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Chapter 9
Blood and Physiological Fluid Evidence:
Evaluation and Initial Examination
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How Biological Evidence Analysis Has Changed Because of DNA Typing
Nature of Blood
Collection, Preservation, and Packaging of Biological Evidence
Test Controls, Substratum Comparison Specimens, and Contamination
Issues
Initial Examination of and for Biological Evidence
Forensic Identification of Blood
Species Determination
Forensic Identification of Body Fluids
Forensic Investigation of Sexual Assault Cases
Blood and Body Fluid Individuality: Traditional Approaches
I. How Biological Evidence Analysis has
Changed Because of DNA Typing
• Prior to the introduction of forensic DNA typing
analysis, blood groups were the genetic markers that
were analyzed from biological evidence (forensic
serology)
• Forensic biology now refers to the preliminary
examination of biological evidence prior to the DNA
typing analysis procedures
II. Nature of Blood
• Blood contains cells, nutrients, chemical messengers, and
ingested substances
• A tube of whole blood will clot producing two fractions: a
yellow serum layer and a dark red clot containing cellular
material
• Anticoagulants prevent
blood clotting yielding a
yellow plasma layer and a
cell fraction that settles to
the bottom of the tube
II. Nature of Blood
• The cellular fraction of blood contains red blood cells
(erythrocytes) and white blood cells (leucocytes)
• White blood cells are the
source of DNA for DNA
typing analysis
• Red blood cells do not
contain any nuclear DNA
III. Collection, Preservation, and Packaging
of Biological Evidence
• Blood or Buccal Swabs from Known Person:
• Blood is drawn into a vacutainer tube containing an
anticoagulant such as EDTA (“purple top” tube)
• Buccal (cheek) swabs are
often used in place of liquid
blood as the known sample
III. Collection, Preservation, and Packaging
of Biological Evidence
Biological Evidence from Scenes:
• Fresh or web blood should be collected on clean, sterile, gauze
and allowed to dry
• Four sampling methods for dried blood:
– Cutting – For stains on objects that are difficult to submit to the lab. The
cut portion should include unstained areas around the bloodstain
– Swabbing – Stain is transferred to a swab which has been moistened with
sterile water or saline.
– Scraping – a sharp instrument is used to scrape the stain off of a surface &
onto clean paper
– Elution – using a small amount of saline or distilled water to dissolve the
dried stain
III. Collection, Preservation, and Packaging
of Biological Evidence
• The most important consideration for
preserving biological evidence from scenes is
to thoroughly dry the item before packaging
and then store in a cool dry environment
• Biological evidence must be packaged in paper
containers that can breathe
IV. Test Controls, Substratum Comparison
Specimens, and Contamination Issues
1. Known (Exemplar or Reference) Control:
– are specimens from a known source
– essential for comparison with DNA profiles from
evidentiary specimens
2. Alibi (Alternative) Known Control:
– From a known source that may be the source of the
evidence
3. Blank Control:
– A specimen known to be free of the item or substance being
tested
IV. Test Controls, Substratum Comparison
Specimens, and Contamination Issues
4. Substratum Comparison Specimens:
• Substratum refers to the underlying material or surface on
which the evidence is found
• A substratum comparison specimen is subjected to the same
testing as the evidence
• The specimen helps to detect interference in lab tests
originating from the evidence surface
• An unstained portion of the evidence underlying material is
collected for this purpose
IV. Test Controls, Substratum Comparison
Specimens, and Contamination Issues
• Evidence may be contaminated in several ways:
– Biological material may have been on a surface before the
biological evidence was deposited
– During scene searching &/or processing activities
– During laboratory examinations &/or manipulations
V. Initial Examination of and for Biological
Evidence
• The initial examination is designed to evaluate stains
for possible evidentiary value
• Activities include:
– Searching for biological stains
– Preliminary tests for physiological fluids
– Positive preliminary tests are then subjected to
confirmatory tests
– Cutting out or transferring stains to swabs for subsequent
examinations
VI. Forensic Identification of Blood
Two categories of identification tests:
• Presumptive or preliminary test
– Used for screening specimens that might contain the substance or
material of interest
– Both false positive and false negative results may be obtained
• Confirmatory test
– Are tests which are entirely specific for the substance or material for
which it is intended
– A positive confirmatory test is interpreted as an unequivocal
demonstration that the specimen contains the substance or material
VI. Forensic Identification of Blood
Presumptive Tests for Blood:
• Presumptive blood tests are used to screen evidence for the
possible presence of blood
• Most are color tests and are based on the peroxidase-like activity
of hemoglobin
• Peroxidase catalyzes the following reaction
• Reduced Dye + peroxide --> Oxidized dye + water
• The presence of hemoglobin catalyzes the reaction, forming a
colored dye product
• Positive presumptive tests do not prove that blood is present
VI. Forensic Identification of Blood
Confirmatory Tests for Blood:
• Older tests included crystal tests such as the
Teichmann and Takayama tests
• Current immunological tests use antibodies specific
for human hemoglobin, thus combining the
confirmatory test for blood with a human species test
• The crystal tests and the immunological tests are
known as direct confirmatory tests
VII. Species Determination
• Tests must be done on blood specimens to determine the
