spermatozoans. They consist of
centrioles one proximal centriole and other distal centriole.

The middle piece: consists of apical part of the apical filament surrounded by
a tightly coiled spiral sheath of elongated mitochondria. The mitochondria
contain oxidative enzymes and provide energy for sperm motility. The tail or
flagellum consists of a central axial filament, thin layer of cytoplasm and an
outer smooth plasma membrane. It is 45-50 µ in length.

8.4.3 Enzymes and Proteins Present in Seminal Fluid

8.4.3.1 Acid Phosphates (AP)
It consists of a group of phosphates with optimal activity is an acidic pH
environment. The greatest forensic importance of AP is that the prostate is
the most abundant source of AP and contributes most of the activity present
in semen. AP levels in semen are not affected by vasectomies.

220
8.4.3.2 Prostate Specific Antigen (PSA) Serology

PSA is a major protein present in seminal fluid at concentration of 0.5-2.0

mg/ml. PSA is produced in the prostate epithelium and secreted into the
semen.
PSA is a protein that has a molecular weight of 30 KDa and is thus also
known as P30. It is responsible for hydrolyzingsemenogelin (Sg), which
mediates gel formation in semen.
PSA is the member of the tissue kalikrein family and is coded by the KLK3
locus located on chromosome 9. In addition to PSA, outer tissue kalkarines
encoded by KLAk 2 and KLK4 loci.
The half-life for PSA in a dried semen stain is about 3 years at 370C. Wet
conditions may reduce half-life.

8.4.4 Examination of Semen and Seminal Stains

8.4.4.1 Physical Examination:

Colour: Thick, yellowish white, glairy, opalescent, secretion having a
characteristic odor known as seminal odor.
Texture: On touch, seminal stains are starchy.
Appearance: Garments sent for forensic examination are usually dirty
having variety of stains, in natural light some stains are reddish coloured,
while others are brown, yellow or faint grey in colour. These are often mixed
with stains of blood vaginal discharge, urine and semen, so as to restrict the
investigation to seminal stains only, preliminary examination is done under
filtered UV light. The fluorescence of the seminal stains is of a bluish white
colour and such stains should be selected for further examination.

8.4.4.2 Presumptive Test:

Acid Phosphate Test: Sodium alpha-naphthyl phosphate method

Reagent Preparations:
Buffer
Glacial Acetic acid 1 ml
Sodium acetate anhydrous 2g
Distilled water 100 ml
Step - 1 Reagent
Buffer 50 ml
Sodium alpha-naphthyl Phosphate, 0.25% (w/v) 126mg
Step - 2 Reagent

Buffer 50 ml
Naphthanildiazo blue B, 0.5% (w/v) 250mg

221
Human Step 1 Reagent and Step 2 Reagent can be made up in bulk and aliquoted into
Identification:
Establishing test tubes and frozen. When needed, one tube of each reagent can be thawed
Identity-II under warm running water for use.

Procedure:

1) Place a small piece (2 x 2 mm) of suspected seminar stain material on

Whatman filter paper or other suitable test paper. Use proper standards
and controls including positive, negative and unstained controls; see
below.

2) Add 1-2 drops of Step 1 Reagent and allow to react for 30 seconds. (No
colour should develop at this stage).

3) Add 1 drop of Step 2 Reagent. Record the result after 10 seconds.

4) A positive reaction is recorded upon rapid development of a purple

colour, which is indicative of semen. This is not a confirmatory test for
semen.

8.4.4.3 Confirmatory Test: Microscopic Examination

Upon obtaining a positive preliminary test for acid phosphates, the suspected
stain can be extracted as follows:

1) Cut a small portion (1 cm2 maximum) of the stain and place in a test
tube.

2) Add a few drops of acidulated (slightly acidic) water to cover the stain.

3) Agitate the stain on a vortex, or in an ultrasonic cleaner bath or

manually, by flicking the tube. This will aid in freeing the spermatozoa
from the dried stain.

4) When the solution is cloudy, withdraw the liquid with a pipette and place
into a disposable 400ul plastic centrifuge tube and centrifuge in a
microfuge for 30 seconds.

5) Carefully withdraw the supernatant, which contains soluble group-

specific substances, enzymes and other solutes from seminal plasma.

6) Collect the button of cellular and other insoluble components from the
tube and place on a clean-labeled microscope slide.

