Rebekah E. Findlay 1202708 Dissertation

School of Science, Engineering and Technology
Abertay University
The degradation of blood evidence on materials
after treatment with stain removers and active
oxygen detergents
Rebekah Elizabeth Findlay
Bachelor of Science with Honours, 2016

i
Table of Contents
List of Tables............................................................................................ iv
List of Figures .......................................................................................... vi
Acknowledgements................................................................................. vii
Abertay University Dundee, Permission to Copy ...................................viii
Abstract.................................................................................................... ix
Abbreviations, Symbols and Notation .......................................................x
Table of Abbreviations...........................................................................x
Table of Symbols...................................................................................x
Table of Notations ................................................................................ xi
1 Introduction........................................................................................1
1.1 General Introduction ....................................................................1
1.2 Blood............................................................................................1
1.2.1 Haemoglobin .........................................................................2
1.2.2 White Blood Cells..................................................................3
1.3 Deoxyribose Nucleic Acid (DNA) .................................................3
1.4 Material........................................................................................4
1.5 Treatment of stains ......................................................................5
1.5.1 Chlorinated cleaning products...............................................5
1.6 Washing of clothing......................................................................6
1.6.1 Powder detergents ................................................................6
1.6.2 Oxygenated washing powder ................................................7
1.7 Presumptive tests ........................................................................8
1.7.1 Kastle-Meyer .........................................................................9
1.7.2 Leucomalachite Green ........................................................10
1.7.3 False positive ......................................................................11
1.7.4 False negatives ...................................................................11
1.7.5 False negative caused by sodium percarbonate .................11
1.8 DNA analysis .............................................................................12
1.8.1 Extraction ............................................................................12
1.8.2 DNA Quantitation ................................................................14
1.8.3 DNA amplification................................................................15
1.8.4 Gel electrophoresis .............................................................15

ii
2 Aims and Objectives ........................................................................17
3 Methods and Materials.....................................................................18
3.1 Fabrics.......................................................................................18
3.2 Pre-treatment chemical compositions........................................18
3.3 Washing detergents chemical compositions ..............................18
3.4 Reagents ...................................................................................19
3.4.1 Kastle-Meyer reagent..........................................................19
3.4.2 Leucomalachite Green ........................................................20
3.5 Procedure ..................................................................................20
3.5.1 Preparation of materials ......................................................20
3.5.2 Blood deposition..................................................................21
3.5.3 Pre-treatment of samples ....................................................21
3.5.4 Washing of samples............................................................21
3.5.5 Sample preparation for presumptive testing........................22
3.5.6 Presumptive testing.............................................................23
3.5.7 Controls...............................................................................23
3.5.8 Recording of presumptive tests results ...............................23
3.5.9 Material preparation for DNA extraction ..............................24
3.5.10 Extraction of DNA................................................................24
3.5.11 UV Quantification ................................................................25
3.5.12 UV Quantification calculation...............................................26
3.5.13 Agarose Yield Gel ...............................................................26
3.5.14 Polymerase Chain Reaction (PCR).....................................27
3.5.15 NuSieve® GTG® Agarose Gel Electrophoresis ..................27
4 Results and Discussion....................................................................28
4.1 Presumptive Testing results.......................................................28
4.1.1 Test Group A – Non-biological powder and Bleach.............29
4.1.2 Test Group B – Bio and Bleach...........................................30
4.1.3 Test Group C – Non-bio and stain remover spray...............31
4.1.4 Test Group D – Bio and Stain remover spray......................32
4.1.5 Controls...............................................................................33
4.2 DNA Analysis.............................................................................34
4.2.1 UV Quantification ................................................................34
4.2.2 Agarose Yield Gel ...............................................................36

iii
4.2.3 NuSieve® GTG® Agarose Gel............................................37
5 Discussion .......................................................................................38
5.1 Interferences from products on presumptive tests .....................38
5.2 Comparison of hydrogen peroxide concentration on testing ......39
5.3 Correlation between washing groups and presumptive tests.....40
5.3.1 Comparison of pre-treatments.............................................40
5.3.2 Comparison of washing powder type ..................................43
5.4 Difference between use of spatula and filter paper....................45
5.5 Sodium percarbonate and presumptive testing..........................46
5.6 DNA analysis .............................................................................47
5.7 Connection between presumptive testing and DNA analysis.....49
6 Conclusions .....................................................................................50
Future Work ............................................................................................51
Reference list..........................................................................................52
Appendix 1 Results from KM and LMG...................................................58
Appendix 2 Photographs of presumptive test results .............................63
Appendix 3 Raw data collected from UV Quantification.........................92
Appendix 4 Results of UV Quantification results.....................................93
Appendix 5 Images of Yield Gel through UV imaging .............................94
Appendix 6 Images obtained at time intervals for NuSieve® Gel...........95

iv
List of Tables
Table 1 Fabrics randomly selected for DNA analysis..............................24
Table 2 - Mean reaction time for KM presumptive tests on Group A using
different testing methods.........................................................................29
Table 3 - Mean reaction times for LMG presumptive tests on Group A
using different testing methods ...............................................................29
Table 4 Mean reaction times for KM presumptive tests for Group B using
different testing methods.........................................................................30
Table 5 Mean Reaction times for LMG presumptive tests for Group B
Table 6 Mean reaction times for KM tests for test group C using different
testing methods.......................................................................................31
Table 7 Mean Reaction times for LMG presumptive tests for Group C
Table 8 Mean reaction times for KM tests for test group D using different
testing methods.......................................................................................32
Table 9 Mean Reaction times for LMG presumptive tests for Group D
Table 10 Results of presumptive testing on control samples ..................33
Table 11 Results of UV quantification Calculations for Amount of DNA
and Range of Purity in control samples ..................................................34
and Range of Purity for test group A.......................................................35
and Range of Purity in test group B ........................................................35
and Range of Purity for test group C.......................................................35
and Range of Purity for test Group D......................................................35
Table 16 Graph comparing 3% and 6% H2O2 used when coupled with KM
presumptive test on samples ..................................................................39
Table 17 Graph comparing times of presumptive testing between bleach
and stain removing spray........................................................................41

