Tanya-Leigh Full Dissertation!

0 | P a g e
Transfer and persistence of semen
onto different materials.
Tanya-Leigh Lane

1 | P a g e
Contents
Acknowledgment.............................................................................................................. 2
1. Abstract ....................................................................................................................... 3
2. Introduction .................................................................................................................. 4
2.1. Statistics of Sexual Assault and Rape within the United Kingdom .................................. 4
2.2. Criminal Offences.................................................................................................. 5
2.3. Evidence Recovery ............................................................................................... 6
2.4. Transfer and Persistence ....................................................................................... 7
2.5. Semen.................................................................................................................. 8
2.6. Acid Phosphatase (AP).......................................................................................... 9
2.7. Prostate Specific Antigen (PSA) ............................................................................11
2.8. Ultra-Violet Light ..................................................................................................12
2.9. Microscopy ..........................................................................................................13
3. Aims and Hypothesis ...................................................................................................14
4. Method........................................................................................................................15
4.1. Materials..............................................................................................................15
4.2. Selection of transfer material.................................................................................15
4.3. Collection and storage of semen samples ..............................................................15
4.4. Assessment of the drying duration of semen ..........................................................16
4.5. Semen transfer from primary to secondary and tertiary sources ..............................16
4.6. Preparation of semen stained material for analysis .................................................16
4.7. Acid phosphatase (AP) assay ...............................................................................17
4.8. Prostate specific antigen (PSA) assay ...................................................................17
4.9. Microscopy ..........................................................................................................18
5. Results........................................................................................................................18
5.1. The effect of material type on the transfer of semen ...............................................18
5.2. The Effects of drying time on semen transfer to secondary and tertiary materials from
cotton underwear ............................................................................................................19
5.3. Comparison of the acid phosphatise test and the prostate specific antigen test ........20
5.4. Microscopy ..........................................................................................................21
5.5. Assessment of dry time ........................................................................................22
6. Discussion...................................................................................................................22
7. Conclusion ..................................................................................................................30
8. Further Work ...............................................................................................................31
9. References..................................................................................................................32
10. Appendices .............................................................................................................38

2 | P a g e
Acknowledgment
I would like to thank my project supervisor Laura Walton-Williams for approving my
project and for her devoted time and effect in the assistance of this. Moreover, a big
thank you as well to Elizabeth Deakin for the persistency during my time in the
laboratory.

3 | P a g e
1. Abstract
This research will examine the possibility of detection of semen onto different materials
at different levels of transfer; secondary and tertiary. To aid this investigation, different
variables are assessed along the way to detect whether this impacts on the transfer
and persistency; timings of which the semen will have come into contact with the
materials and the materials chosen for each level of transfer. To gain results, a mixture
of presumptive and confirmatory tests are used; Acid phosphate (AP), Prostate-
Specific Antigen (PSA) and microscopy. This information will hopefully be used to help
aid sexual assaults and rape cases on whether or not the DNA evidence can be
detected even if the victim hasn’t reported the rape immediately after the incident and
regardless of the victim’s actions afterwards in relation to movements

4 | P a g e
2. Introduction
2.1. Statistics of Sexual Assault and Rape within the United Kingdom
Official Statistics of crimes are produced by statisticians in the Ministry of Justice,
Home Office and the Office for National Statistics. The information given includes the
victims’ experience, the police role in recording and detecting the crimes, how criminal
justice agencies deal with an offender once identified and the criminal histories of sex
offenders. The information provides an overview of sexual offending across England
and Wales and it has been identified that official statistics help to provide a fuller
picture of offences and offenders through the criminal justice system (Home Office,
2013).
However, the statistics given can be incorrect as it is difficult to give direct comparisons
due to self-completed questions, police recorded crime figures being based on
offences per victim, different periods of time and not being the same victim or offender
or the same offences (Home Office, 2013). Also, the source of the information can be
unreliable and the statistics can only be an approximation due to a lot of incidents
going unreported; only 15 per cent of victims reported the incident due to;
embarrassment, fear of not being believed, the thought of the police not being able to
do much, not worth reporting or believing it to be a private or family matter. Figures
between the courts and police will differ (Home Office, 2013).
For statistics on sexual offences, 2005 was the first full year following the introduction
of the Sexual Offences Act 2003. Sexual offences have been recorded into 2
categories;

5 | P a g e
• Most serious sexual offences covering all rape, attempted rape and sexual assault
offences
• Other sexual offences, which includes sexual activity with minors (excluding rape and
sexual assaults), exposure and voyeurism (Home Office, 2013).
From the ‘Crime Survey for England and Wales’ in 2009/10, 2010/11 and 2011/12, on
average 2.5 per cent of females and 0.4 per cent of males have been said to be a
victim of a sexual offence including attempts within the previous twelve months. The
figures amount to around 473,000 adults being victims of sexual offences; 404,000
being females and 72,000 of males on average per year. These statistics cover all
incidents of sexual offences within both categories (Home Office, 2013).
Around one in twenty females aged sixteen to fifty nine reported being a victim of a
most serious sexual offence since the age of sixteen (Home Office, 2013). The
number of reports for the most serious sexual offences of ‘rape’ was 16,000 and for
‘sexual assault’, 22,100 offences were accounted for. Seven thousand people reported
that they had encountered exposure or voyeurism and 5800 informed the police of
sexual activity. Approximately 90 per cent of the victims knew their perpetrator in the
category of serious sexual offences unlike the other sexual offences (Home Office,
2013). These statistics show that is doesn’t matter what age or gender a person is,
they can still encounter such a traumatic experience and that they will only report the
crime if it is a serious offence rather than a minor one (Home Office, 2013).
2.2. Criminal Offences
Criminal offences that have a sexual nature like sexual assault, rape, internet grooming
and trafficking are all covered by the Sexual Offences Act 2003 and all carry their own

6 | P a g e
consequences (Home Office, 2002). Over time the definition of sexual offences have
been reformed due to the law being ‘archaic, incoherent and discriminatory’ (Home
Office, 2002). Before the Criminal Justice and Public Order Act 1994, rape could only
be committed against a woman (Elliot and Quinn, 2012) and now, the definition of rape
and sexual assault means the offence can be committed by both genders and come
into two separate categories; Rape is defined as a person (A) commits an offence if (a)
he intentionally penetrates the vagina, anus or mouth of another person (B) with his
penis, (b) B does not consent to the penetration, and (c) A does not reasonably believe
that B consents. Whereas sexual assault has a slightly different definition; A person (A)
commits an offence if (a)he intentionally touches another person (B), (b)the touching is
sexual, (c)B does not consent to the touching, and (d)A does not reasonably believe
that B consents (The National Archives, 2012). If a person is convicted of rape it
carries the maximum penalty of life imprisonment unlike sexual assault where the
offender can be given the statutory maximum penalty in the Magistrates' court OGL.
(2014) which is; up to 6 months in prison, a fine of up to £5,000, a community sentence
or 10 years imprisonment in the Crown Court. However, if the offender is under 18, the
offence comes within section 91 of the Powers of Criminal Courts (Sentencing) Act
2000 (The National Archives, 2012).
2.3. Evidence Recovery
When recovering, analysing and identifying bodily fluids it is crucial that it is done with
precision and accuracy as it may be the only chance of doing so as there may be little
evidence left behind which could be due to many reasons including; the victim having
washed the clothes worn during the incident, had a shower, didn’t report the crime
straight after the incident occurred or that the perpetrator used a condom during the
assault or rape and was very organised when leaving the crime scene (Slaughter and
Brown, 1992). Although, there may be other evidence in conjunction with the incident

