Chapter 3 Biological material – collection, characterization and storage
3 Biological material –
collection, characterization
and storage
The sensitivity and evidential power of DNA profiling have impacted on the way
in which crime scenes are investigated. Because only a few cells are required for
DNA profiling, crime scene investigators now have a much wider range of biological
evidence to collect and also have a much greater chance of contaminating the scene
with their own DNA.
Sources of biological evidence
The human body is composed of trillions of cells and most of these contain a nucleus,
mature red blood cells being a notable exception. A wide variety of cellular material
can be recovered from crime scenes (Table 3.1).
Each nucleated cell contains two copies of an individual’s genome and can be used,
in theory, to generate a DNA profile under optimal conditions [1–3]. In practice,
15 or more cells are required to generate consistently good-quality DNA profiles
from fresh material [4, 5]. Forensic samples usually show some level of degradation,
and with higher levels of degradation, more cellular material is required to produce
a DNA profile. If the material is very highly degraded then, even with the high
sensitivity of DNA profiling, it may not be possible to generate a DNA profile.
The biological material encountered most often at scenes of crime is blood
(Figure 3.1). This is mainly because of the violent nature of many crimes and also
because it is easier to visualize than other biological fluids such as saliva
Other frequently encountered samples include seminal fluid, which is of prime
importance in sexual assault cases; saliva, which may be found on items either held
in the mouth, such as cigarette butts and drinking vessels, or on bite marks, or in close
proximity to the mouth when speaking, such as the inside of masks or phones; and
epithelial cells, deposited, for example, as dandruff and in faeces.
With the increase
in the sensitivity of DNA profiling the recovery of DNA from epithelial cells shed
on touching has also become possible [6]. Door handles, steering wheels and knife handles are examples were no biological material is visible but is highly likely to be
present. Hairs are naturally shed, and can also be pulled out through physical contact
and can be recovered from crime scenes. Naturally shed hairs tend to have very little
follicle attached and are not a good source of DNA, whereas plucked hairs or hairs
removed because of a physical action often have the root attached, which is a rich
source of cellular material.
Table 3.1 Types of biological material that can be recovered from a crime scene.
The DNA profiles generated from crime scene material are compared against reference
profiles that are provided by suspects (or to a collection of reference samples held on
a DNA database), and in some cases, the victims
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(a) (b)
Figure 3.1 Blood is the most common form of biological material that is recovered from crime
scenes. (a) Large volumes of blood can be collected using a swab; if the blood is liquid then a
syringe or pipette can be used. (Picture provided by Allan Scott, University of Central Lancashire.)
(b) Blood on clothing is normally collected by swabbing or cutting out the stain. (Picture provided
by Elizabeth Wilson)
The four most common nucleated cell types that are recovered from scenes of crime
are white blood cells, spermatozoa, epithelial cells and hair follicles (Figure 3.2).
(a) (b) (c) (d)
Collection and handling of material at the crime scene
The high level of sensitivity that makes DNA profiling an invaluable forensic tool can
also be a potential disadvantage. Contamination of evidential material with biological
material from another source, such as an attending police officer or scene of crime
officer, is a very real possibility. It is vital that the appropriate care is taken, such
as maintaining the integrity of the scene and wearing full protective suits and face
masks during the investigation of the scene [7–9] (Figure 3.3). Improper handling of
the evidence can have serious consequences. In the worst cases, it can cause cross-contamination, lead to sample degradation and prevent or confuse the interpretation
of evidence.
Identification and characterization of biological evidence
Locating biological material is necessary before collection for further analysis can
occur. Furthermore, identification of the source of the material, for example demon-strating that a stain is blood, can be a vital piece of information in a given case,
even before any DNA analysis is undertaken.
Searching for biological material, both at the crime scene and in the forensic laboratory is performed primarily by eye. In the laboratory low-power search microscopes
may help to localize stains and contact marks.
