FORENSIC SEROLOGY

[ANA 431- INTRO TO FORENSIC SCIENCE]

DR CYRIL AGBOR
OUTLINE

1. Introduction
2. Body fluids
• Body Fluid Compartments
• Composition of Body Fluid
3. Blood
• Blood cells
• Plasma
• Blood typing
4. Forensics of Blood
5. Forensics of Semen
6. Saliva Forensics
7. Vaginal Fluid
8. Urine
 INTRODUCTION

Definition:
Forensic serology is the study, detection,
identification and characterisation of body
fluids and their relationships to a crime
scene
Basic Concepts
• Evidence
▪ Direct Evidence
▪ Circumstantial Evidence
[either physical or biological in nature]

• Physical Evidence
Fingerprints, Footprints, Shoe prints, Tire impressions,
Tool marks, Fibres, Weapons, Bullets, and Shell casings
• Biological evidence
Body fluids **
Hair,
Plant parts, and
Natural fibres.
 Significance of Forensics of Body Fluid

• Body fluids are Source of Biological evidences

• Link suspect(s) and victim(s) to each other
And to the scene
• Include or exclude potential suspect(s) or
Victim(s)
• Knowing identity of fluid can influence
outcome of a case

• Establish crime scene

• Identify weapon(s) used for a particular crime

• Corroborate case circumstances

 Significance of Forensics of Body Fluid

• Managing trace evidences no matter how small

• Preservation of DNA evidence

• Eye witness account and wrongful convictions

 BODY FLUID

Body Fluid compartments

• Intercellular fluid compartment
• The extracellular compartment
• Interstitial fluid
• Plasma
• Lymph
• Trans-cellular fluid
* Cerebrospinal fluid of the CNS,
* Aqueous and vitreous humours of the eye,
* Synovial fluids of the joints.
* Other body fluids – Semen, saliva, gastric juice, tears, urine, sweat
• Detectable body fluids in crime scene

▪ Blood

▪ Semen

▪ Saliva

▪ Sweat

▪ Urine

▪ Vaginal fluid
 BLOOD
• Components of blood:
❑ Blood cells [ 45% ]
• Red blood cells (Erythrocytes)
• White blood cells (Leukocytes)
• Platelets (Thrombocytes)

❑ Plasma [55%]
• Plasma proteins
• Hormones
• Dissolved gasses
• Electrolyte
• Nutrient
 • Erythrocytes are red blood cells. They are responsible for oxygen
distribution. Red blood cells are most numerous; 5 to 6 million per mm3

• Leukocytes are the white blood cells; they are responsible for “cleaning”
the system of foreign invaders. White blood cells are larger and less
numerous; 5,000 to 10,000 per mm3

• Thrombocytes or platelets are responsible for blood clotting. Platelets are

tiny, cellular fragments; 350,000 to 500,000 per mm3

Serum is the liquid that separates from the blood when a clot is formed.
Characteristic of Red Blood Cell
• Red blood cell can readily squeeze through the
narrow passage of capillaries.
• They have nuclei during their early stages of
development, but extrude nuclei as a cell mature,
for haemoglobin.
• Haemoglobin has affinity for oxygen
• They do not reproduce. Lack nuclei, and cannot
synthesis mRNA.
• Are destroyed in about 120 days after production.
• Glycocalyx on cell membrane of RBCs determines
blood type and transfusion compatibility
 Blood Typing

Two systems of classification exist,

1. ABO SYSTEM
2. RHESUS SYSTEM

• ABO System:
Around 1900, Karl Landsteiner discovered that there are four
different types of human blood based on the presence or
absence of specific antigens found on the surface of the red
blood cells.
• Rhesus System: In 1940, Landsteiner and Weiner reported
the discovery of the Rh factor by studying the blood of the
Rhesus monkey.
• Rhesus positive (Rh+) individuals - Rh antigen present
• Rhesus negative (Rh-) individuals - No Rh antigen
ANTIGEN – ANTIBODY REACTION
Antigen—a substance that can stimulate the body to make antibodies.
Certain antigens (proteins) found in the plasma of the red blood cell’s
membrane account for blood type.
Antibody—a substance that reacts with an antigen
Agglutination—clumping of red blood cells; will result if blood types
with different antigens are mixed
Antigen + Antibody = Agglutination
ANTIGEN – ANTIBODY REACTION… cont..
Agglutination……
 FORENSICS OF BLOOD

Unknown stain at a crime scene and Questions to be answered:

• Is it blood?