7 | P a g e
that could aid the investigation such as; lubricants, restraints, transportation to or from
places, duration of the encounter and injuries obtained (Summers, et al, 2001) It is said
that the victim’s body is the main source of evidence (Hazelwood and Burgees,
2009). The main examination would be of the hymen, the entire vulva and perineal
area for lacerations and bruises that occur with forced penetration (Sommers, et al,
2001). Moreover, during the collection of bodily fluids; blood, semen, sweat, saliva and
vaginal fluid, with semen being the second most common bodily fluid to blood, it can
help with identifying whether or not the crime committed was rape or sexual assault as
well as examining any injuries they may have received and foreign hairs left behind
(Hazelwood and Burgees, 2009).
2.4. Transfer and Persistence
Transfer and persistence of an object can be indirect or a direct transfer and depending
on which transfer it is depends on how complicated and the potential limits of an
interpretation can be within an investigation. The persistence is influenced by the
activity done during and after the incident, location and time of collection (Houck and
Siegel, 2010) The fabric type will also influence the transfer and persistence depending
on the absorbency rate, absorbent capacity, surface properties and the structure of the
fabric (Kamath, et al 2004).
Locard’s (1953) principle is that ‘every contact leaves a trace’ and when investigating
criminal offences like sexual assault and rape the physical contact between the
perpetrator(s) and the victim is the main source of evidence; bodily fluids; as there are
no potential witnesses to the offence (McGrath, 2011) so collection of evidence is
crucial in identifying and prosecuting perpetrators. Primary transfer is when a
substance comes into direct contact with another surface by touch although, this can

8 | P a g e
occur even if the substance hasn’t directly touched that surface, for example, a window
breaking and glass shards getting embedded into a jumper, even though that jumper
hasn’t come in contact with that window (Robertson, 1999). Secondary transfer is when
another surface/material comes into contact with that jumper and the glass is
transferred to the other surface. Tertiary is when a third contact happens. The amount
of times a surface/object can come into contact with another can be endless but the
more times the contact happens the less likely it is that anything can be detected
(Robertson, 1999).
An experiment was carried out by Lehmann et al (2013) where blood; wet and dry; was
deposited onto both porous and non-porous cotton and glass. The results gathered
were that if the blood was wet on a non-porous surface it could be transferred up to
four times but the biological source was unable to transfer further than the primary or
secondary transfer if it was dry. Unlike a porous surface, the blood could travel through
to the sixth substrate, wet or dry. (Lehmanna, et al, 2013). A case which proved tertiary
transfer can happen was Dirk Greineder’s (2010). The morning of his wife’s murder, he
had shared a towel with his wife; his DNA had transferred onto the towel he had used
to dry his face to which his wife used afterwards to then dry her face. Later, his wife got
attacked by an intruder wearing gloves and it therefore looked like Mr Greineder had
committed the crime due to his DNA being transferred from the towel to her face.
(Pyrek, 2007)
2.5. Semen
A typical semen sample has a grey-opalescent appearance but this depends on the
sperm concentration (Cambridge University, 2003) and what state the sample is in; if
it’s wet. However, if the sample is dry it can appear crusty and contains no moisture

9 | P a g e
with a white colouration (Maloney. M, 2012). Premature ejaculation is the seminal fluid
that can occur before the male actually ejaculates. The appearance of it is similar to the
actual ejaculation fluid but is slightly more watery but still has evidential value. Semen
is a mixture of spermatozoa and the seminal fluid from the testis and epididymis which
at the time of ejaculation is also combined with the secretions from the prostate gland,
seminal vesicles that nourish the sperm including fructose which feeds the sperm,
fibrinogen which coagulates the fluid together and prostaglandins; that helps the sperm
penetrate the cervix but helps alkaline the fluid (<pH 7), urethral and bulbourethral
glands which in turn produces the compound of viscous fluid (Cambridge University,
2003). The average ejaculation for a male is approximately 1 - 5 ml and contains
anything from 10-50 million sperms cells but this can be dependent upon age, any
medical conditions, genetic background, diet, and other habits such as smoking.
(Lewis, 2003). Even taking these factors into consideration, there are some males
within the population that have conditions like Azoospermia; no sperm count,
Oligospermia; low sperm concentration and Asthernospermia; poor sperm motility that
will have effect on the results depending on the method used for testing. (Raju and
Iyengar, 1964) Semen can be analysed by an expert that can testify to a scientific
certainty that individual samples came from certain individuals. (Bigbee and Spalding,
2001).
2.6. Acid Phosphatase (AP)
The acid phosphatase (AP) reagent was developed in 1957 by Stuart Kind and is used
as a presumptive test for seminal fluid but will need a confirmatory test or microscopy
to confirm the results given due to the number of potential false positive reactions
including vaginal material, faecal material, foods and beverages. AP can detect semen
in the vagina up to 72 hours post-coitus. (Peonim et al, 2013) The AP reagent consists
of sodium acetate, acetic acid, α-naphthyl phosphate disodium salt and brentamine fast

10 | P a g e
black K salt. When In the presence of acid phosphatase, α-naphthyl phosphate is
hydrolysed to produce α-naphthol. This then combines with the fast black K to produce
a purple azo dye. (Simmons et al, 2014) AP can be applied directly or indirectly but
both can give results. An indirect test is done by swabbing the area of interest and an
extract is made from this then applied to the filter paper. The reagent is then added to
the filter paper. The direct screening is done using a dampened filter/blotting paper or
swab is pressed onto the semen sample then tested with the chemical reagent which
will change colour from orange to purple if there is acid phosphate present. Depending
on the time taken for the colour change it take place within the 2 minute window given,
how dark or light the colour change is and the alleged case circumstances helps to
determine whether or not the colour change is due to the presence of semen. (Lewis et
al, 2013) Semen can be detected for up to 15 minutes or more in dilutions up to 1 in
400 using the press test method and in dilutions up to 1 in 1000 using the direct
method. (Redhead and Brown, 2013).
Acid phosphatase reaction is a reliable chemical test to identify seminal stains
particularly in cases of Azoospermia, Oligospermia and Asthernospermia where the
microscopic detection of spermatozoa is very difficult (Raju and Iyengar, 1964).
The Acid Phosphate test has been scrutinised for the time length needed for the
reaction to occur. Lewis et al (2013), Peonimv (2013) and Redhead and Brown (2013),
each have different variations in timings to reveal how long it takes for the AP reaction
to give a positive result. This ranges from immediately adding the acid phosphatase
chemical to up to 15 minutes. Peonim (2013) said that the longer the reaction is left to
develop it increases the chance of getting a false positive result.

11 | P a g e
2.7. Prostate Specific Antigen (PSA)
The prostate-specific antigen was introduced in 1971 for the use with rape cases. It is
regarded as a useful confirmatory test in rape investigations because it can persist at
detectable levels in the vagina for up to 48hours post-coitus and although female bodily
secretions such as breast milk and sweat do contain PSA, the levels are usually below
the limit of detection so likelihood of a false positive is low (Peonim et al, 2013).
Overtime, the enzyme-linked immunosorbent assay (ELISA) was introduced into the
PSA detection which is a widely used technique as it’s a rapid one-step
immunochromatographic assay that has improved the accuracy of testing (Peonim et
al, 2013)
Prostate-specific antigen (PSA) is a protein within the seminal fluids and the main
function is to liquefy the seminal fluid; serine protease. PSA has a concentration of 0.5–
3.0 mg/ml (Gallo et al 2013). The PSA test works by adding a semen sample to the
sample window where it travels to the glass fibre pad membrane that has two
monoclonal murine anti-PSA antibodies as active compounds. One of the antibodies is
immobilized at the test region on the membrane. The upstream control region and the
region of the internal standard contain immobilized polyclonal goat anti-mouse
antibodies which bind the anti-PSA-antibodies in the sample at the control and internal
standard region, showing two pink/purple bands. (Peonim et al, 2013) If the PSA reacts
with the monoclonal antibody, meaning a positive result; a third pink/purple band will
appear between the control and internal standard region, if not, giving a negative result,
a pink/purple band will only occur in the control region (t-line). If the test is invalid, no
line will appear. The assay is optimized to detect 4 ng/ml of PSA although a positive
result can be detected in as low as 0.5ng/ml. The more intense the t-line colour is,