The use of either chemical or physical
methods can be used to detect biological materials. Alternative light sources (ALSs)
using both infrared and ultraviolet light can provide a contrast between the fluores-cence of proteins in the body fluid and the background substrate. Chemical methods
use either the production of light or a colour change reaction.
These techniques have been developed to enable crime scene investigators and forensic biologists to
utilize the inherent properties of biological evidence to both locate and characterize the material. When characterizing material there are two categories: presumptive
and confirmatory. A range of presumptive and confirmatory tests is available that
aids the identification of the three main body fluids encountered: blood, semen and
saliva. Ideally, tests should be safe, inexpensive, simple to carry out, use a very small
amount of the sample, be quick to perform and provide a simple indication of the
presence or absence of a body fluid. The test should not affect the ability to carry
out subsequent DNA profiling.
Figure 3.3 It is standard practice for scene of crime officers to wear full overalls, shoe covers,
gloves and face masks when collecting biological evidence from a scene of crime. Even with
these precautions it is possible for crimes to be contaminated by forensic investigators, and it
is becoming common for the DNA profiles of police officers and crime scene investigators to be
stored on a database; any profiles recovered from the scene of crime can be checked against this
elimination database to rule out the possibility of a profile coming from an investigating police or
crime scene officer
Presumptive tests can give false positives; however, in many circumstances, when
the type of biological material is not of critical importance for a case, a positive
result with a presumptive test will be sufficient information to move on to DNA
analysis – which itself acts a confirmatory test for human biological material. In other
circumstances, when the origin of the material is important, as is often the case with
offences of a sexual nature, a confirmatory test is required that will unambiguously
identify the biological material.
Blood
Blood is composed of liquid plasma, which contains soluble proteins, lipids, glucose, hormones, metabolites and salts, and the cellular component – red blood cells
(erythrocytes), white blood cells (leucocytes) and platelets (thrombocytes).
In many cases blood will be clearly visible to the naked eye; however, if it is against
a dark background or if the bloodstain has been cleaned, its detection may not be
so straightforward. To help localize bloodstains an ALS, emitting at a wavelength
of 415 nm light, will enhance bloodstains, which maximally absorb light at this
wavelength and appear much darker than under white light [10, 11].
Searching a crime scene or items recovered from a crime scene for blood can
also be aided by the use of luminol (3-amino-phthalhydrazide) dissolved in alkaline
solution containing hydrogen peroxide or sodium perborate [12]. This solution can
be sprayed on a wide area and will become oxidized and emit light by chemilumines-cence in the presence of haemoglobin and hydrogen peroxide (Figure 3.4) [13, 14].
It is necessary to be able to darken the area that is being searched in order that the
chemiluminescence can be detected. Luminol can also be used in the more controlled
environment of the forensic laboratory and can be particularly useful when searching
clothing for trace amounts of blood. Fluorescein sprays can be used as an alternative
to luminol: the detection levels are similar to luminol, but it has the advantage that it
can be used in lighter environments, although an alternative light source, at 450 nm,
is required for detection [15]. In both cases, the presence of haemoglobin produces a
light blue light that fades after approximately 30 seconds. A solution can be reapplied
and the fluorescence activated a couple of times until the haemoglobin is saturated.
Other presumptive tests are available that, like luminol and fluorescein, take
advantage of the peroxidase-like activity of the haem group, which is abundant
as part of the haemoglobin molecule within red blood cells. The haem catalyses
the hydrolysis of hydrogen peroxide, which in turn leads to the oxidation of the
target chemical compound, resulting in a colour change. Commonly used chemical compounds include leucomalachite green (LMG) (colourless to blue-green) [13], phenolphthalein (Kastle–Meyer reagent) (colourless to pink) and tetramethylbenzidine (TMB) (colourless to green) [16–19] (Figure 3.5).
Any biological material that contains peroxidase activity, such as some plant
extracts, or any material that leads to the hydrolysis of hydrogen peroxide can also
result in a false positive [20, 21].