7) Fix in dilute H2SO4 acid and dry. It is now ready for staining.

TANK BUFFER Tris 0.208 M 25.2 g.

(pH 9.1) EDTA (free acid) 0.0086 M 2.5 g.
Boric acid 0.0307 M 1.9 g.
Distilled water 1L
GEL BUFFER Same as tank buffer
(pH 9.1)
222
 Serology
SUPPORT MEDIUM 1% AGAROSE, mr = 0.25, E25 or Sigma Type II.
AMOUNT 3.5 ml for a 1” x 3” slide / 7 ml for a 2” x 3” slide
REQUIRED
2% STOCK 10 grams of agarose is added to 500 ml distilled water in a
AGAROSE large flask. Heat to boiling on a constant stirring hot plate
until all the agarose is dissolved. Pour into a plastic
sandwich box and store upside down in the refrigerator.
Blocks of 2% stock will be weighed out for later use.
1% AGAROSE 50 grams of Melt stock agarose and gel buffer together
2% stock and pour 3.5 ml quantities into 10 x 75 test
agarose tubes. Allow to cool, cork and store at
o
50 ml of gel 4 C.
buffer
TEMPERATURE Room
&CONDITIONS Temperature.
VOLTAGE AND DAY: 120V for 30 minutes (approximately 30 mA for 1x
DURATION 3” slide)
STAIN (0.1% Naphthalene Black 1.2g
AMIDO BLACK) 10B 1L
Methanol 200 ml
Glacial acetic acid 1L
Distilled water
DESTAIN Methanol 1L
SOLUTION Glacial acetic acid 200 ml
Distilled water 1L
CONTROLS Liquid semen diluted 1:50, 1:100, 1:200 with saline.
Extract of unstained control (whenever possible to obtain).
Saline black run against anti-P30.
Cell free extract of questioned stain run against saline
instead of anti-P30.

Check Your Progress 4

10) What is the forensic significance of semen found in crime scene?

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223
Human 11) Explain the test for semen identification.
Identification:
Establishing
Identity-II ……………………………………………………………………………
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12) Describe the Structure of Spermatozoa.

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8.5 PATTERNS OF BLOODSTAINS

Bloodstain patterns are defined as a deposition of blood on a surface or by a
surface contacting blood is known as bloodstain. And a grouping or
spreading of bloodstains that indicates through regular or repetitive form,
order, or arrangement the manner in which the pattern was deposited is
known as bloodstain pattern. Bloodstains are frequently found at various
types of crime scene, like homicide, assault, robbery, child abuse, rape, etc.
In addition, forensic scientists are often called upon to examine clothing,
weapons, vehicles and other physical evidences to identify whether a victim’s
or a suspect’s blood has been transferred or not to those items. We come to
know that the bloodstain pattern analysts can examine the blood evidence left
behind and draw conclusions as to how the blood may have been shed. From
what may appear to be a random bloodstains distribution at the crime scene.
The analysts can categorize the stains by gathering information from spatter
patterns, transfers, voids and other impressions that can help to investigators
in reconstruct the sequence of events that occurred after bloodshed.

Figure 6: Blood spatter patterns appear on a wall

Source: Courtesy of National Forensic Science Technology Center (NFSTC), Largo, Florida
224
Bloodstain pattern analysis (BPA) is the interpretation of bloodstains at a Serology

crime scene in order to recreate the actions that caused the bloodshed. The
analysts can examine the size, shape, distribution and location of the
bloodstains patterns and can be confirm or refute assumptions concerning
events and their sequence.

BPA uses principles of biology (behaviour of blood), physics (cohesion,

capillary action and velocity) and mathematics (geometry, distance, and
angle) to assist investigators in answering questions such as:

Where did the blood come from?

What caused the wounds?
From what direction was the victim wounded?
How were the victim(s) and perpetrator(s) positioned?
What movements were made after the bloodshed?
How many potential perpetrators were present?
Does the bloodstain evidence support or refute witness statements?

8.6.1 Types of Blood Stains

The bloodstains and patterns are classified on the basis of their physical
features like size, shape, location, concentration and distribution, etc. There is
a variety of way to classify bloodstain patterns, but the aim of classification is
the overall analysis. Classification sets the steps for the analyst to define
more efficiently the origin of any given stain.