12 | P a g e
indicates the higher concentrations of PSA (Peonim et al, 2013). It is said by Gallo et al
(2013), that Prostate-specific antigen can be detected in dried samples of semen for up
to thirty years although cannot be detected in stains that have been washed multiple
times. PSA can be used as a marker to whether a condom was used and how effective
it was. (Snead, 2013).
2.8. Ultra-Violet Light
Common methods for searching for bodily fluids are; Visual, Physical, Chemical and
Fluorescence. With these examinations, it is able to examine all aspects of bodily
fluids. As the observation of semen by the naked eye or under white light prior to any
staining or light source can be difficult, using an alternate light source such as Ultra-
Violet light can direct attention to a latent stain at a crime scene (Virkler et al, 2009)
which provides a rapid, non-destructive way of screening and can examine large items
and a multiple number of items. UV light is a source of electromagnetic waves
measured in nanometres (nm). Depending on the fluorescence, semen could be
generated with wavelengths from 350 nm to 500 nm caused by the excitation of
molecules and depending on the light source used, fluorescence can be different
colours. (Kobus et al, 2002). UV light is seen as a presumptive testing due to the
mixed opinions of the technique; false positives were found using the ultra-violet light to
detect fluorescence on human skin. Two of these were easily identifiable with the
naked eye on areas of thickened or dried skin and one was thought to be hand-cream.
Thus, UV Light is not specifically designed to fluorescence semen alone. (Wawryk and
Odell, 2005).Moreover, depending on the fabric type the semen was distributed onto,
the absorbency and wetness of the semen, depends on the fluorescence of the stain or
if it is fluorescence at all. (Kobus et al, 2002).

13 | P a g e
2.9. Microscopy
“Microscopy is defined as the application of any enlarged-image process enabling
visualization of objects nominally invisible to the unaided human eye” (McCrone, 1993).
A confirmatory test for the presence of spermatozoa in semen is microscopic
examination. If the AP test previously came back positive, a fabric sample can be taken
or swabbed and be placed into a micro centrifuge tube with distilled water. This will
remove any of the sperm, if present, from the fabric or swab head. The resulting
supernatant can then be centrifuged to form a sperm pellet which can then be mounted
onto a slide for examination. However, the sperm tails are lost during this technique
resulting in sperm heads without tails. (Simmons et al, 2014)
Before the sperm heads/tails can be placed under the microscope, they need staining
using either haematoxylin and eosin (H&E) staining; this will stain the sperm heads
purple, and the remaining cellular material a pink colour or the ‘Christmas tree stain’
which uses nuclear fast red and picroindigocarmine to stain sperm a green and red
colour (Virkler and Lednev, 2009) so they can be seen under the 400x magnification.
They can then be mounted on a slide and the presence and concentration of any
sperm heads (with or without tails) can be recorded (Simmons et al, 2014). If the AP
test has given a positive result but no sperm heads are present during microscopic
examination, this could indicate a potential false positive AP reaction, or it may be a
result of an oligospermic or azoospermic semen sample. In these situations, alternative
confirmatory tests can be used including the choline test and prostate specific antigen
(PSA). (Simmons et al, 2014)

14 | P a g e
3. Aims and Hypothesis
The direct transfer of semen often occurs in cases of rape. However, due to the delay
between the crime and its reporting, it is often the case that semen analysis must be
performed on secondary and tertiary materials following semen transfer that must be
relied upon for evidence gathering. In this regard, reliable methods for the analysis of
semen stains on to secondary and tertiary materials is of substantial importance to the
forensic sciences. The acid phosphatase assay, a presumptive test, and the prostate
specific antigen test and microscopic analysis, both confirmatory tests, have been
shown to have some merit when investigating cases of rape and are routinely used for
analysis of semen from direct transfer. However, controversy exists in the forensic
literature with regard the most reliable presumptive and confirmatory tests for semen
analysis that would produce useable data with which to build a case against the
accused. These tests are dependent on the transfer and persistence of semen and little
research has been done to compare and contrast these methods. The aim of this
experiment was therefore to determine if it is possible to detect seminal fluid after
secondary and tertiary transfer using a variety of materials to simulate secondary and
tertiary transfer from cotton underwear. It is hypothesised that secondary transfer will
produce significantly greater positive test results compared to tertiary transfer. Further
it is hypothesised that the confirmatory prostate specific antigen test and microscopy
will provide significantly greater positive tests that the presumptive acid phosphatase
test.

15 | P a g e
4. Method
4.1. Materials
Semiquant PSA assay kit was obtained from Seratec Gbhm, Germany. Underwear
samples were obtained from a high street store (Primark, Stoke-on-Trent, UK).
Microscopy was performed using a Nikon Eclipse E200 light microscope (Nikon, Surry,
UK) and microscopy images were captured using a Nikon digital viewer (Nikon, Surry,
UK) connected to a Nikon Eclipse E200 microscope. Semen samples were obtained
from a healthy 27 year old volunteer. All other chemicals and reagents used in this
project were supplied by Sigma-Aldrich, Poole, UK.
4.2. Selection of transfer material
It was important to establish the most common underwear fabric worn by females in
order to increase the relevance of this research. In this regard a questionnaire was sent
to a commercial supplier of women’s underwear (Boux Avenue Ltd, Manchester, UK) to
enquire about their most popular underwear fabric. Another questionnaire was then
given to 10 females aged from 18 to 31 years to determine what their preferred fabric
or material for underwear. However, subjective analysis of the gussets of collected
underwear samples showed that a common material was used for this purpose and this
material was selected as the most appropriate for assessment of semen transfer. All
underwear samples were made of 100% cotton.
4.3. Collection and storage of semen samples
Each semen sample was collected in and stored in a plastic sterile air-sealed
container. Semen samples were stored in a fridge at 5 °C and used within 2 days of
collection.

16 | P a g e
4.4. Assessment of the drying duration of semen
To help with gaining an insight on what timings could be used within the research
project for the transferability of semen, the drying duration of semen was assessed. A
deposit of 1 ml of semen was pipetted onto the gusset of cotton underwear that lay
upon blue towel and left until completely dry. The underwear was pressed with blue roll
every half hour and the degree of drying was determined using subjective appraisal of
the degree of moisture present on the blue towel. When no moisture was observed on
the blue towel following pressing the semen was considered to have dried.
4.5. Semen transfer from primary to secondary and tertiary sources
Semen (1 mL) was pipetted onto a 19.6 cm2
sample the selected primary material
(underwear gusset). The contaminated gusset was then put into contact with 19.6cm2
of secondary and tertiary material (see table 4.1.). These secondary and tertiary
samples of materials were placed in contact with the primary material in the order from
lowest to highest: primary (underwear gusset), secondary, tertiary, on a flat and even
surface with an added weight of approximately 63 kg. This was left for 30 min of
contact time. Each material was then analysed separately using presumptive and
confirmatory testing (see section 3.7., 3.8. and 3.9.).
4.6. Preparation of semen stained material for analysis
Test material (1cm3
) was added to 500 µl of phosphate buffered saline and the
contents were then mixed using a vortex mixer for 1 min and left to stand in a test tube
rack for 30 min. The contents was then centrifuged at 1300 g (14800 rpm) and the
supernatant collected for analysis.