v
Table 18 Graph comparing times of presumptive testing between bleach
and stain remover ...................................................................................42
Table 19 Comparison of washing powder type on reaction time for KM
testing in the presence of bleach ............................................................43
Table 20 Graph comparing washing powder type on reaction times for
KM in the presence of stain removing spray ...........................................44
Table 21 NuSieve gel results for control samples ...................................47
Table 22 NuSieve gel 1 results for groups A and B ................................47
Table 23 NuSieve® Gel 2 results for Group C and D..............................48

vi
List of Figures
Figure 1 - Structure of haemoglobin, obtained from anatomy and
physiology.................................................................................................3
Figure 2 - Structure of fibres, obtained from Rowe 2006. .........................4
Figure 3 - Reaction of KM. Adapted from Winchester, Blood detected by
chemical methods .....................................................................................9
Figure 4 - Reaction of LMG. Adapted from Winchester Blood detection. 10
Figure 5 - Gel electrophoresis apparatus and results, obtained from
Butler, 2010.............................................................................................16
Figure 6 - 1ml of Blood dropped on fabrics in order of denim, cotton,
polyester and wool ..................................................................................21
Figure 7 Yield gels obtained from Bio-Rad UV imager............................36
Figure 8 NuSieve® Gel Image from Groups A, B and positive control....37
Figure 9 NuSieve® Gel Image from Group C, D and negative control....37
Figure 10 Control reaction of KM on neat bleach....................................38
Figure 11 Control reaction of LMG on neat bleach .................................38

vii
Acknowledgements
I would like to say a massive thank you to my supervisor Darren Phillips
that helped me through this project and put up with my many questions
and confused moments, as well as Doug Lester who helped guide me
through DNA analysis.
I would also like to say thank my mum, Rose Findlay for putting up with
me through this time and always being there to support me and Anna
May Lang for always being there for me to cry on.
I would like to say a special thanks to all the lab techs at Abertay,
especially Maurice Lindsay, Max Larg, Louise Milne, Hazel Boyle, David
Flyn and Morag Steele! You were all amazing and I promise to sing to
you any time you want.

viii
Abertay University Dundee,
Permission to Copy
Author: Rebekah Elizabeth Findlay
Title:
The degradation of blood evidence on materials
after treatment with stain removers and active
oxygen detergents
Qualification:
Bachelor of Science with Honours in Forensic
Science
Date of Submission: 18/04/2016
I agree that a copy may be made of the whole or any part of the above-
mentioned project report without further reference to the undersigned
Or
A copy shall not be made of the whole or any part of the above
mentioned project report without the written consent of the
undersigned.
Signature:
R Findlay
Address: 80Albany Street,
Dunfermline,
Fife,
Scotland,
KY12 0RA
Date: 18/04/2016

ix
Abstract
With many crimes that involve heavy blood loss it is typical behaviour that
the person who committed the crime would try and clean up after
themselves. Clothing may be washed using a combination of chlorinated
cleaning products and washed with an active oxygen containing
detergent. This may impact the presumptive testing and DNA analysis to
follow. Blood was added to four separate substrate types then washed
using different products and in all oxidising bleach detergent was added.
It was found that negative presumptive tests were obtained for these
materials when tested using a method adopted by forensic laboratories,
however did not have an effect on DNA analysis unless chlorinated
bleach was present.

x
Abbreviations, Symbols and Notation
Table of Abbreviations
Abbreviation Meaning
Bio Biological
CO2 Carbon dioxide
DeoxyHb deoxyhaemoglobin
H2O2 Hydrogen peroxide
Hb Haemoglobin
Non-bio Non-biological
O2 Oxygen
OxyHb Oxyhaemoglobin
PCR Polymer chain reaction
RBC Red Blood Cells
WBC White blood cell
UV Ultra-violet
qPCR Real-time quantitative PCR
Table of Symbols
Symbol Meaning
α Alpha
β Beta
= Equal to
≠ Not equal to
% Percent

xi
Table of Notations
Notation Meaning
cm Centimetre
g Gram
ml Millilitre
mg Milligram
M Molar
mm Millimetre
µL Microliter
º Degree
ºC Degrees Celsius
nm Nanometre

1
1 Introduction
1.1 General Introduction
When a crime scene or suspect is investigated it is a priority during the
investigation to piece evidence together to help explain the events that
took place. Blood is one of the most common evidence types to come
across at a crime scene and can be found on many different substrates
but whether or not these stains are visible they can undergo presumptive
testing. This can be done at a crime scene by using a Kastle-Meyer (KM)
mini test kit for both hard to find stains and stains that may not be visually
identifiable as blood. Materials that can be removed from a scene such as
clothing are examined and tested within a laboratory using
Leucomalachite Green (LMG) or KM and may undergo further
examination through DNA analysis. However, a recent development in
stain removing detergents has caused interference with oxidising
haemoglobin presumptive tests. The addition to detergents of sodium
percarbonate or active oxygen as it is commercially known to remove
stains has caused false negatives in presumptive tests for blood however
they do not seem to affect the ability to obtain a DNA profile (Castelló et
al. 2009). Nevertheless, if a combination of sodium percarbonate and
chlorinated bleach is used this may destroy all blood evidence.
1.2 Blood
Blood is a needed component in the body that transports oxygen and
nutrients around the body as well as a defence system against illness
(Gordon-Smith 2013). It is transported through the body by contractions
of the heart and travels through a series of arteries, arterioles, capillaries,
venules and veins which lead back to the heart (Mosby's Dental
Dictionary 2013). The blood then goes to and from the lungs allowing for
the collection of oxygen and release of carbon dioxide from the body
(Uzoigwe 2006). Blood is made up of several components, red blood cells
(RBC), white blood cells (WBC) and platelets which are suspended in
plasma. Although plasma makes up more than half of the blood
compositions the next largest component is RBCs.