species of origin
• Species origin tests are done using immunological
methods which involve the interaction of antigens and
antibodies
• Hemoglobin from human red blood cells can be used as
the antigen to produce anti-human hemoglobin serum
• Specific antiserum can be used to test for the presence of
antigens in unknown specimens
VII. Species Determination
• Common immunological species tests include the Ouchterlony
method
• Extracts of the bloodstain to be analyzed are tested with specific
antisera
• If the bloodstain contains the antigens corresponding to the
specificity of the antiserum, a visible precipitate (precipitin) is
obtained
VIII. Forensic Identification of Body Fluids
1. Identification of Semen:
• Semen is a mixture of specialized cells, called spermatozoa,
suspended in a fluid known as seminal plasma
• UV light causes semen stains to fluoresce, and is therefore
used to locate
stains
• Both presumptive and
confirmatory tests for
semen stains are available
VIII. Forensic Identification of Body Fluids
Presumptive Test for Semen:
• The AP test is a color test based on the detection of acid
phosphatase, an enzyme from the prostate gland that is found in
high concentration in human semen
Confirmatory Test for Semen:
• A commonly used approach is to use a microscope to detect
spermatozoa in smears made from dried stains
• When no sperm are found, immunological methods are used to
detect the presence of a prostate gland protein called p30 or PSA
VIII. Forensic Identification of Body Fluids
2. Identification of Vaginal Secretions, Saliva, and Urine:
• There are no reliable methods for identifying human vaginal
material
• Presumptive tests for saliva are based on the presence of the
enzyme amylase
• There are no confirmatory tests for saliva
• Presumptive tests for urine are based on the presence of urea and
creatinine
• There are no confirmatory tests for urine
IX. Forensic Investigation of Sexual Assault
Cases
1. Coordination of Effort – SANEs and SARTs
• The medical examination of complainants in sexual assault cases
is performed by specially trained sexual assault nurse examiners
(SANE)
• Forensic nurses take a lead role in the coordinated response by the
sexual assault response team (SART)
• Complainants are taken to a medical facilities or a SANE/SART
facility to attend to their medical needs and to collect relevant
evidence using a sexual assault evidence collection kit (”rape
kit”)
IX. Forensic Investigation of Sexual Assault
Cases
2. The Forensic Scientist’s Role:
• Sexual assault evidence collection kits are forwarded to the
forensic lab for examination
• The forensic scientist’s primary role is the analysis of the
physical evidence
• If semen is present it helps to establish the corpus delicti
• If semen or other fluids are found, DNA typing is conducted to
determine if there is a match to a suspect or an exclusion
IX. Forensic Investigation of Sexual Assault
Cases
3. Medical Examination:
• Medical evaluation and treatment of sexual assault
victims initially involves recording the history of the
events, tending to any injuries, and documenting any
injuries, bruises, or contusions
• This is followed by evidence collection, which includes
clothing, vaginal swabs, pubic hair combings, any stains
on the skin surface, and a known control (blood or buccal
swab)
IX. Forensic Investigation of Sexual Assault
Cases
4. Sexual Assault Evidence Collection Kits:
• Sexual assault evidence collection kits contain a variety of
containers and envelopes plus a detailed set of instructions on how
to use them
• Not every container/envelope is used in every case
IX. Forensic Investigation of Sexual Assault
Cases
5. Types of Sexual Assault Cases
• There are three types of sexual assault cases:
unknown offender (identification cases), known
offender (consent cases), and sexual assaults
involving children
• DNA profiling is helpful in identification cases but
not in consent cases
• State laws define the age of consent, thereby
differentiating between an adult and child
IX. Forensic Investigation of Sexual Assault
Cases
6. Drug Facilitated Sexual Assault:
• Several drugs are commonly encountered as “date
rape” drugs: rohypnol, GHB, & ketamine
• All are depressants with amnestic effects, and are
often used along with alcohol
• These types of cases require toxicological analysis of
the evidence
X. Blood and Body Fluid Individuality:
Traditional Approaches
1. The Classical or Conventional Genetic Markers:
• 5 categories of classical genetic markers: blood groups,
isoenzymes, plasma (serum) proteins, hemoglobin
variants, and HLA
• The first blood group markers were ABO, discovered in
1901 by Karl Landsteiner
X. Blood and Body Fluid Individuality:
Traditional Approaches
• ABO markers were first applied to criminal cases involving
bloodstains by Dr. Leon Lattes of Italy in 1913
• Isoenzymes are enzymes which occur in multiple molecular forms,
reflecting differences in the gene that code for the enzyme
• Similarly, there are common variants of the protein hemoglobin
X. Blood and Body Fluid Individuality:
Traditional Approaches
2. How Does Typing Genetic Markers Help
“Individualize” a Biological Specimen?
• A gene is a region of DNA that codes for a particular
protein or enzyme
• Because chromosomes are paired (maternal and paternal),
and there is one gene on each chromosome, the genes are
paired
• A gene locus is the location on a chromosome where a
particular trait is determined
X. Blood and Body Fluid Individuality:
Traditional Approaches
• The genes making up a pair at a given locus are called alleles
• The alleles may be the same (homozygous) or different
(heterozygous)
• Population genetics looks at how often alleles found at a given
locus occur in a population
• A portion of a large population is sampled and tested to
determine the frequency of a particular allele
• Statistics are used to estimate the frequency of an allele in the
entire population