17 | P a g e
4.7. Acid phosphatase (AP) assay
A brentamine spot test based on the reactivity of acid phospahatse in semen sample
with alpha-naphthyl acid phosphate and brentamine fast blue. All testing was
performed after the semen sample residues had been transferred to a filter paper using
the indirect method. This involved adding a 5 µl of the supernatant prepared in section
3.6. Directly to a dry filter paper. A purple discolouration of the filter paper indicated a
positive. Negative and positive controls were performed using 5 µl saline buffered
saline or neat semen, respectively.
4.8. Prostate specific antigen (PSA) assay
A 1 ml sample of the supernatant prepared in section 3.6. Was added to the test well of
a Semiquant PSA assay kit result (see figure 3.8). Material that could not be cut, such
as the ceramic tile, was swabbed and the end of the cotton swab was then used for
analysis. A negative test was indicated by a colour change in the control and internal
standard area of the kit only. A positive test was indicated by a colour change in all
three test areas on the assay kit.
Figure 3.8. The Semiquant prostate specific antigen (PSA) assay is a chromatographic
immunoassay. It can be used for the rapid detection of PSA in human body fluids, including
semen. The Semiquant assay contains two monoclonal murine anti-PSA antibodies as active
compounds. One of these antibodies is immobilised in the test region of the kit. The control
region and the internal standard region contain immobilised polyclonal goat anti-mouse
antibodies. Samples are loaded into a glass fibre well. Here monoclonal murine anti-PSA
antibodies will bind to any PSA in the sample. Capillary action transfers the complex to the test
line where it will bind with the monoclonal antibodies at the test line causing a purple
decolouration. The control lines and internal standard lines are independent of the presence of
PSA in the sample, indicating only the correct application of the sample. The lower limit of

18 | P a g e
detection, based on the amount of antibody at the internal standard region, is set at 4 ng/mL
PSA.
4.9. Microscopy
A sample of supernatant prepared in section 3.6. (5 µl) was added to the microscope
slide and left for 5 minutes to dry on the hot plate at 90 o
C. Haematoxylin stain was
added to the microscope slide, making sure the supernatant was covered, and this was
left to dry for a further 2 min at 90 o
C .Excess haematoxylin stain was gently washed
away with distilled water and the slide stained with eosin stain, which was left to dry at
90 o
C for 30 sec. The eosin stain was then washed off with distilled water, before
allowing the slide to dry for a further 1 min. Once the slide had cooled, Depex was
added to the slide to cover the stained area and the slider was left until the Depex was
dry. The slide was then observed through a Nikon Eclipse E200 microscope at a
magnification of x 400. A Nikon digital viewer was used to capture the image.
5. Results
5.1. The effect of material type on the transfer of semen
The main finding of this study was that the transfer of semen to tertiary materials (see
table 1 for material classification) was significantly lower when compared to the transfer
to secondary materials (P=0.001) (figure 4.1) at the t=0 min time point. However, at the
t=120 min time point, no differences existed between the transfer rate of semen to
secondary or tertiary materials (P=1.000) (figure 4.1.1.).
0
5
10
15
20
Secondary material Tertiary material
PositiveAPTestFrequency
**

19 | P a g e
Figure 4.1. A comparison of the semen transfer to secondary and tertiary materials at the t=0 min time
point using the acid phosphatase (AP) test.A 1 mL sample of semen was transferred to cotton underwear
and immediately brought into contact with secondary and tertiary materials placed on top of one another.
Following 30 min of contact time the transfer of semen was assessed using an acid phosphatase assay.
From the data 2 × 2 contingency tables were constructed and a 2-tailed Fisher's exact test performed on
the data. The results of the analysis showed that there was a significant difference (P=0.001) between the
transfer of the semen between the secondary and tertiary material. *P<0.05, **P<0.01, ***P<0.001.
Figure 4.1.1. A comparison of the semen transfer to secondary and tertiary materials at the
t=120 min time point using the acid phosphatase (AP) test. A 1 mL sample of semen was
transferred to cotton underwear. The sample was allowed to air dry over a period of 120 min,
before being brought into connect with secondary and tertiary materials place on top of one
another. Following 30 min of contact time the transfer of semen was assessed using an acid
phosphatase assay. From the data 2 × 2 contingency tables were constructed and a 2-tailed
Fisher's exact test performed on the data. The results of the analysis showed that there was no
significant difference (P=1.000) between the transfer of the semen between the secondary and
tertiary material. *P<0.05, **P<0.01, ***P<0.001.
Table 4.1. Classification of the secondary and tertiary materials used to measure the rate of
semen transfer from cotton underwear. Secondary materials were brought into direct contact
with the semen soaked cotton underwear, while tertiary materials were brought into contact with
semen only through indirect transfer through secondary materials. All secondary and tertiary
materials were approximately 19.6 cm2.
5.2. The Effects of drying time on semen transfer to secondary and
tertiary materials from cotton underwear
A significant effect was found for drying time on the transfer rate of semen from cotton
underwear to different materials. Increasing the drying time from 0 to 120 min
significantly decreased the transfer rate of semen from cotton underwear to secondary
0
2
4
6
8
10
12
NegativeAPTestFrequency
Secondary materials Tertiary materials
Cotton pyjama bottoms Ceramic tile
Nylon tights Felt car boot lining
Denim jeans

20 | P a g e
materials (P=0.001). However, there was no significant effect of drying time on the rate
of semen transfer from cotton underwear to tertiary materials (P=1.000) (figure 4.2.).
Figure 4.2. A comparison of the semen transfer to secondary and tertiary materials at the t=0
min versus the t=120 min time point using the acid phosphatase (AP) test. A 1 mL sample of
semen was transferred to cotton underwear. For the t=0 min treatment, cotton underwear
soaked in semen was brought into immediate contact with secondary and tertiary materials. For
the t=120 samples, secondary and tertiary materials were only brought into contact with the
cotton underwear soaked in semen following a 120 min drying period. Sampling of the materials
(n=1 to 3) for semen contamination was then performed using an AP assay. From the data 2 ×
2 contingency tables were constructed and a 2-tailed Fisher's exact test performed on the data.
The results of the analysis showed that there was no significant difference (P=1.000) between
the transfer of the semen to tertiary material between the t=0 min and t=120 min time points.
However, a significant decrease in the transfer of semen to secondary material, as indicated by
an increase in the number of negative AP test results, between the t=0 min and t=120 min time
period was observed (P=0.001). *P<0.05, **P<0.01, ***P<0.001.
5.3. Comparison of the acid phosphatise test and the prostate specific
antigen test
Another key finding of this study was that the number of false negative tests for the acid
phosphatise assay was significantly higher when compared to the prostate specific
antigen assay for secondary and tertiary materials combined (P=0.006), and for tertiary
materials in isolation (P=0.010), but not secondary materials in isolation (P=1.000) (figure
4.3.).
0
99
10
0
2
4
6
8
10
12
t=0 min t=120 min
Secondary Tertiary
**
*

21 | P a g e
Figure 4.3. The number of false negative acid phosphatise (AP) test results for various
conditions. False negative results for the presumptive AP test were calculated by comparison to
the confirmatory prostate specific antigen (PSA) test at both t=0 min and t=120 min. The
increase in the number of false negative results on the AP test in comparison to the PSA test
were calculated and the results entered into a 2 × 2 contingency table (n=1 to 3). From these
tables a 2-tailed Fisher's exact test was performed on the data. The results of the analysis
showed that there was a significant increase in false negative test results for the PA test in
comparison to the PSA test (P=0.006), in all data combined [AP(C)]. For tertiary materials
[AP(T)], a significant increase in the number of false negatives results occurred between the
PSA and AP test (P=0.0102). However, there was no significant increase in the number of false
negatives for the AP test when compared to the PSA test for secondary materials (P=1.000)
[AP(S)]. *P<0.05, **P<0.01, ***P<0.001.
5.4. Microscopy
Microscopy was used to assess the presence of semen on the tertiary and secondary
materials brought into contact with the semen soaked cotton underwear. Sperm heads,
indicative of the transfer of semen, were only found on samples from tights and cotton
pyjamas, following secondary transfer (see figure 4.4.1 and 4.4.2.). No sperm heads
were detected on samples taken from tertiary materials (see figure 4.4.3.).
0
1
2
3
4
5
6
7
8
9
AP (C) AP (T) AP (S)
Increaseinnumberoffalsenegativetests
**
*