2
RBCs, also known as erythrocytes, have a distinctive biconcave shape
which is believed to help increase the surface area to volume ratio
allowing for better diffusion of gaseous elements (Uzoigwe 2006). The
average life span of a RBC is 120 days before it is removed from
circulation and more are required to be made (Lledó-García et al. 2012).
The main constituent of a RBC is haemoglobin (Hb) which is responsible
via the haem for the distribution of oxygen (O2) around the body as well
as the removal of carbon dioxide (CO2). RBC’s do not contain a nucleus
making it impossible to obtain DNA from them, but it is still possible to be
found in blood by means of WBCs which are a source of DNA (Hashmi
2007).
WBCs are the bodies defence mechanism against illness and help detect
foreign pathogens within the body. The final component in blood is the
platelets which are also referred to as thrombocytes these have many
roles in the body but the main use is to help clot blood when a person has
been injured to try and reduce blood loss (Golebiewska and Poole 2014).
1.2.1 Haemoglobin
Hb is an essential element in RBCs. Its main function is to transport O2
from the lungs to the body as well as interact with CO2 (Schechter 2008,
Shadrina, English and Peslherbe 2012). Hb is a tertiary structure formed
by symmetric pairing of four polypeptide chains, two α-globins and two β-
globins which can be seen in Figure 1. The four chains all connect to a
haem group that contains an iron atom at its centre (Dickson et al. 2015,
Jones et al. 2014). This structure allows small ligand molecules such as
O2 to bind reversibly within the ferrous haem iron in an α-helical globin
fold located ~9 Å below the protein surface (Kapoor, Mandal and
Bhattacharyya 2009). The gas can then diffuse within the globin matrix
and be transported (Dickson et al. 2015, Jones et al. 2014).

3
Figure 1 - Structure of haemoglobin, obtained from anatomy and
physiology
Once the iron in the haem has been oxygenated the Hb is in a state
known as oxyhaemoglobin (oxyHb) and in doing so the oxygen gives up
an electron to the iron creating a new molecule (Wittenberg et al. 1970).
A maximum of four O2 molecules can be attached to a single haem group
at one time. Once released the Hb returns to its original state as
deoxyhaemoglobin (deoxyHb).
1.2.2 White Blood Cells
WBCs, also known as leucocyte are the frontline defence for the body
against illnesses. There are many different types of WBCs and each has
a specialised process inbuilt to recognise foreign pathogens and to ingest
or degrade them (Cavenagh 2007) as well as the ability to kill pathogens
and produce a specific pathogen antigen (Gordon-Smith 2013). This cell
contains a nuclease which in turn has DNA within.
1.3 Deoxyribose Nucleic Acid (DNA)
DNA can be found in WBCs and is unique to each individual. Erwin
Chargaff found that %A=%T and %C=%G however %A+T ≠ %C+G this
indicates that each individual will have a unique DNA sequence
(Forsdyke and Mortimer 2000). As this is unique to each individual, it is
highly improbable that two people will share the same base pair pattern
unless they are monozygotic twins, in which they are indistinguishable (Li
et al. 2013). However, between siblings and even half-siblings, there is a
chance that their profiles will partially match due to sharing loci that was
passed down from their shared parent (Buckleton and Triggs 2006).

4
1.4 Material
Fabrics are categorised based on how they are produced; either synthetic
or natural. Natural fibres are those produced in nature either originating
from an animal or plant. In either instance with the exception of silk which
in its natural form is a complete strand they must be made into a yarn
before production into a sheet of material in contrast to synthetic
materials that are produced in a continuous strand and put into fabric in
this form (Rowe 2006).
Fibres that originate from animals such as wool are similar in structure to
that of human hair (Komboonchoo and Bechtold 2009). Wool is a protein
rich complex in a cylindrical form encased within cuticles resulting in a
smooth texture (Vsevolodov, Golichenkov and Latypov 2014). Fibres
produced by plants however have a different structure as they are mainly
composed of cellulose. The fibre grows in a cylindrical form and later
becomes flattened and twisted as the walls become thicker. Cotton has
the purest form of cellulose in a material in addition to being one of the
most popular materials available (Ramamoorthy et al. 2015. Kljun et al.
2014). Synthetically manufactured fibres in contrast are made by a
polymerisation process in a continuous manner. The structure of these
fibres are highly organised and give a crystalline structure (Rowe 2006)
which can be seen in Figure 3.
Figure 2 - Structure of fibres, obtained from Rowe 2006.

5
Research by Cox (1990) found that material type has an effect on how
much blood is retained; cotton and wool which are natural fibres preserve
blood stains more than materials such as acetate which is synthetic due
to the texture of the surface of the fibres (Cox 1990). Polyester, a
common synthetic material, is an example of this. The structure is smooth
and cylindrical throughout the fibre so blood cannot as easily adhere to it
(Rowe 2006).
1.5 Treatment of stains
Materials can become subject to staining and often an active effort is
made to try and remove them. There are several methods to do this but
for this project the following methods have been investigated: the use of
bleach and a common stain remover spray. Stain removers are now
commercially available and are a common household item (Horjan,
Barbaric and Mrsic 2016). Stain removal treatments are usually
accompanied by later washing the clothes.
1.5.1 Chlorinated cleaning products
Chlorinated bleach is a common household cleaner which main
constituent is sodium hypochlorite (NaOCl), this chemical has an
oxidising effect on other chemicals and cause the colour of chromophores
to change (Egan et al. 2006). NaOCl is contained in most household
bleaches and cleaners and are often used to clean up after a crime has
been committed. Bloodstains are usually cleaned up using this type of
cleaner, as they have a reputation to hinder forensic tests as they cause
damage to biological material in addition to degrade DNA that is present
(Passi et al. 2012). Although they have an effect on DNA analysis, the
result of presumptive tests should not be affected by the presence of
bleach (Castelló et al. 2009) but research by Cox (1990) suggests it has
an effect on the retention of blood within a fabric.