Figure 3.4 Luminol, sprayed onto recovered objects or at the crime scene, gives out a blue
fluorescence on contact with blood; a limitation is that the test has to be carried out in a dark
conditions. The detection of potential bloodstains using this method requires further confirmatory
analysis to avoid false positives
(a) (b) (c)
Figure 3.5 In the presence of haem and hydrogen peroxide the chemical colour change can be
seen. To perform a Kastle–Mayer (KM) test a piece of filter paper is folded in half and then half
again to make a corner (b). This is rubbed gently against the blood, transferring a trace of the
dried stain to the filter paper. A drop of KM solution (should give no change by itself) followed by
a drop of H2O2 leads to a pink/purple colour reaction in the presence of haemoglobin (c)
Confirmatory tests
The Teichman and Takayama crystal tests, which are based on the formation of
haem-derived crystals, were developed in 1853 and 1912, respectively [17], but,
along with other microscopic and spectroscopic techniques, are rarely used now
[22]. Two relatively new confirmatory tests offer some advantages over previous
tests: messenger RNA (mRNA) analysis allows the origins of several different types
of biological material to be identified; and flow immunochromatographic strip tests
offer a simple and sensitive test for human blood.
mRNA expression analysis has been shown to be a viable confirmatory test
[23–27], even in bloodstains that are several months old [28]. The targets that are
amplified are blood-specific, such as the β-spectrin (SPTB) [23, 25, 27], porphobilinogen deaminase (PBGD) [27], δ-aminolevulinate synthase (ALAS2) [25] and
the alpha locus 1 (HBA) marker [29]; the methodology can also be used to differentiate between menstrual and vascular blood, targeting the menstrual blood-specific
matrix metalloproteinase-7 (MMP-7) transcript [24, 30, 31]. The major drawback of
mRNA technology is that it requires specialist techniques and is a more complex
process than DNA profiling itself; however, the extra probative value of identifying
the origin of the blood will be very valuable in some cases.
An antibody-based lateral flow immunochromatographic strip test is relatively easy
to perform. The test involves reacting the suspected bloodstain with a glycophorin A
antibody (glycophorin A is found in the membranes of red blood cells). The mixture
is applied to and migrates along a membrane; if blood is present a visible complex
is formed with an immobilized capture antibody (Figure 3.6) [32].
IDENTIFICATION AND CHARACTERIZATION OF BIOLOGICAL EVIDENCE
Figure 3.6 The lateral flow immunology test shows two bands in a positive test whereas in a
negative test only one band is present. Positive tests are shown from left to right for blood, semen,
saliva. A test is also available for urine
Semen
Semen comprises mature sperm (spermatozoa) suspended in a fluid secreted from
the prostate gland, seminal vesicles, Cowper’s gland and the glands of Littre´
[16]. The positive identification of semen can be extremely important evidence to
support an allegation of sexual assault and both presumptive and definitive tests are
commonly used.
Presumptive test
As with blood, ALS can be a powerful technique to locate and presumptively
identify semen. Semen produces strong photoluminescence over a range of
wavelengths – exposed to UV light it will emit blue photoluminescence that will
be visible to the naked eye [10, 11, 33]; as with other ALS-based methods false
positives are detected [11, 33, 34].
A simple, and commonly used, test involves assaying for the presence of the
enzyme seminal acid phosphatase (SAP), which is present in high concentrations in
seminal fluid [16]. Other body fluids, such as saliva and vaginal secretions, contain
the enzyme albeit in significantly lower concentrations and so can give a positiveresult [35]. The presence of SAP is tested for by its ability to catalyse the hydrolysis
of organic phosphate, for example α-naphthyl phosphate, which following hydrolysis
will react with Brentamine Fast Blue (a diazonium salt chromogen), leading to a
colour change [22]. Other biological material containing acid phosphatases can lead
to false positives, such as plant material and vaginal secretions, although the reaction
with semen is usually stronger, and therefore the colour change faster, than with other
material. SAP is quick, simple and safe to perform. As it is an enzyme-based test,
old stains may give a slower reaction, and therefore a longer colour change, and in
some case no reaction may occur if the enzyme no longer functions.