22 | P a g e
Figure 4.4.1. Detection of sperm heads on tights using light microscopy. Samples were taken
from materials brought into contact with semen soaked cotton underwear and stained with
haematoxylin stain and counterstained with eosin stain. Once the slide had cooled, Depex was
then added to the slide. Sperm heads on the tight can be seen stained red, indicating semen
contamination from the cotton underwear.
Figure 4.4.2. Detection of sperm heads on cotton pyjamas using light microscopy. Samples
were taken from materials brought into contact with semen soaked cotton underwear and
stained with haematoxylin stain and counterstained with eosin stain. Once the slide had cooled,
Depex was then added to the slide. Sperm heads on the tight can be seen stained red,
indicating semen contamination from the cotton underwear.
Figure 4.4.3. Detection of sperm heads on a tile using light microscopy. Samples were taken from
materials brought into contact with semen soaked cotton underwear and stained with haematoxylin stain
and counterstained with eosin stain. Once the slide had cooled, Depex was then added to the slide. Sperm
heads are absent from the sample, indicating that the transfer of semen from the secondary material was
not successful.
5.5. Assessment of dry time
It was found that semen dries within a time frame of approximately four and half hours.
6. Discussion
Evidence recovery is paramount in forensic science. In this regard, accurate and
reliable measurements of semen contamination are important. The direct transfer of

23 | P a g e
semen often occurs in rape cases (Horswell et al, 2004), yet it is the secondary and
tertiary transfer that may have to be relied upon to create evidence for a case
(Christian et al, 2000). However, controversy exists about the methodologies
surrounding measurements of secondary and tertiary transfer rates for semen.
Therefore the aims of this study were to assess the transfer rates of semen from cotton
underwear to a range of materials through secondary and tertiary transfer. In this
regard, the main findings of this study were that the transfer of semen from cotton
underwear to secondary materials occurs at a significantly higher rate that when
compared to the transfer of semen from cotton underwear to tertiary material. Further,
it was observed that the presumptive acid phosphatase test produces a significantly
higher amount of false negative tests compared to the confirmatory prostate specific
antigen test.
A primary finding of this research was that transfer of semen from cotton soaked
underwear was significantly greater to materials brought into direct contact with the
underwear (secondary materials) compared to those brought into contact with
secondary sources (tertiary sources). Examination of the different transfer rates to
secondary and tertiary materials using the acid phosphatise test showed significantly
greater numbers of positive tests for secondary transfer materials when these tests
were performed without allowing the semen to dry. These results may be explained in
terms of the volume of semen diminishing as increasing numbers of materials are used
for its transfer. In this regard it would be expected that secondary sources would be
contaminated with the highest amounts of semen, and the semen volumes would
decreased as tertiary transfer occurred. It is therefore likely that the semen present in
tertiary sources dropped below the lower level of detection for the acid phosphatise
test. This highlights the requirement for semen samples to be taken spatially as close
to the source of the crime as possible.

24 | P a g e
Other researchers have assessed the reliability of the acid phosphatase test and
shown it to be a reliable test when measuring the direct transfer of semen (Schiff, 1978;
Ricci and Hoffman, 1982). Further, very dilute solutions as low as 1 drop of a 2 %
solution of semen in distilled water readily respond to the acid phosphatase assay even
after a period of one year (Raju and Iyengar, 1964). However, the usefulness of the
test for the detection of semen on secondary and tertiary materials is more
controversial. In this regard, the acid phosphate test is not semen specific, meaning it
can give false positives through detection of other substances that may have come into
contact with the secondary or tertiary material during the day, including breast milk,
saliva and vaginal secretion, urine, faeces and certain foods (Lewis et al, 2013). Also, a
high enough concentration of semen on the secondary material is required to produce
a positive test using the acid phosphatise assay, and this is not assured. How much of
the semen from the primary sources contaminates the secondary tertiary materials may
therefore have a bearing on the detection rates using the acid phosphatise test due to
its lower sensitivity compared to other similar tests (Redhead and Browne, 2013).
Further, other conditions can affect the transfer of semen from primary to secondary
sources inclusion the pressure applied, the location and the number of contacts
(Houck, 2010).These factors may explain differences in the literature regarding transfer
rates from primary to secondary and tertiary sources.
Addition of a drying time of 120 min following the application of semen of cotton
underwear significantly increased the number of negative tests produced by the acid
phosphatise test, with regard to the secondary materials. This increase in the number
of negative tests annulled the significant difference between the semen transfer rates
to the secondary and tertiary materials. These results suggest that the drying of semen
on materials decreases the usefulness of the acid phosphatise tests at detecting the
presence of semen contamination. This therefore highlights the importance of the

25 | P a g e
temporal component in the gathering of evidence from a crime scene to allow samples
to be collected in the best condition possible.
The effects of the drying time of the detection rates of semen are interesting, as they
may highlight methodological problems with the acid phosphatise test. For example,
Davidson and Jalowiecki (2012) showed that significant increases in the reliability of
the acid phosphatise test can be observed through wetting both the semen stained
garment and the test paper, when compared to wetting the test paper alone. This
suggests that wetting of the semen is an important step in the successful transfer of the
spermatozoa from the garment to the test strip and that drying is detrimental. This is in
agreement with Goray (2010) who reported increased detection of semen as the
moisture content of the deposits was increased. In this regard, the quantity of semen
present following direct transfer may have an influence on the detection of semen on
secondary and tertiary materials, due to the extended drying time that this would
create. For example, Oorschot’s (2014) and Houck’s (2010) findings showed an
improved detection of semen with increasing volume, which may have resulted from
extending the drying time. Lomas (2014) also reported increases in drying time for
semen as semen volumes on garments was increased. Factors that dilute the semen
within the garments may also negatively affect the reliability of the acid phosphatise
test (Redhead and Browne, 2013). Further, animal experiments have shown that
dilution of the sperm is an important component in the longevity of the quality of the
spermatozoa (Varner et al, 1987; Love et al, 2015).
The acid phosphatase assay is a presumptive colorimetric test, the results given being
qualitative (Green, 2000). Because food, faeces and urine, and particularly female
bodily fluids such as vaginal secretions and sweat may contain acid phosphatase
activity (Diamandis, 1997; Peonim et al, 2013) contamination by these factors can
decrease the reliability of the test. Further, while acid phosphatase activity is known to

26 | P a g e
be greater in semen than in any other bodily fluid (Green, 2000), seminal acid
phosphatase levels vary between males (Redhead et al, 2013), the size of the testis
can influences the total number of spermatozoa per ejaculate (Andersen et al., 2000)
and the amount of seminal fluid produced differ from the first ejaculation to last (Cooper
et al. 1993). These factors can therefore increase the likelihood of obtaining a false
positive or false negative acid phosphatase test. Confirmatory tests would therefore be
required to increase the reliability of the data in addition to any acid phosphatase tests
performed. Additionally, there may be instances where there is a positive acid
phosphatase reaction, but no sperm heads are present during microscopic
examination. This may indicate a potential false positive acid phosphatase reaction.
However, it may also be a result of an oligospermic or azoospermic semen sample. In
such circumstances, alternative confirmatory tests including the choline test and
prostate specific antigen assays may be used. Evidence suggests that of these, the
semi-quantitative colorimetric prostate specific antigen assay may be more sensitive
than the choline test (Simmons, 2014).
In study the prostate specific antigen assay was used was used as a confirmatory test
to the acid phosphatase test. A key finding in this regard was that the number of false
negative results increased significantly with use of the acid phosphatase assay
compared to the prostate specific antigen assay for tertiary material transfer, and
tertiary and secondary material transfer combined. However, for secondary material
transfer, no significant differences between the two assay types was observed. These
data may reflect the increased reliability of the acid phosphatase test on secondary
compared to tertiary transfer, due to the aforementioned increase in semen volume
following secondary in comparison to tertiary transfer. As transfer passes from the
secondary to tertiary stages, semen volume decreases and thus relative drying time
decreases, and this increases the risk of false negatives when using the acid