6
1.6 Washing of clothing
The washing of clothes is a common part of daily life (Zhang, Yang and
Lu 2014). Clothes were cleaned for centuries by hand but since the
creation of the fully automated washing machine in the 1960s this has
become the most popular method adapted to clean clothing (French-
Fuller 2006). Clothes washing has proved to be effective in removing
stains but this can be problematic for forensic scientists investigating as it
may wash away some forensic evidence, such as residues, blood and
fibres. Regardless, when presumptive tests for blood are run positive
result can be obtained when washed with detergent even after several
washes (Cox 1990, Mushtaq, Rasool and Firiyal 2016). It is also possible
to obtain a DNA profile on porous materials such as clothing after
washing. However, the temperature the washing is done at can cause
proteins to denature which could impact the result (Cox 1990).
As previously discussed fibres have different structures and can be
affected by cleaning processes. The structure of the fibres reflects on
which detergent can be used to clean it. Research by Rowe (2006) shows
that natural fibres, contain areas where detergents can become attached
or stuck to during the washing process as they contain many different
chemicals within them that water can attach to. Whereas synthetic fibres
have a smoother, linear finish making it difficult for chemicals or even
blood to attach.
1.6.1 Powder detergents
In 1907 washing powders were invented by Henkel in Germany, and they
have been widely used and evolved throughout the world for decades
becoming a part of people’s daily lives and viewed as indispensable
(Zhang, Yang and Lu 2014). Most synthetic household detergents are
formulated using over 25 different ingredients. These are categorised into
four groups: bleaching agents, builder, surfactants and auxiliary agents
(Khanmohammadi et al. 2007).

7
The types of product that are used are based on a person’s preference.
The main product to be added is the cleaning agent which helps
breakdown dirt and stains. Powdered detergents are a popular cleaning
agent that is used and they are split into two distinct types: biological (bio)
or non-biological (non-bio).
Non-biological detergents contain no enzymes and are classed as
sensitive as they are soap based rather than enzyme based. This was the
first type of detergent introduced to a commercial market in 1907
(Advantage Business Media 2015). In contrast, biological washing
powder contains an enzyme called protease. The introduction of enzymes
to washing powders was made in the 1960s and over 15 different strains
of protease have been used in detergents since (Maurer 2004). Protease
is the main enzyme used as it is non-toxic to the body and has been used
medically to breakdown blood clots proving that its use will not irritate or
cause harm to the body (Gacko and Głowiński 1998). Protease has
proven its use to help break down biological stains and dirt on clothing.
Observations by Maurer (2004) shows that biological detergent
containing protease will target proteins in blood on clothing but if the
protein becomes denatured by aging, heat or oxidation the enzyme
cannot target it as well. This can affect results from forensic tests but
show that blood evidence is not completely removed by the enzyme.
There is debate to whether or not these detergents should be used at a
higher temperature, but this does not change its ability to clean. In either
instance the temperature does not have an effect on the yield of DNA
during analysis (Nasiri et al. 2005).
1.6.2 Oxygenated washing powder
Oxygenated washing powders are a recent development in the removal
of stains such as blood from clothing during the process of washing.
Originally hydrogen peroxide (H2O2) in its pure form was used as an
active oxygen component in cleaners and is now replaced by sodium
percarbonate.

8
Sodium percarbonate is a solid per-oxygen compound that is easily
stored and environmentally friendly (Khanmohammadi and Kargosha
2005). The chemical itself has an alkaline pH and contains the solid state
of H2O2 which is loosely attached to it, which is released when in aqueous
solution. It is a cost effective and readily available for use in detergent
where the release H2O2 acts as a bleaching agent for stains (McKillop
and Sanderson 1995). Due to this ability it is widely known as a bleaching
or oxidising agent (Khanmohammadi and Kargosha 2005). Maurer (2004)
has shown that when sodium percarbonate and a detergent are used to
clean clothing they denature the blood that is present and makes it less
accessible to enzymatic degradation which effects presumptive testing.
1.7 Presumptive tests
When an item is suspected of being contaminated with blood at the scene
of a crime it is taken into a forensic laboratory where it undergoes
presumptive testing for blood. The two leading tests that are carried out
are Kastle-Meyer (KM) and Leuchomalachite Green (LMG). Both work
based on peroxidase activity within the Hb found in RBCs. Once the test
solution has been added to a sample H2O2 is applied. The haem structure
pulls apart the hydrogen peroxide which results in a water molecule and
an oxygen radical being formed (Williams 2012), as the tests are colour
catalytic reactions they are dependent on this taking place (Johnston et
al. 2008). There are many factors that will affect these tests but the
duration of time between blood being deposited and testing has
extremely little to no effect on the result (Cox 1990).
The sensitivity of these tests has been subject to much research. The
results however have been variable which could be due to inconsistency
in the technique. Research by Thorogate (2008) demonstrates the tests
sensitivity but shows that they are not species specific and must be
determined through other means. It has been observed that the sensitivity
of KM and LMG is largely decreased when applied to filter paper that has
been in contact with a blood stain and that it is recommended to test
directly onto the stain itself (Webb, Creamer and Quickenden 2006).

9
A study was conducted to see the effectiveness of presumptive tests on
the detection of blood on washed fabrics, it was found that KM was
sensitive to the tests and would give positive results however LMG
showed to give much less positive results. Overall, both tests were only
able to detect blood on 50% of washed fabrics (Mushtaq, Rasool and
Firiyal 2016). Although they have been found to be useful in identifying
the presence of blood they are prone to giving false positive and false
negative reactions which can lead to false identification as well as
effecting subsequent DNA analysis (Li et al. 2014).
1.7.1 Kastle-Meyer
KM, also known as phenolphthalein, has been considered as a medico-
legal technique for the detection of blood since the 1920s (Glaister 1926).
The KM is an alkaline solution that when in the presence of blood turns
bright pink (Virkler and Lednev 2009) and the reaction for which can be
seen in Figure 4. Oxidation however, is not specific to this reaction and
false positives can occur, for example this can take place in the presence
of bleach if it is not left to evaporate. In case work, false positives are
reduced by indirectly testing the stain on filter paper. The use of filter
paper is further recommended as KM causes damage to DNA (Tobe,
Watson and Daéid 2007).
Figure 3 - Reaction of KM. Adapted from Winchester, Blood detected
by chemical methods