• Is it human blood?

• Whose blood is it?

Reasons for forensic identification of blood
• Invisibility of some stains to the naked eye
• Similarity in appearance to other substances
• A stain could contain multiple body fluids from
more than one donor
Forensic Techniques for Blood Identification
Presumptive Tests
Confirmatory Tests
 Presumptive Tests
Presumptive tests uses chemical reactions that are
linked to a color indicator, to screen the fluid sample as
blood. It does not identify the origin of the blood as
human or others.

• Visual Examination - Alternate Light Source (ALS)

• The Luminol Test

• The Phenolphthalein Test

• Precipitin Test
 • Alternate Light Source (ALS)

• Devices use light emitting diodes

• Produce high-intensity light of varying
wavelengths
• Used to identify blood trace on dark
background
• Care must be taken as UV rays can affect DNA
thereby destroying evidence
 Forensic ALS Photography System

Moveed ALS Forensic Camera

 The Luminol Test

• A sensitive chemiluminescence test for blood

stain BEFORE AFTER

• Haemoglobin is capable of converting luminol

to a coloured compound in the presence of
H2O2

• Emits blue glow on its own in a dark scene

• Will not damage potential DNA evidence

• Give false positive result with blood in urine,

copper containing compounds and bleaches

• Detect blood stain diluted up to 300,000 times

 The Phenolphthalein Test

• Also known as Kastle-Meyer test,

sensitive to haemoglobin based blood
• A presumptive blood test, first
described in 1903
• Detect blood as dilute as 1 part in
10,000
• Gives pink colour in alkaline solution
(H2O2) if blood is present
• The presence of copper or nickel salt
gives a false positive result.
 • Precipitin Test

• Used to determine the species origin of a

questioned bloodstain
• Investigators use the precipitin test to
determine whether the blood is of animal
or human origin
• Antisera is derived from rabbits that have
been injected with the blood of a known
animal.
• Formation of precipitin (antigen - antibody)
complex confirms the blood is human
 Confirmatory Tests For Blood

A confirmatory test is needed to identify the specific body

fluid as being human blood.

• Microscope examination

• Crystal tests

• Spectroscopic methods

• Immunological tests

• Chromatographic methods
 Confirmatory Tests For Blood Cont….

Microscope examination
• Tests involves the identification of blood
cells by directly visualizing them under
the microscope.
• Scanning Electron Microscope (SEM)
which allows scientists to study the
morphology of an unknown stain
• Its chemical composition can be analysed
using an Energy Dispersive X-ray (EDX)
Analyser
 Confirmatory Tests For Blood Cont….

Crystal tests
• Teichman and Takayama crystal tests
• Teichman: Formation of brown, rhombic
crystals after heating with reagents
• Takayama: Yields needle-shaped crystals in
positve result when dried blood is heated
• A positive result yields needle-shaped
crystals
 Confirmatory Tests For Blood Cont….

Spectroscopic methods
• Derivatives of haemoglobin have a characteristic

strong absorbance band called Soret Band

• Soret Band has peak wavelength around 400 nm

• Interference can come from water submersion,

sunlight exposure, heating, and rust

• Detect bloodstains up to 10 years old

 Confirmatory Tests For Blood Cont….

Chromatographic methods
• Used in separation of haemoglobin and its derivatives

• An alcoholic benzidine spray reagent is used to detect

spots in conjunction with a hydrogen peroxide

• Hematoporphyrin component was visualized

because of its fluorescence property

 BLOOD SPATTER

Factors Determining
Bloodstain pattern
• Terminal Velocity
• Directionality
• Angle of Impact
The shape of a blood stain:
• Round--if it falls straight down at a 90 degree
angle.
• Elliptical--Blood droplet elongates as the angle
decreases from 90 to 0 degrees.
• The angle can be determined by the following
formula:
width = sine of the impact angle
length
IMPACT
• The more acute the angle of impact,
the more elongated the stain.
• 90 degree angles are perfectly round
with 80 degree angles taking on a
more elliptical shape.
• At about 30 degrees the stain will
begin to produce a tail.
• The more acute the angle, the easier
it is to determine the direction of
travel.
AREA OF INTERSECTION
The location of the blood
source can be determined by
drawing lines the various
blood droplets to the point
where they intersect. This is
the blood’s origin.
AREA OF CONVERGENCE
• The area of convergence is
the point of origin; the spot
where the “blow”
occurred.
• It is determined by drawing
a line from the area of
intersection straight up to
where to where the angle
of impact would intersect
FORENSICS OF SEMEN
• Semen detection is in needed rape evidence
Presumptive Methods
Alternate Light Source (ALS)
• Wood’s Lamp (WL) is a specific device that
emits wavelengths from about 320–400 nm
• Simple and non-destructive method