27 | P a g e
phosphatase assay. However, the higher sensitivity of the prostate specific antigen
assay negates these factors and thus decreases the risk of a false negative result.
Other authors have investigated the use of the prostate specific antigen assay for use
in the detection of semen stains. For example, Simich et al (1999) used the Tandem-E
PSA immunoenzymetric assay to detect prostate specific antigen in 100 % of the
forensic casework fabric samples and 80 % of the forensic casework vaginal swabs,
with lower limits of detection of 1.77 ng/ml. Although these authors used an
immunoenzymatic, rather than immunochromatographic assay, the principles for the
two tests can be considered to be broadly similar (Talthip, 2007; Hochmeister, 1999).
Other authors have compared the acid phosphatase assay with prostate specific
antigen assay and found that results from the prostate specific antigen assay were
more reliable compared to the acid phosphatase test when samples were analysed
after 48 hours (Khalidi et al, 2004). However, although the accuracy of the prostate
specific antigen assay is regarded as close to that of a various ELISA techniques
(Talthip, 2007; Hochmeister, 1999), the prostate specific antigen assay may still
produce false positives (Peonim et al, 2013).
It was noted during the analysis of the results from the prostate specific antigen test kit
that weak colouration was sometimes present on the T-line The manufacturers of the
kit (Seratec) state that “any visible T-line (strong or weak coloured) indicates a positive
result”, with claimed sensitivity to the 1 ng/ml level of prostate specific antigen.
However, a strong colouration of the T-line shows that the semen level of the prostate
gland is distinctively higher than 4 ng/ml. With this test being a colorimetric test, the
results are subjective to the observer and the environmental conditions to some extent.
There are other methods used for PSA detection like Abacus OneStep ABAcard which
is inexpensive and can also give semi-quantitative results (Snead et al, 2012). Based
on the subjectivity involved in the interpretation of the strength of the colourimetric

28 | P a g e
change, the prostate specific antigen test was used as a qualitative test, and this may
be one weakness of this study.
Microscopy is a confirmatory test often used in conjunction with the prostate specific
antigen test and the acid phosphatise test. Microscopy confirms the presence of the
sperm heads and tails visually and this produces good reliability (Virkler et al, 2009). Of
course, the use of microscopy also relies on the semen originating from an individual
who is not azoospermic (Virkler et al, 2009). In the results presented here, there was a
trend for the detection of spermatozoon heads on secondary materials only, supporting
the other observations that transfer of sperm to the tertiary materials was limited.
However, sperm heads were only detected on tights within the secondary material
grouping, despite positive prostate specific antigen and acid phosphatise test results in
other materials.
Cambridge (2003) suggested that the seminal fluid should ideally be analysed
microscopically as soon as liquefaction has occurred, for as little as one hour is enough
duration to see significant a degradation of the semen quality. This could potentially
give a partial explanation to why the presence of spermatozoon heads were not
detected reliably by microscopy despite the detection of prostate specific antigen or
acid phosphatase contamination of secondary, and to a lesser extent, tertiary
materials. However, other authors have shown microscopy to be a useful and reliable
tool in the detection of semen (Christian et al, 2000; Wilson, 1974; Henkel et al, 2007),
and so the reason for the lack of confirmatory positive tests from microscopic analysis
of samples is not clear. It is possible that methodological differences may explain the
discrepancies. For example, Christian et al (2000) used Papanicolaou differential
staining as used in the method reported here, but subsequent analysis under × 1000 oil
immersion to indentify spermatozoa.

29 | P a g e
The fibre type within the material could also potentially explain the reason for the
detection of sperm heads on the tight, while on other materials sperm heads were not
detected. Natural and man-made fibres can have quite different physical properties on
account of weave pattern, fibre thickness and elasticity of fibre type. Fibres can also be
spun, woven or regenerated to produce very different fibre structures and textures.
Materials can therefore potentially have a wide range of characteristics that affect the
properties (Robertson, 1999). As tights are of a stretchy and loose nature, with a thin
ad elastic fibre type, this may have facilitated significantly greater transfer or retention
of semen compared to other more course, less elastic and more tightly woven
materials. This may then have facilitated the detection of a significantly larger number
of sperm heads due to higher semen volume
Another consideration with regard the generation of false negative results is the
consistency of the semen. The collection of semen and its use needs to be rigorously
controlled to provide reliable and consistent results, but this is not always possible due
to inconsistencies in the semen volume and consistency. During the course of
analysing samples, it was noted that some samples of semen were observed to have a
more watery consistency while others that were of a more thick and jelly like texture.
This may relate to the storage time of the semen. For example, upon immediate
ejaculation semen will be in a semisolid form, but over time the semen will become
thinner to produce a more heterogeneous mixture in a liquefaction process.
(Cambridge, 2003). Over the course of roughly 15 minutes at room temperature for
example, it has been reported that liquefying will be complete (Cambridge, 2003). The
deterioration of the sample then occurs in other ways, as for example proteins are
removed from their optimal temperature range. The length of time between ejaculation
and use of the sample may therefore have a significant baring on the outcome of the
transfer rates from primary to secondary sources.

30 | P a g e
Further, the colour was more diverse from that the grey-opalescent colour described by
Cambridge (2003), including slight shades of yellow or green colour, which may reflect
changes to the dietary intake of the donors. For example, Kasperczyk (2014) reported
that zinc intake can have a significant effect on the concentration of seminal fluid.
Afediche et al (2014) reported that increases in dairy consumption was associated with
higher sperm concentration and a higher sperm motility. In fact, seminal fluid can have
a high concentration of sperm cells but a low volume of ejaculation and vice versa, all
depending on the dietary components (Partyka, et al. 2014). Because the diet of most
individuals varies over time, it may be that semen consistency also varies. Even using
a single donor for semen analysis could therefore lead to inconsistencies in the semen
quality over time, and this may have a significant effect on the detection of semen
following secondary and tertiary transfer.
7. Conclusion
The results presented here demonstrate that the detection of semen from secondary
and tertiary materials is complex. Presumptive tests such as the acid phosphatase can
be a useful tool in the detection of semen stains in secondary and tertiary semen
transfer, but the duration of time between the crime and the use of the test is
paramount. However, as the drying time increases the number of false negatives
increase when compared to the more reliable prostate specific antigen test. Microscopy
has been reported to be a reliable tool for the analysis of semen, and so the
inconsistent findings reported here may be due to methodological differences in
comparison to previous work.

31 | P a g e
8. Further Work
After the completion of this project, many limitations were encountered which could
provide a means for further work.
As there were only a small amount of repeats conducted in this research project and
limited material types, it would be of greater value to the forensic outlook to conduct
many more repeats as well as using other donors to see how much variation there is in
the consistency of different donors. This will support the microscopy results if research
was conducted into Azoospermia, Oligospermia and Asthernospermia.
Further research could look into the use of other tests for the traceable amount of
semen i.e. choline, luminal and the Christmas tree staining, as it seems the AP and
PSA test results were questionable.
Due to the subjectivity of qualitative data, a method of providing numerical data for the
current tests could be developed.
The environmental conditions in which a semen sample could be found in could be
examined i.e. temperature changes and humidity. Oorshot, 2014 had looked into the
transfer of blood at different temperature so it would be useful to look into other bodily
fluids as there are different climates throughout the world and in all circumstances in
every rape/sexual assault cases.
The different methods of contact that can occur could affect the identification of semen
at the time of analysis e.g. passive, pressure, and friction with secondary and tertiary
materials due to different movements of rape and sexual assault victims after an
incident. This study could be repeated however with adaptations that accommodate the
above factors.