10
Research conducted by Vandewoestyne (2015) showed that blood stains
that are hardly visible or invisible could be detected using KM in samples
diluted to 1 in 500. However, investigations by Cox shows that KM is
sensitive to blood diluted 1 in 10,000 (Cox 1990) which contradicts their
later work which states that KM is sensitive to blood diluted to 1 in
10,000,000 (Cox 1991).
1.7.2 Leucomalachite Green
LMG works optimally in an acidic environment and forms a blue-green
colour when in the presence of blood after the addition of H2O2 which can
be seen in Figure 5 (Virkler and Lednev 2009). The use of this test has
been found to have no effect on DNA typing if the solution is neutralised
before using the reagent otherwise DNA can be damaged due to the
acidity of the solution, but this should be kept to a minimum as loci starts
to get damaged (Tsukada et al. 2011). Due to this it is recommended that
samples should be indirectly tested for blood (Tobe, Watson and Daéid
2007).
Figure 4 - Reaction of LMG. Adapted from Winchester Blood
detection.
Studies have been conducted to investigate the sensitivity of LMG and
found it to be less sensitive than KM as it could detect blood to a limit of 1
in 5000 (Cox 1991). However, sensitivity values are marginally different
between papers but are consistent in that values don’t vary widely (Webb,
Creamer and Quickenden 2006). An investigation found that contrary to
other research that there was little difference on the results of KM and
LMG tests (Vennemann et al. 2014).

11
1.7.3 False positive
It is possible that presumptive tests react with other substances that are
present that may oxidise the reactions when testing for blood. KM has a
tendency to result in more false positives than LMG. False positive with
KM can be produced by the presence of some foods, bleach, semen and
some metals; in contrast LMG gives far less false positives and occur
before the addition of H2O2, therefore could not be mistaken as a reaction
for blood (Cox 1991). KM conversely, gave a positive reaction after the
addition of H2O2 which could be falsely identified as blood (Tobe, Watson
and Daéid 2007, Cox 1991).
1.7.4 False negatives
As well as false positives, false negative reactions can occur; caused by
the oxidation of haemoglobin due to the presence of a reducing agent
before presumptive tests are carried out. This is a recent problem caused
by the presence of sodium percarbonate in detergents. (Castelló et al.
2009).
1.7.5 False negative caused by sodium percarbonate
The presence of sodium percarbonate in detergents allows for effective
stain removal however interferes with forensic tests. This interference is
highlighted by Castelló et al (2009), where it is found to give false
negative reactions for the presence of blood. The presumptive tests
require the addition of hydrogen peroxide to catalyse the reaction which
would result in a positive. However, sodium percarbonate and water
results in a significant amount of H2O2 being produced and in turn has an
inhibiting effect on the haemoglobin as this depletes its ability to
breakdown hydrogen peroxide. Therefore the haemoglobin will become
exhausted and will not react resulting in a negative result (Castelló et al.
2009). Even with a negative result for the presence of blood, DNA can
still be extracted from the sample (Castelló, Francés and Verdú 2012).

12
1.8 DNA analysis
DNA analysis is carried out by obtaining a genotype and comparing it to a
known sample (Petersen and Kovacs 2014). This is completed by
profiling short tandem repeats (STR) which are used in forensics for the
purposes of human identification. Protocol for this analysis is comprised
of extraction, quantitation, amplification through PCR, and gel
electrophoresis (Brevnov et al. 2009)
Research conducted by Harris et al. (2006) has shown that 80% of
samples that gave a false negative presumptive result for blood, once
subjected to subsequent DNA extraction were able to provide a DNA
profile. This however was only found in porous materials. When the
results were compared to non-porous surfaces the DNA profiles achieved
were unreliable. Chlorinated bleach was found to be the most degenerate
to DNA. It is believed that the hypochlorite in these products is what
causes the DNA to denature (Harris et al. 2006)
1.8.1 Extraction
Before a DNA profile can be analysed, it must first be extracted. Despite
the fact there is always a possibility to obtain a DNA profile from a
biological sample, there are many methods that can be used. The method
of extraction however must take into consideration the substrate and
sample type. Research by Brownlow (2012) showed that varying
quantities and quality of DNA was extracted from different fabric types but
was also dependent of the extraction method (Brownlow, Dagnall and
Ames 2012).
The first extraction methods do be employed was organic phenol-
chloroform extraction which was later followed by Chelex® 100, but both
had many downfalls. Over the years’ modern approaches have
developed and solid-phase extraction methods have gained popularity.
The first company to produce materials for this method was Qiagen and
have gained popularity (Butler 2010).

13
1.8.1.1 Solid-phase DNA extraction
Laboratories have implemented the use of solid phase DNA extraction
methods. Popularity has been gained due to their ability to remove PCR
inhibitors and to a certain degree allow some automation (Idris and
Goodwin 2015). A micro-device is utilised comprising of magnetic silica
particles with an optimised volumetric flow rate and elution buffer,
resulting in a 15-fold increase in DNA concentration and a 50-fold
decrease in volume. This method is now recommended over organic
phenol chloroform and chelex extraction methods and gives a much more
rapid sample preparation (Reedy et al. 2010).
1.8.1.2 Qiagen
The most commonly encountered kits in forensic laboratories are created
by Qiagen, Inc. (Valencia, CA). For over a decade Qiagen has introduced
automated systems for solid-phase extraction. The first of which was
silica based presented in a QIAamp® spin column and has proven to be
an effective means of extracting DNA (Butler 2010). This approach allows
nucleic acid to be absorbed into the silica support system composed of
small glass beads accompanied by the presence of highly concentrated
chaotropic salts. These salts break the hydrogen bonds within water,
triggering proteins to denature and DNA to become thermodynamically
stable. In acidic solutions the absorption of DNA is ~95% and
contaminants are washed away, in contrast when in alkaline solution, with
lower salt concentration the DNA will elute from the silica (Butler 2010).
Qiagen has created several kits for the extraction of DNA. A frequently
used kit is the QIAamp® Blood Mini Kit, which uses QIAamp® silica-gel
membrane to specifically bind DNA when in the presence of a guanidine-
based lysis buffer and removes PCR inhibitors through washing. The
QIAamp® spin column have been found in a study by Greenspoon
(1998), to extract DNA more consistently than that of Chelex® 100 from
surfaces. Issues caused by freezing DNA samples have been resolved
with the use of the spin column allowing for the appropriate loci to be
identified after the sample has been thawed (Greenspoon et al. 1998).