As we discussed earlier many texts are classified bloodstain as Low, Medium

and High velocity spatter based upon their size distribution and hence the
force necessary for their production. Bloodstains are classified based on their
appearance and mechanism of transfer onto a surface. It was also classified
into three basic types: passive stains, transfer stains and projected or impact
stains.

Passive Stains

Passive stains include drops, flow, splashes and pools, was typically resulting
from gravity acting on an injured body. These stains are subdivided into:

a) Drops– These stains occur when blood drips passively under the
influence of gravity. They may occur singly, in clusters or provide a trail
and thereby indicate movement and its direction. However, direction
may be difficult to determine if the victim moves slowly and therefore
the drops fall at 90 degree resulting in the stains being more or less oval.
A classic situation is when you cut yourself and leave a drip trail from
the place where the accident occurred to the nearest washbasin..

225
Human
Identification:
Establishing
Identity-II

Figure: 6.1 Single drop of blood on a wooden Figure 6.2 Bloodstain from single
floor drop of blood.

Source: Courtesy of John Black, Ron Smith & Associates

b) Flows-These occur when blood flows passively to produce small rivers

or streams of blood. These can be useful for determining orientation at
the time of attack or movement after death. For example, if a person is
stabbed in the chest whilst standing a trickle of blood will head vertically
down the body. If, however, the person was lying on the floor the flow
will veer to whichever side of the body is angled to the floor.

Figure 6.3: Showing the blood flow on surface

Source: http://www.knoxforensics.com/shoot.php

c) Pooling - A bloodstain resulting from an accumulation of liquid blood

on a surface.

226
 Serology

Figure: 6.4. Pooling bloodstain from surface

Source: https://www.kindpng.com/imgv/hbJTwwi_blood-clip-art-bloodstain-blood-stain-
transparent-png/

Transfer Stains:

Transfer result from objects coming into contact with existing bloodstains
and leaving wipes, swipes or pattern transfers behind such as a bloody shoe
print or a smear from a body being dragged. These stains are subdivided into:

a) Wipe Pattern -An altered bloodstain pattern resulting from an object

moving through a pre-existing wet bloodstain.

Figure :6.5. Numerous wipe pattern created by the victim feet moving in pre-existing
blood pool.
Source:https://www.chegg.com/flashcards/forensics-final-blood-spatter-anaylisis

b) Swipe Pattern - A bloodstain pattern resulting from the transfer of blood

from a blood-bearing surface onto another surface, with characteristics
that indicate relative motion between the two surfaces.

227
Human
Identification:
Establishing
Identity-II

Figure 6.6.Swipe pattern created by bloody hairs.

Source: https://www.chegg.com/flashcards/forensics-final-blood-spatter-anaylisis

c) Transfer Stain - These occur when wet blood is transferred from one
object to another. For ex., when a bloody knife is wiped onto clothing, a
bloody hand leaves prints on doorknobs or weapons and when a person
stepping in blood leaves behind impressions of their footwear.

Figure 6.7.Transfer bloodstain made by footwear.

Source: Courtesy of John Black, Ron Smith & Associates

Projected or Impact Stains

These stains are created when a blood source is subjected to an external force
greater than gravity. Impact stains result from blood projecting through the
air and are usually seen as spatter, but may also include gushes, splashes and
arterial spray.

Blood spatter is categorized as impact spatter (created when a force is applied

to a liquid blood source) or projection spatter (caused by arterial spurting,
expirated spray or spatter cast off an object). The characteristics of blood
spatter depend on the speed at which the blood leaves the body and the type
of force applied to the blood source.These stains are subdivided into:

228
a) Cast-off - This results when an object swung in an arc flings blood onto Serology

nearby surfaces. This occurs when an assailant swings the bloodstained

object back before inflicting another blow. Analysts can tell the direction
of the impacting object by the shape of the spatter (tails point in the
direction of motion).

Figure 6.8: Cast-off blood stains on the celling caused by blunt force instrument
Source: https://www.practicalhomicide.com/Research/LOmar2007-2.htm

b) Expirated spatter – It is usually caused by blood from an internal injury

mixing with air from the lungs being expelled through the nose, mouth or
an injury to the airways or lungs. Expirated spatter tends to form a very
fine mist due to the pressure exerted by the lungs moving air out of the
body. Small air bubbles in the drops of blood are typically found in this
type of spatter.