Confirmatory tests
The most commonly used confirmatory test for semen is visualization of the
spermatozoa following staining; commonly used dyes are haematoxylin and eosin
(Figure 3.2b) and Christmas tree stain, which stains heads red and tails green [36].
Another marker for the identification of semen is the protein P30, which is a
prostate-specific antigen (PSA) [37, 38]. The advantage of using PSA compared with
the reaction involving acid phosphatase is that PSA is produced independently from
the generation of sperm and therefore it can be used for both spermic and azoospermic
samples; it is also very sensitive and resistant to degradation, even in cadavers [39].
Detection of PSA is most common with the use of the immunochromatographic strip
test, using antibodies raised against human PSA [40–42] (Figure 3.6).
Another confirmatory test is mRNA analysis, detecting the semen-specific protamine (PRM)1, PRM2 and kallikrein 3 (PSA) genes [24, 28, 29].
Saliva
Saliva is a fluid produced in the mouth to aid in swallowing and the initial stage of
digestion. A healthy person produces between 1 L and 1.5 L of saliva every day and
can transfer saliva, along with epithelial cells sloughed off from the buccal cavity,
in a number of ways. Transfer may be by contact, such as on food products when
eating, drinking vessels, cigarette butts, envelopes or in oral sexual assaults. Transfer
may also be by aerial deposition of saliva such as on to the front of a mask when
worn over the head or on to a telephone when talking into the mouthpiece.
Presumptive test
As with blood and semen ALS can be used to locate saliva; stains appear blue-white
when viewed under UV light. Most presumptive tests for saliva make use of the
enzyme α-amylase, which is present at high concentrations and digests starch and
complex sugars. The α-amylase enzyme hydrolyses α1–4 glycosidic bonds in glucose polymers, such as glycogen and starch. The digestion of starch can be assessed
using the starch–iodine test: a sample of the evidential stain is incubated with a starch solution; iodine is then added and if the starch has been broken down the
solution will be clear, whereas if starch is still present the colour will be blue. The
technique is not widely used as more sensitive assays have been developed using
modified starch that is covalently linked to a dye, such as cibachron blue or procion
red, to form an insoluble complex [43–45]; in the presence of α-amylase activity the dye is released from the complex and becomes soluble. The release of the
dye causes a colour change that can easily be detected, either in solution or by its
ability to migrate through an otherwise impermeable barrier, such as paper [43–45]
(Figure 3.7). Amylases are present in other body fluids such as sweat, vaginal fluid,
breastmilk and pancreatic secretions; however, amylase is present in saliva at concentrations greater than in other body fluids [44, 46]. The process takes at least
30 minutes to complete and, unlike the tests for blood and SAP for semen, can only
be performed in the laboratory. The test for saliva is only used in specific tests, such
as oral sexual assault cases.
Confirmatory tests
Until recently there were no readily used confirmatory tests for saliva. As with blood
and semen, antibody tests, using lateral flow strips, have been developed that are specific for saliva [47–49]. mRNA can also be isolated from saliva; detection of several
transcripts can provide confirmation that a stain contains saliva [23, 24, 50, 51].
Epithelial cells
The success in finding biological material depends upon the search method employed
and also on the integrity and state of the scene. In the UK, biological material is found at approximately 12% of inves-tigated crime scenes; this figure can go up
significantly if the crime scene is exhaustively searched [58].