Seminal Acid Phosphatase (SAP) Test

• SAP is an enzyme secreted by the prostate gland
• It is found in a concentration about 400 times
higher than any other body fluid
 Presumptive Methods .. Cont…

• SAP gives purple colour with a combination of

combination is alpha-naphthyl phosphate and
Brentamine Fast Blue
• SAP versus VAP
• Enzyme can degrade when exposed to heat,
mould, putrefaction, or chemicals

• Test for Seminal Zinc

• It detected semen on stains that were 25 years old
• Better and less degradable marker than SAP
• Zinc test paper-strip are used for quick results
 Presumptive Methods .. Cont…

Test for Seminal Choline

• A florescence microscopic test
• Gives brown needle-like crystals with a solution of
iodine and potassium
• Negative for other body fluids including vaginal fluid
as well as semen from other species

Test for Spermine (SPM)

• Amongst all polyamines, SPM is highest in semen

• High performance liquid chromatography (HPLC) is
used to detect spermine
• SPM is not found in urine, blood, sweat, breast milk,
and saliva
 Presumptive Methods .. Cont…

Trace metal detection

• This method is classified as presumptive
• Trace elements occur in varying proportions among
different body fluids
• Chlorine for instance, was the largest peak detected
in the semen samples
• Calcium can be used as an identification marker
 Confirmatory tests for Semen

Microscopic identification
• Semen is the only body fluid which possesses
sperm cells
• Characteristic colour of red head and green
tail with ‘Christmas tree’ reagent
• Azoospermic donor will not test positive
using this technique
 Confirmatory tests for Semen.. Cont..

Test for prostate-specific antigen (PSA)

• Most reliable confirmatory test even for
azoospemic donors

• Other body fluids contain a very low level of PSA

and do not interfere

• Detecting PSA on laundered fabrics or

decomposed cadaver is a challenge
 FORENSICS OF VAGINAL FLUID

Forensic identification of vaginal fluid

• Common in scenes involving sexual assault
• Fluid is not very well defined as constituents keep changing.
• Neonate – Paediatrics – Puberty – Pregnancy – Menopause

Presumptive tests
• Test for glycogenated epithelial cells
• Vaginal Peptidase Test
• Test for Oestrogen receptors
• Lactic acid / citric acid ratio
 FORENSICS OF SALIVA
Saliva is a watery substance formed in the mouth of animals,
secreted by the salivary glands
Human saliva comprises:
• 99.5% water
• Electrolyte
• Mucus
• White blood cells
• Epithelial cells (which can be used to extract DNA)
• Glycoprotein
• Enzymes (such as amylase and Lipase)
• Antimicrobial agents such as secretory IgA and Lysosome
• Presumptive tests for saliva are available

• No confirmatory test for saliva exist yet

• Saliva lack of solid particles in sample

• Saliva stains will appear bluish-white when being viewed under an
ultraviolet light
• Presumptively test for saliva is based on the activity of amylase
• Amylase coded by the AMY1 locus on chromosome found in saliva,
breast milk, and sweat.
• Amylase found in the pancreas, semen, and vaginal secretions is coded
by the AMY2 locus
• A radial diffusion assay has been used to distinguish sources of AMY1
and AMY2
 FORENSICS OF URINE
Factors associated with Urine test in forensic
• Low sensitivity
• False positive results

Presumptive tests for Urine

• Urine will also fluoresce when exposed to
ultraviolet light
• Microscopic identification of crystalline materials
and epithelial cells
• Test for Urea
• Creatinin gives a red coloured product (creatinine
picrate) with picric acid
 FORENSICS IDENTIFICATION OF SWEAT

• Sweat is the least common body fluid found at

crime scenes

• Contains less urea and creatinine compared to

urine
• SEM coupled with Energy Dispersive Xray (EDX) can
be used to measure trace metals concentration

• G-81 monoclonal antibody has affinity for sweat-

specific protein and it is using ELISA technique.
THANK YOU