14
1.8.2 DNA Quantitation
After extraction, quantitation of the isolated DNA is the next step in DNA
analysis. PCR methodology is the dominant analytical technique for the
quantitation of DNA and has become indispensable; samples from a
crime scene may contain low volumes which will need amplification
before it can be analysed as well as assessing degradation and quality of
the sample (Nicklas and Buel 2003).
This stage helps provide information about the DNA present in the
unknown sample, the data of which can be kept and used to successfully
obtain better quality results by preserving the sample for further analysis
(Martins et al. 2015). Older methods such as ultra-violet (UV) light
detection and dyes were used to quantify the DNA present however this
could not take into account species to which the sample originated from.
PCR based methods have now been developed and are human-specific,
which gives precise quantitation for STR analyses (Nicklas and Buel
2003). This is necessary before amplification through PCR can take place
as if there is too much DNA results can become difficult to interpret and if
there is too little there will be a loss of alleles (Butler 2010).
1.8.2.1 DNA quantitation methods
PCR based techniques have become the main method of DNA
quantitation. Real-time quantitative PCR (qPCR) allowed for different
target lengths to be simultaneously quantified permitting a degradation
ratio to be calculated along with a quantitative determination of the quality
of the DNA template as well as an assessment of PCR inhibition (Swango
et al. 2007). A nuclease assay, TaqMan, is used and attaches to the DNA
on the 5’ end causing it to quench and another dye attaches to the 3’ end.
The probe is degraded by polymerase as the PCR primer elongates
resulting in a fluorophore to be generated. Fluorescing is directly
proportional to the cycle number and volume of DNA that is present
(Nicklas and Buel 2003). Once completed the sample is amplified.

15
1.8.3 DNA amplification
DNA amplification better known as PCR is an enzymatic process which
targets specific fragments of DNA and causes the regions to replicate
several times. Each assay has to contain the template DNA, primers,
nucleotides and DNA polymerase (Garibyan and Avashia 2013). This
process has three mains steps: denaturing, annealing and primer
extension.
Denaturing is a result of the sample being heated, breaking the bonds
between base pairs causing the double strand to split into two single
strands. The sample is then cooled and a forward and reverse primer
consisting of a short DNA sequence anneal to the DNA strand at the flank
of the sequence that is to be copied which acts as a target. The
temperature in then increased once again which allows for the DNA
polymerase to extended upon the primers, producing a copy of each DNA
template. This cycle is then repeated several times which increase the
number of DNA present in the sample. This process is carried out in a
thermal cycler to accurately go between temperatures at the appropriate
times. This process can also be multiplexed allowing for several samples
to be run at a single time (Butler 2010)
1.8.4 Gel electrophoresis
Once the sample has been amplified the sample must be separated to
allow analysis to be carried out. This method was developed in the 1960s
for the use in molecular biology (Slater 2009). Gel electrophoresis is still
used as a method to separate DNA fragments into their bands. There are
several methods that allow this, but one of the most common approaches
is through the use of an agarose slab gel. This is a simple method that
can be easily made and is cost-effective. The agarose gel once made up
is left to set with a comb at the cathode end of the gel which produces
wells were the sample is placed. A buffer solution is added to the
apparatus after the gel has set. The samples are put into the wells of the
gel and the apparatus turned on.

16
This causes the DNA to separate through the gel as the DNA is pulled
towards the anode as DNA is negatively charged. The further the sample
moves through the gel the smaller the fragment is. When the apparatus is
turned off distinctive bands can be identified in the gel. These are
compared to the DNA ladder on the far side of the gel which identifies
how many base pairs are in that region, this can be seen in Figure 5.
These bands can then be compared to that of other samples and if two
are consistent or partially consistent in the case of paternity cases it is
possible to positively identify the person from which the sample
originated.
Figure 5 - Gel electrophoresis apparatus and results, obtained from
Butler, 2010.

17
2 Aims and Objectives
This experiment was conducted to find out if using a combination of
chlorinated cleaning products and washing with an active oxygen
containing detergent would have a detrimental effect on both presumptive
testing for blood and DNA analysis on porous materials.
This may occur when a person has committed a crime such as a murder
and try to clean their clothing after the incident to remove evidence that
they were involved.
Blood would be stained onto material and subjected to stain removing
treatments and cleaning detergent containing oxygen bleach. Once
completed materials would undergo presumptive testing using KM and
LMG followed by DNA analysis.
Hypothesis:
 When presumptive tests are carried out after being treated with an
oxygen beaches a false-negative result should be observed.
 Subsequent DNA analysis will be hindered by the presence of
bleach.
 Presumptive tests carried out on colour catchers used in washing
may give a positive reaction.
 DNA might be achieved from colour catchers used when materials
were being washed.