Fig.6.9.Expirated bloodstain caused by mouth injury

Source:https://static1.squarespace.com/static/55dfd21ee4b0718764fb34cc/t/56de10882b8dd
e6e3795ef39/1457393809358/Bloodstain+Pattern+Analysis.pdf

c) Arterial spray – It refers to the spurt of blood released when a major

artery is severed. The blood is propelled out of the breached blood vessel 229
Human by the pumping of the heart and often forms an arcing pattern consisting
Identification:
Establishing of large, individual stains, with a new pattern created for each time the
Identity-II heart pumps.

Figure: 6.10.arterial bloodstain on wall surface

Source: https://www.journalijdr.com/sites/default/files/issue-pdf/7128.pdf

d) Impact Pattern -Impact spatter is generated by the application of some

force to a blood source resulting in the random dispersion of smaller
drops of blood. The blood source may be on the skin’s surface, as in the
case of a beating, or it may be exposed at the moment of impact, as in the
case of a gunshot injury. The resulting impact pattern may consist of
thousands of bloodstains.

Figure: 6.11.impact pattern develop in wall surface.

Source: https://www.thinglink.com

e) Splash Pattern - Splash patterns occur when a volume of blood is

projected into a scene with minimal force characterised by a large central
stain exhibiting minimal distortion. There will be very little satellite
230
 spatter present. They most often appear as large-volume patterns. Serology

Figure 6.12: Splash pattern created on surface.

Source: https://reekoscience.com/science-experiments/miscellaneous/splatter-blood-for-
blood-spatter-analysis

Check Your Progress 5

13) Define blood stain and its patterns.

……………………………………………………………………………
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14) Discuss different type of blood stain.

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15) What information you can get from bloodstain pattern analysis.

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231
Human
Identification: 8.7 SUMMARY
Establishing
Identity-II
Identification of body fluids is one of the most important components of
Forensic Science as it plays a vital role in criminal investigation.
Identification and individualisation of a criminal can also be done by the
body fluids present on the crime scene such as Blood, semen, saliva, urine,
sweat etc. Chemical test can confirm that the blood found is human or not,
further it can be analyses the species of origin.

Bloodstain identification can be done by using various sensitive methods.

Individualisation of bloodstain can be done by serum protein and Enzyme
polymorphisms. Biological evidence can facilitate individualisation of
criminals with the help of advanced DNA profiling techniques. Semen or
seminal fluid is a body fluid secreted by gonads of male during process of
ejaculation. It is one of the major evidence found in the criminal cases related
to sexual offences such as rape, sodomy etc. In microscopic examination the
identification of spermatozoa is a confirmatory evidence for the presence of
semen in a suspected stain.

Saliva, a watery fluid secreted into mouth can be encountered in the

investigation of criminal cases like sexual offences etc. Urine is a liquid
byproduct of body secreted by the kidneys through the process called
urination. It generally consists of water, organic and inorganic substances.
Sweat is a transparent bio-fluid with low toxicity and slightly acidic in nature
with pH. Examination of sweat stains is usually required in forensic practice
in order to determine the blood group of an individual from his wearing
apparel. Bloodstain patterns can reveal a great deal about position and
movement during the crime. It can link a victim to suspect.

8.8 REFERENCES
Bhasin, M.K., Fuhramann, W., (1972) Geographic and ethnic distribution of
some red cell enzymes. Humangenetik. Vo.. 14, Pp. 204-223.

Chattergee, C.C. (1951) Human Physiology, Vol. –II, Publication- Medical

Allied Agency, Calcutta.

Gaensslean, R.E. (1992). Sourcebook in forensic serology, immunology and

biochemistry. (NIJ) 810 Seventh Street NW, Washington, DC 20531, United
States.

Gunn, A. (2009). Essential of forensic biology (2nd ed.). Liverpool John

Moores university, UK: Wiley-Blackwell.

Harris, H. (1969) Genes and isozymes. (Review lecture). Proc. Roy. Soc.
Lond. B., Vol. 174, Pp. 1-31.

Jackson, A.R.W., & Jackson, J.M. (2011). Forensic science. Essex: Pearson
Education Limited.
232
James, S.H., John, J.N., & Bell, S. (Eds.). (2015) Forensic science: An Serology

introduction to scientific and investigative techniques. CRC Press.