32 | P a g e
9. References
1. Achiff, A. F. Reliability of the acid phosphatise test for the identification of
seminal fluid. Journal of Forensic Sciences. 23(4): 833-844
2. Afediche. M, Bridges. N, Williams. P, Gaskins. A, Tanrikut. C, Petrozza. J,
Hauser. R and Chavarro. J. (2014). Dairy intake and semen quality among men
attending a fertility clinic. Fertility and Sterility. 101 (5), 1280-1287
3. BBC. (2011). BBC - Food and semen taste (the experiment). Available:
https://www.youtube.com/watch?v=P1k1Dy1mVeQ. Last accessed 05/03/2015
4. Bigbee. P and Spalding. R (2001). Practical Aspects of Rape Investigation A
Multidisciplinary Approach. 3rd ed. USA: CRC Press. 34-45
5. Cambridge University (2003). WHO Labortory Manual for the Examination of
Human Semen and Sperm-cervical Mucus Interaction. 4th ed. Cambridge:
World Health Organisation. 1-5
6. Christian, C. W., Lavelle, J. M., De Jong, A. R., Loiselle, J., Brenner, L. And
Joffe, M. 2000. Forensic evidence findings in prepubertal victims of sexual
assault. Pediatrics. 106(1): 100-104
7. Cooper. T, Noonan. E, Eckardstein. S, Auger. J, Baker. H, Behre. H, Haugen.
T, Kruger. T, Wang. C, Mbizvo. M and Vogelsong. K. (2010). World Health
Organization reference values for human semen characteristics. Human
Reproduction Update. 16 (3), 231-245
8. Davidson, G. and Jalowiecki, T. B. 2012. Acid phosphatise screening – wetting
test paper or wetting fabric and test paper. Science and Justice. 52(2): 106-111
9. Diamandis, E. P. 1997. Nonprostatic sources of prostate-specific antigen.
Urologic clinics of North America. 24(2): 275-82
10. Elliot. C and Quinn. F (2012). Criminal Law. 9th ed. Essex: PEARSON. 173-174
11. Gallo. M, Snead. M, Black. C, Brown. T, Kourtis. A, Jamieson. D, Carter. M,
Penman-Aguilar. A and Macaluso. M. (2013). Optimal methods for collecting

33 | P a g e
and storing vaginal specimens for prostate-specific antigen testing in research
studies. Contraception. 87 (6), 830–835
12. Green. W. (2000). CLINICAL FORENSIC MEDICINE | Sexual Assault and
Semen Persistence. Encyclopedia of Foresic Sciences. 1 (1), 397-403
13. Greig. A. (2013). Neighbor of Ariel Castro confesses to kidnap, rape, and
murder of two women Read more: http://www.dailymail.co.uk/news/article-
2467091/Ariel-Castros-neighbor-Elias-Acevedo-confesses-kidnap-rape-
murde. Available: http://www.dailymail.co.uk/news/article-2467091/Ariel-
Castros-neighbor-Elias-Acevedo-confesses-kidnap-rape-murder.html. Last
accessed 05/03/2015.
14. Hall. W. (Unknown). First Aid for Emotional Trauma. Available:
http://www.theicarusproject.net/files/trauma_first_aid_fact_sheet12-08.pdf. Last
accessed 05/03/2015
15. Hayden. S, Blanchard. T, Brinsko. S, Varner. D, Hinrichs. K and Love. C.
(2015). The “dilution effect” in stallion sperm. Theriogenology. 83 (4), 772-777
16. Hazelwood. R and Burgees. A (2009). Practical Aspects of Rape Investigation.
4th ed. USA: CRC Press. 6-35
17. Henkel, R., Schreiber, G., Sturmhoefel, A., Hipler, U., Zerman, D. H. and
Menkveld, R. 2007. Comparison of three staining methods for the
morphological evaluation of spermatozoa. Fertility and Sterility. 89(2): 449-455
18. Hochmeister, M. N., Budowle, B., Rudin, O., Gehrig, C., Borer, U., Thali, M. And
Dirnhofer, R. 1999. Evaluation of prostate-specific antigen (PSA) membrane
test assays for the forensic identification of seminal fluid. Journal of Forensic
Sciences. 44(5): 1057-1060
19. Home Office (2002) Protecting the Public; Strengthening Protection Against Sex
Offenders and Reforming Law on Sexual Offences, London: HMSO

34 | P a g e
20. Home Office. (2013). An Overview of Sexual Offending in England and
Wales. Available: https://www.gov.uk/government/statistics/an-overview-of-
sexual-offending-in-england-and-wales. Last accessed 12/11/2013
21. Horswell. J and Fowler. C (2004). The Practice of Crime Scene Investigation.
USA: CRC Press. 3-11
22. Houck.M and Siegel.J (2010). The fundamentals of Forensic Science. 2nd ed.
London: Academic Press. 29-47
23. Kobus. H, Sileniek. E, Scharnberg. J. (2002). Improving the Effectiveness of
Fluorescence for. Journal of Forensic Science. 47 (4), 1-5
24. Kamath. M, Dahiya. A and Hegde. R. (2004). ABSORPTION
PROPERTIES. Available: http://www.engr.utk.edu/mse/Textiles/Absorption.htm.
Last accessed 08/10/2014
25. Kasperczyk. A, Dobrakowski. M, Horak. S, Zalejska-Fiolka. J and Birkner. E.
(2015). The influence of macro and trace elements on sperm quality.Journal of
Trace Elements in Medicine and Biology. 30 (1), 153-159
26. Khalidi, N., Miras, A, Botti, K., Benali, L., Gromb, S. 2004. Evaluation of three
rapid detection methods for the forensic indentification of seminal fluid in rape
cases. Journal of Forensic Sciences. 49(4): 749-752
27. Lehmanna. V, Mitchell. R, Ballantyne. K and Oorschot. R. (2013). Following the
transfer of DNA: How far can it go?. Forensic Science International: Genetics
Supplement Series. 4 (1), 53-54
28. Lewis. J, Baird. A, McAlisterc. C, Siemieniuk. A, Blackmore. L, McCabe. B,
O'Rourke. P, Parekh. R, Watson. E, Wheelhouse. M and Wilson. N. (2013).
Improved detection of semen by use of direct acid phosphatase
testing. Science & Justice. 53 (4), 385–394
29. Lewis. R. (2003). Medical Article. Male Factor Infertility. 5 (1), 1-7

35 | P a g e
30. Lomas. N (2014) The Secondary Transfer of Semen following Vaginal
Seepage. BSc Forensic Investigation Honours Degree. Stoke-On-Trent:
Staffordshire University
31. Maloney. M (2012). Death Scene Investigation Procedural Guide. USA: CRC
Press. 256-257
32. McGrath (2011). Rape Investigation Handbook. 2nd ed. London: Elsevier Inc.
231-249
33. OGL. (2014). Criminal Courts. Available: https://www.gov.uk/courts/magistrates-
courts. Last accessed 13/10/2014
34. Oorschot. R, Goray. M, Eken. E and Mitchell. R. (2009). Impact of relevant
variables on the transfer of biological substances. Forensic Science
International: Genetics Supplement Series. 2 (1), 547–548
35. Oorschot. R, McArdle. R, Goodwin. W and Ballantyne. K. (2014). DNA transfer:
The role of temperature and drying time. Legal Medicine. 16 (3), 161-163
36. Partyka. A, Ochota. M, Niżański. W and Prochowska. S. (2014). Effect of
dilution rate on feline urethral sperm motility, viability, and DNA
integrity. Theriogenology. 82 (9), 1273-1280.
37. Peonim. V, Worasuwannarak. W, Sujirachato. K, Teerakamchai. S, Srisont. S
and Chudoung. U. (2013). Comparison between prostate specific antigen and
acid phosphatase for detection of semen in vaginal swabs from raped
women. Journal of Forensic and Legal Medicine. 20 (6), 578–581.
38. Pyrek. K (2007). Forensic Science Under Siege: The Challenges of Forensic
Laboratories and the Medico-Legal Investigation System. USA: Elsevier. 95-99.
39. RAINN. (2012). National Sexual Assault Online Hotline. Available:
https://ohl.rainn.org/online/resources/how-long-to-recover.cfm. Last accessed
05/03/2015.