18
3 Methods and Materials
3.1 Fabrics
Four substrate groups: wool, cotton, denim and polyester were obtained.
Wool came in the form of a 100% wool blanket and obtained from a
charity shop. A 100% cotton bed sheet was obtained from the home
department of Asda. The 100% Polyester specimen came in the form of a
blanket and was obtained from Primark. The denim used was produced
by New Look and consisted of 80% Cotton, 18% polyester and 2%
elastin. A colour sheet (Tesco) was added to each wash.
3.2 Pre-treatment chemical compositions
The bleach used in this experiment was manufactured by Domestos and
contained sodium hypochlorite solution, C12-C18 alkyl dimethylamine
oxide and sodium hydroxide.
A stain remover spray was used that was obtained from vanish and
contained: aqua, hydrogen peroxide, laureth-7, sodium C14-17 Alkyl sec
sulfonate, laureth-3, alkyl hydroxyethyl dimethyl ammonium chloride,
tetrasodium iminodisuccinate and parfum.
3.3 Washing detergents chemical compositions
The non-biological washing powder used was produced by Tesco and
contained: sodium carbonate, sodium carbonate peroxide, alkyl
benzenesulfonic acid, Sodium Sulphate, aqua, C10-C14 alkyl
benzenesulfonic acid, zeolite, sodium silicate tetracetylethylenediame,
C14-C15 pareth-7, sodium acrylic acid, maleic acid copolymer, parfum,
cellulose gum, bentonite, (1-hydroxyethylidene) diphosphonic, sodium
salt acid, polyaromatic ester, sodium sufate, sodium silcoal, paraffin wax,
sodium chloride, sodium glycolate, alcohols C12-14 ethoxylated,
disodium phosphonate, sodium acetate, and ci fluorescent brightener
260.

19
The biological washing powder was obtained from Tesco as well and
contained: sodium carbonate, sodium carbonate peroxide, sodium
sulphate, aqua, C10-C14 alkyl benzenesulfonic acid, zeolite, sodium
silicate, tetracetylethylenediame, C14-C15 pareth-7, sodium acrylic acid,
maleic acid copolymer, parfum, cellulose gum, bentonite, diphosphonic,
sodium salt acid, polyaromatic ester, sodium sufate and sodium silcoal,
paraffin wax, sodium chloride, sodium glycolate, calcium carbonate,
cellulose, kaolin, polyethylene glycol, dextrin, alcohols C12-14
ethoxylated, disodium phosphonate, titanium dioxide, subtilisin
concentrate, hydroxyethyl cellulose, sucrose, propylene glycol, sodium
acetate, protease, mannanase, zinc sulphate, lipase concentrate,
pectate lyase, mica, amylase, peg-90m, hydrated silica, calcium oxide, ci
fluorescent brightener 260.
The oxygenated stain remover used in the washes as well was produced
by Vanish-Oxi action and is the original powder of this. This contains:
sodium carbonate peroxide, sodium carbonate, sodium sulphate,
tetraacetylethyleendiamine, sodium C10-13 alkyl benzenesulfonate,
disodium disilicate, sodium C12-18 alkyl sulphate, aqua, pareth-5, zeolite,
subtilisin, parfum, mannanase, amylase and lipase.
3.4 Reagents
Each reagent required to carry out testing was prepared before testing.
3.4.1 Kastle-Meyer reagent
A stock solution was produced by dissolving 2g of potassium hydroxide
pellets (Fisher Scientific) in 100ml of distilled water. Phenolphalein
(Fisher scientific), zinc powder (Fisher Scientific), anti-bumping granules
(Sigma Aldrich) and the dissolved solution of zinc hydroxide were placed
in a 250ml round bottom flask. The flask was fitted with a condenser and
placed in a stirring heated mantel. The mixture was refluxed for 2 hours
until the solution became colourless. Zinc powder was added to an amber
bottle and the mixture added through filtration to remove excess zinc
powder and stored in a fridge. Excess zinc powder was discarded of by
dissolving it in 2M hydrochloric acid (Fisher Scientific).

20
The working solution of Kastle-Meyer reagent was produced by using
20ml of stock KM solution and mixed with 80ml of ethanol (Fisher
Scientific). This was made fresh every time it was required and kept cool.
3.4.2 Leucomalachite Green
A stock solution of acetic acid was produced by diluting 600ml 95% acetic
acid (Fisher scientific) in 300ml distilled water and stored in a large screw
top bottle at room temperature. Leucomalachite green working solution
was made by dissolving 10mg of leucomalachite green powder (Sigma
Aldridge) in 10ml of the acetic acid stock solution in a beaker.
3.5 Procedure
Once the four types of substrates were prepared they were divided into
four categories and processed dependant on their group.
A. Pre-treated with chlorinated bleach and washed with active oxygen
detergent and non-biological washing powder.
B. Pre-treated with chlorinated bleach and washed with active oxygen
detergent and biological washing powder.
C. Pre-treated with stain remover and washed with active oxygen
detergent and non-biological washing powder.
D. Pre-treated with stain remover and washed with active oxygen
detergent and biological washing powder.
As can be seen above each group was different and the method altered
based on the requirements for that group.
3.5.1 Preparation of materials
Each fabric was cut into 10cm x 10cm squares and stored in plastic zip
lock bags until they were needed. Materials were left in their natural form
and without being washed before testing begun. Sterile laked horse blood
(TSC Bioscience) was acquired and stored in a fridge until required.

21
3.5.2 Blood deposition
Each fabric sample was placed onto a white tile. 1ml of laked horse blood
was deposited from a burette 10cm above the fabric. The blood on the
fabrics can be observed in Figure 7. This was then transferred to a plastic
sheet and left for one hour to partially dry before being relocated to a
washing line and left to dry overnight at room temperature.
3.5.3 Pre-treatment of samples
Each group of fabrics were treated depending on their group. Group A
and B underwent stain removal with chlorinated bleach (Domestos). Each
stain was treated with 1ml of bleach and hand washed under tap water
until the stain was hard to see and washed shortly after.
Group C and D were treated with stain removing spray (Vanish Oxi
Action), this was carried out in accordance with instructions on the bottle.
The stain was treated by spraying three times on each side of the stain
and left for 10 minutes before being washed.
3.5.4 Washing of samples
Each group of fabrics were washed separately in accordance to the
washing technique designated to that particular group. Each group was
washed at 30ºC, on a mixed material setting and 800 spin cycles in a
BEKO VM6112W washing machine. All samples were placed in a net
garment bag and prior to washing had a colour catcher added. The
washing cycle ran for 90 minutes. Once washed the materials were hung
up on a washing line and left to dry at room temperature.
Figure 6 - 1ml of Blood dropped on fabrics in order of denim,
cotton, polyester and wool