BIOLOGICAL MATERIAL – COLLECTION, CHARACTERIZATION AND STORAGE
(a) (b) (c)
Figure 3.7 Location of saliva using Phadebas paper. (a) Even with appropriate lighting the
identification of saliva stains can be difficult. (b) The article being examined is moistened using
sterile DNA-free water and Phadebas paper is placed on top with the carbohydrate dye-coated
side in contact with the fabric; a glass plate holds the paper in contact. (c) α-amylase breaks down
the carbohydrate-dye complex, and the dye migrates through the paper and can be visualized
The methods used for collection will vary depending on the type of sample. Dry
stains and contact marks on large immovable items are normally collected using a
sterile swab that has been moistened with distilled water [59, 60]; in other cases,
scraping or cutting of material may be more appropriate. Lifting from the surface
using high-quality adhesive tape is an alternative method for collecting epithelial
cells [52]. Liquid blood can be collected using a syringe or pipette and transferred to
a clean sterile storage tube that contains anticoagulant (ethylene-diamine tetraacetic
acid (EDTA)), or by using a swab or piece of fabric to soak up the stain, which
should be air dried to prevent the build up of microbial activity [8]. Liquid blood
can also be applied to FTA paper, which is impregnated with chemicals to prevent
the action of microbial agents and stabilize the DNA. (FTA paper was developed
by Flinders Technology Associates.)
Smaller movable objects, such as weapons, which might contain biological material
are packaged at the scene of crime and examined in the controlled environment of
the forensic laboratory. The same range of swabbing, scraping and lifting techniques
as used in the field can be employed to collect the biological material. Clothing taken
from suspects and victims presents an important source of biological evidence. This
is also analysed in the forensic biology laboratory, where stains and contact areas
can be recorded and then cut out or swabbed.
Sexual and physical assault
Following sexual assaults, the victim should be examined as soon after the event as
possible. Semen is recovered by a trained medical examiner using standard swabs;
fingernail scrapings can be collected using a variety of swabs; combings of pubic
and head hair are normally stored in paper envelopes. The samples collected by
the medical examiner are dependent on the nature of the allegation and information
given by the alleged victim. Contact marks, for example bruising caused by gripping
or bite marks, can be swabbed for DNA. The same types of evidence (except semen)
can be taken after cases of physical assault [8].
Reference samples
In order to identify samples recovered from the scene of crime, reference samples are
needed for comparison. Reference samples are provided by a suspect and, in some
cases, a victim. Traditionally, blood samples have been taken and these provide
an abundant supply of DNA; however, they are invasive and blood samples are a
potential health hazard. Buccal swabs that are rubbed on the inner surface of the
cheek to collect cellular material have replaced blood samples in many scenarios.
In some circumstances plucked hairs may be used, but this source of material is not
commonly used.
BIOLOGICAL MATERIAL – COLLECTION, CHARACTERIZATION AND STORAGE
Figure 3.8 FTA cards can be used to store both blood and buccal cells. The cellular material lyses
on contact with the card. The DNA binds to the card and is stable for years at room temperature
FTA cards can be used to store both buccal and blood samples (Figure 3.8). The
FTA card is a cellulose-based paper which is impregnated with chemicals that cause
cellular material to break open; the DNA is released and binds to the card. The chemicals on the card also inhibit any bacterial or fungal growth and DNA can be stably
stored on FTA cards for years at room temperature as long as the card remains dry.
Storage of biological material
Biological material collected for DNA analysis should be stored in conditions that
will slow the rate of DNA degradation, in particular low temperatures and low
humidity. A cool and dry environment limits the action of bacteria and fungi that find
biological material a rich source of food and can rapidly degrade biological material.
The exact conditions depend on the nature of the samples and the environment in
which the samples are to be stored. Buccal swabs and swabs used to collect material at
a crime scene can be stored under refrigeration for short periods and are either frozen
directly or dried and then stored at −20 ◦C for longer term storage. Blood samples
will normally be stored at between −20 ◦C and −80 ◦C. Buccal and blood samples
collected using FTA cards can be stored for years at room temperature. Some items
of evidence, like clothing, can be stored in a cool dry room; in temperate regions
of the world DNA has been recovered from material stored at room temperature
for several years [59]. When samples are not frozen, for example clothing, they are
stored in acid-free paper rather than plastic bags, to minimize the build up of any
moisture. Once the DNA has been extracted from a sample, the DNA can be stored
short term at 4 ◦C but should be stored at −20 ◦C to −80 ◦C for long-term storage.
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