36 | P a g e
40. Raju. P and Iyengar. N. (1964). Acid Phosphatase Reaction as a Specific Test
for the Identification of Seminal Stains. Journal of Criminal Law and
Criminology. 55 (4), 522-524.
41. Rape Crisis. (2009). Offenders. Available: https://rainn.org/get-
information/statistics/sexual-assault-offenders. Last accessed 05/03/2015
42. Rape Crisis. (2015). Rape Trauma Syndrome. Available:
http://rapecrisis.org.za/information-for-survivors/rape-trauma-syndrome/. Last
accessed 05/03/2015.
43. Redhead. P and Brown. M. (2013). The acid phosphatase test two minute cut-
off: An insufficient time to detect some semen stains.Science & Justice. 53 (2),
187–191
44. Ricci, R. A. and Hoffman, S. A. 1982. Prostatic acid phosphatise and sperm in
the post-coital vagina. Annals of Emergency Medicine. 11(10): 530-524
45. Robertson. J (1999). Forensic Examination of Fibres. 2nd ed. USA: CRC Press.
100-102.
46. Seratec. (2013). SERATEC PSA Semiquant. Available:
http://www.seratec.com/docs/user_instructions/psm400f_multi.pdf. Last
accessed 6/3.2015.
47. Simich, J. P., Morris, S. L., Klick, R. L. And Rittenhouse-Diakun, K. 1999.
Validation of the use of a commercially available kit for identification of prostate
specific antigen (PSA) in semen stains. Journal of Forensic Science. 44(6):
1229-1231
48. Simmons. R, Deacon. P, Phillips. D and Farrugia. K. (2014). The effect of mark
enhancement techniques on the subsequent detection of
semen/spermatozoa. Forensic Science International. 244 (1), 231–246
49. Slaughter. L and Brown. C (1992). Colposcopy to establish physical finding in
rape victims. American Journal of Obstetrics and Gynecology, 166, 83-86

37 | P a g e
50. Snead. M, Kourtis. A, Black. C, Mauck. C, Brown. T, Penman-Aguilar. A,
Melendez. J, Gallo. M, Jamieson. D, Macalusod. M. (2013). Effect of topical
vaginal products on the detection of prostate-specific antigen, a biomarker of
semen exposure, using ABAcards . Contraception. 88 (3), 382–386.
51. SOMMERS. M, SCHAFER. J, ZINK. T, HUTSON. L and HILLARD. P. (2001).
Injury Patterns in Women Resulting from Sexual Assault.Trauma, Violence, &
Abuse. 10 (2), 241-257
52. Talthip, J. 2007.An autopsy report case of rape victim by the application of PSA
test kit as a new innovation for sexual assault investigation in Thailand. Journal
of the Medical Association of Thailand. 90(2): 348-351
53. The National Archives. (2012). Sexual Offences Act 2003. Available:
http://www.legislation.gov.uk/ukpga/2003/42/contents. Last accessed
13/10/2014
54. Varner. D, Blanchard. T, Love. C, Garcia. M and Kenney. R. (1987). Effects of
semen fractionation and dilution ratio on equine spermatozoal motility
parameters. Theriogenology. 28 (5), 709-723
55. Virkler. K and Lednev. I. (2009). Analysis of body fluids for forensic purposes:
From laboratory testing to non-destructive rapid confirmatory identification at a
crime scene. Forensic Science International. 188 (1-3), 1–17
56. Wawryk. J and Odell. M. (2005). Fluorescent identification of biological and
other stains on skin by the use of alternative light sources. Journal of Clinical
Forensic Medicine. 12 (6), 296-301
57. Wilson, E. F. 1974. Sperms morphological survival after 16 days in the vagina
of a dead body. Journal of Forensic Sciences. 19(3): 561-564

38 | P a g e
10. Appendices
1. Raw results for all materials using the Acid Phosphate test and Prostate
Specific antigen test with immediate transfer.
2. Raw data for the Comparison of Secondary and Tertiary material transfer rates
using AP test.
using AP test referring to a delay in initial contact of cotton underwear.
using AP test at 120 minutes.
5. Raw data for the comparison of false negative test results between AP and PSA
test at different timings.
6. Protocol for PSA testing.
7. Protocol for Preparation of an H+E slide staining.

39 | P a g e
Appendix 1: Raw results for all materials using the Acid Phosphate test and
Prostate Specific antigen test with immediate transfer
Positive Negative
Pyjamas 100 0
Tile 33 67
Pyjamas 100 0
Car Boot
Lining 66 34
Tights 100 0
Car Boot
Lining 100 0
Tights 100 0
Tile 100 0
Denim 100 0
Tile 0 100
Denim 100 0
Car Boot
Lining 0 100
Positive Negative
Pyjamas 100 0
Tile 100 0
Pyjamas 100 0
Car Boot
Lining 66 37
Tights 100 0
Car Boot
Lining 100 0
Tights 100 0
Tile 100 0
Denim 100 0
Tile 100 0
Denim 100 0
Car Boot
Lining 0 100
100
33
100
66
100
100
100
100
100
0
100
0
0
67
0
34
0
0
0
0
0
100
0
100
PERCENTAGE
MATERIALS USED
ACID PHOSPHATE
TEST
Positive Negative
100
100
100
66
100
100
100
100
100
100
100
0
0
0
0
37
0
0
0
0
0
0
0
100
PERCENTAGE
MATERIALS USED
PROSTATE SPECIFIC
ANTIGEN TEST
Positive Negative

40 | P a g e
Appendix 2: Raw data for the Comparison of Secondary and Tertiary material transfer
rates using AP test.
Comparisonof SecondaryandTertiarymaterial transferratesusingAPtestat t=0
SecondaryMaterial TertiaryMaterial
Positive Test 18 9
Negative Test 0 9
0
2
4
6
8
10
12
14
16
18
20
Secondary Material Tertiary Material
PositiveAPTestFrequency
Material Type
**

41 | P a g e
Appendix 3: 1.Raw data Comparison of Secondary and Tertiary material transfer rates
using AP test referring to a delay in initial contact of cotton underwear.
Comparisonof SecondaryandTertiarymaterial transferratesusingAPtestat t=0 comparedto t=120
t=0 min
Secondary Material TertiaryMaterial Secondary
Positive Test 18 9
Negative Test 0 9
0
99
10
0
2
4
6
8
10
12
t=0 min t=120 min
Secondary Tertiary
**

42 | P a g e
Appendix 4: 1.Raw data for the Comparison of Secondary and Tertiary material
transfer rates using AP test at 120 minutes.
Comparisonof Secondaryand Tertiarymaterial transferratesusingAPtestat t=120
SecondaryMaterial TertiaryMaterial
Positive Test 1 0
Negative Test 9 10
0
2
4
6
8
10
12

43 | P a g e
Appendix 5: Raw data for the comparison of false negative test results between AP and
PSA test at different timings.

44 | P a g e
Appendix 6: Protocol for PSA testing.

45 | P a g e
Appendix 7: protocol for Preparation of an H+E slide staining.

Tanya-Leigh Full Dissertation!

Recommended

Recommended

More Related Content

What's hot

What's hot (18)

Viewers also liked

Viewers also liked (20)

Similar to Tanya-Leigh Full Dissertation!

Similar to Tanya-Leigh Full Dissertation! (20)

Tanya-Leigh Full Dissertation!