James, S.H., Kish, P.E., & Sutton, P. (Ed.). (2005). Practical aspects of
criminal & forensic investigation. Boca Raton, Fla: CRC Press. xxv, 542 pp.

Khoo, J.C., Russell, P.J. (1972). Isoenzyme of adenylate kinase in human

tissue. Biochimica et Biophysica Acta (BBA)- Enzymology. Vol. 268, Issue
1:Pp-98-101.

Mickelsen, O., & Keys, A. (1943). The composition of sweat with special
reference to the vitamins. Journal of Biological Chemistry.

Modi, J.P. (2016). Modi’s medical Jurisprudence. New Delhi, Nexis Lexis
(Elsevier).

Reddy, K.S.N. (2014) Forensic medicine and toxicology (33rd ed.). New
Delhi, Jaypee Brothers.

Richard, L. (2014) Forensic biology. Wiley. New York, USA.

Scientific working group on bloodstain pattern analysis. [online] Available

at: [http://www.swgstain.org]

White, P.C., (2004). Crime scene to court: The essentials of forensic science.
Cambridge: The Royal Society of Chemistry.

Wilson Jr, D.E., Povey, S., Harris, H. (1976) Adenylate kinases in man:
Evidence for a third locus. Annals of Human Genetics. Vol. 39, Issue 3, Pp.
305-313.

8.9 ANSWERS/HINTS TO CHECK YOUR

PROGRESS
1) Species origin test. Refer to section 8.1.1 for detail.

2) Two common confirmatory tests on bloodstains are the Teichmann and

Takayama tests. Refer to section 8.1.2 for detail.

3) The potential for the individualisation of blood is based on typing of

proteins and enzymes. Blood proteins and enzymes have the quality
polymorphism or of being iso-enzymes, which means they exist in
several forms and variants. Refer to section 8.1.5 for detail.

4) Salivary fluid is an exocrine secretion consisting of approximately 99%

water, containing a variety of electrolytes (sodium, potassium, calcium,
chloride, magnesium, bicarbonate, phosphate) and proteins, represented
by enzymes, immunoglobulin’s and other antimicrobial factors, mucosal
glycoproteins, traces of albumin and some polypeptides and
oligopeptides of importance to oral health.

5) Swabbing, cutting, scraping.

233
Human 6) After collection of saliva sample we perform starch- iodine test for
Identification:
Establishing examination of saliva sample.
Identity-II
7) Urine consists of water, organic and inorganic substances.Water alone
forms about 95% of it and other substances form only 5%. The organic
substances are mainly nitrogenous compounds but small amounts of non-
nitrogenous compounds are also present.

8) Examination of urine stains. Refer to section 8.3.4 for detail.

9) Determination of origin of urine stain by precipitin method has not been

found satisfactory. However, Popielski, Brzecka and Szczecin (1963)
described a method of concentrating urinary proteins and thereafter
subjecting that concentrated urine proteins to precipitin test by agar gel
double diffusion method for the determination of species.

10) Seminal fluid contains the “flavin” that causes semen to fluorescence
under the ultra-violet light. Prostatic fluid accounts for approximately
30% the ejaculation. This portion of the semen contains high
concentration of acid phosphatase (AP) and prostate specific antigen
(PSA). Both are very useful to the forensic analyst for detection of semen
in case of rape, unnatural offence, hanging, etc. It is an alkaline fluid
with pH=7.4.

11) Examination of semen. Refer to section 8.4.4 for detail.

12) Human spermatozoa have three distinct regions: Head, Middle piece,
Tail. Figure 4, section 8.4.2 for detail.

13) Bloodstain patterns are defined as a deposition of blood on a surface or

by a surface contacting blood is known as bloodstain.

14) The bloodstains and patterns are classified on the basis of their physical
features like size, shape, location, concentration and distribution, etc.
There is a variety of way to classify bloodstain patterns, but the aim of
classification is the overall analysis. Refer to section 8.6.1.

15) Bloodstain pattern analysis (BPA) is the interpretation of bloodstains at a

crime scene in order to recreate the actions that caused the bloodshed.
The analysts can examine the size, shape, distribution and location of the
bloodstains patterns and can be confirm or refute assumptions
concerning events and their sequence.

234