22
Groups A and C were washed using non- biological washing powder. The
materials were washed based on the recommendations on the box. As
the materials were heavily stained and in a soft water area 145ml of
powder was added to a single wash and a single scoop (60ml) of the
Vanish Oxi-Action powder was added.
Groups B and D were washed with biological washing powder. The
materials were also washed according to recommendations made on the
packaging and 145ml of the powder was added along with 60ml of
Vanish-Oxi Action powder.
3.5.5 Sample preparation for presumptive testing
3 of each fabric were used per group per presumptive test. In total each
group contained 24 samples and a colour catcher. During the testing
period three different testing methods were employed. The first and
second techniques involved the use of a stainless steel spatula to scrape
the material and applied to moistened filter paper. The third set method
used the filter paper directly which was quartered and then scraped on to
the material.
To reduce contamination each sample was placed on a white tile which
was washed with ethanol between tests and left to evaporate. The
spatula used in the first two testing methods was sterilised between each
use by dipping into ethanol and being left to evaporate.
Each sample was designated a unique code so that it could be easily
identified. This was done by appointing the number of testing technique,
the letter from washing group and the first letter of the fabric followed by
which repeat it was. For example, 1AW1, refers to testing method one,
Wash group A, Wool, Repeat 1.

23
3.5.6 Presumptive testing
All samples once on filter paper had 2 or 3 drops of KM or LMG working
solution added. The first testing technique used 3% H2O2 which was
applied after the reagent. Testing Methods 2 and 3 used 6% H2O2 instead
and applied in the same manner.
3.5.7 Controls
Controls were made of each individual component involved in the
washing methods. Controls of biological washing powder, non-biological
washing powder, Vanish Oxi-Action, bleach and stain remover spray
were placed neat on filter paper. A positive control of blood and negative
control of no blood were also tested. Control tests were run using both
6% H2O2 and 3% H2O2.
3.5.8 Recording of presumptive tests results
As hydrogen peroxide was added to each sample a timer was started, the
time for a reaction to take place was recorded. If no reaction occurred
within 60 seconds it was recorded as a no reaction but was left for a
further minute. All filter paper results were photographed and can be
found in Appendix 2.

24
3.5.9 Material preparation for DNA extraction
At random one of each material and a colour catcher was selected to
undergo DNA extraction. In total 22 extractions took place. The fabrics
chosen can be found in Table 1.
Table 1 Fabrics randomly selected for DNA analysis
Group Material Presumptive
Test
Repeat Code
A Colour Catcher LMG ACC
Denim LMG 3 AD
Cotton LMG 3 AC
Polyester LMG 1 AP
Wool LMG 1 AW
B Colour Catcher KM BCC
Denim KM 2 BD
Cotton KM 3 BC
Polyester LMG 1 BP
Wool KM 2 BW
C Colour Catcher KM CCC
Denim KM 1 CD
Cotton KM 3 CC
Polyester LMG 3 CP
Wool LMG 2 CW
D Colour Catcher LMG DCC
Denim LMG 1 DD
Cotton KM 1 DC
Polyester KM 3 DP
Wool KM 2 DW
3.5.10 Extraction of DNA
Extraction was carried out using QIAamp DNA Mini Kit (Qiagen) and
following protocol for DNA purification from dried blood spots.
3 small squares roughly 3mm x 3mm were removed from each sample
and placed into a 1.5ml microcentrifuge tube with 180µl of buffer ATL and
incubated at 85ºC for 10 minutes and then centrifuged. 80µL of
proteinase K stock solution was then added to this and mixed using a
vortex and incubated further at 56ºC for 1 hour and then briefly
centrifuged. To this 200µL of Buffer AL was added and mixed by
vortexing and incubated at 70ºC for a further 10 minutes. 200µL of
ethanol was added to the sample, vortexed then centrifuged.

25
The 600µL of solution created was removed and put into a QIAamp Mini
Spin Column in a 2ml collection tube without wetting the rim and
centrifuged for 1 minute. The QIAamp Mini Spin column was removed
from the collection tube and put into a new one. The used collection tube
was then discarded with the filtrate.
500µL of buffer AW1 was added to the spin column and centrifuged for 1
minute. The QIAamp spin column was removed and added to a new
collection tube where buffer AW2 was added and centrifuged for 3
minutes. The spin column was then added to another collection tube and
centrifuged for a minute. The QIAamp spin column was added to another
collection tube and 150µL buffer AE was added and incubated at room
temperature for 1 minute before being centrifuged.
The spin column was then removed from the collection tube and
discarded. The 150µL of filtrate left in then removed from the collection
tube and placed in a 1.5ml microcentrifuge tube and frozen at -20ºC.
3.5.11 UV Quantification
50µL of the extraction sample was removed and placed into a 1.5ml
microcentrifuge tube and diluted with 950µL of distilled water after which
was placed into a shaker overnight at 37ºC.
A UV spectrophotometer was set to 260nm (A260) and calibrated with
Buffer AE, each sample was placed into a cuvette and put into a UV
spectrophotometer and had its absorbance value detected. The
wavelength was then set to 280nm (A280) and re-calibrated with Buffer
AE. The concentration and total amount of DNA as well as purity was
then calculated from the obtained numbers found by the machinery.

26
3.5.12 UV Quantification calculation
DNA concentration: Undiluted volume of DNA (µg/ml) x A260 absorbance
Amount of DNA present: Concentration (from above) x sample volume.
The purity of the sample was then calculated in the ratio of A260:A280, in
the region of 1.8-2.0. The purity was first found by:
Absorption A260 / Absorption A280
The range was found by dividing the purity first by 1.8 and also by 2.0.
This was the percentage range in which there was pure DNA.
3.5.13 Agarose Yield Gel
A stock solution of TBE (tris boric acid ethylene diamine tetra acetic acid)
buffer was first produced by combining 110g boric acid, 18.6g EDTA
(ethylene diamine tetra acetic acid) and 216g tris (hydroxymethyl) amino
ethane and made up to 1L with distilled water and mixed using a
magnetic stirrer until solution was clear. A 1L working solution was made
by diluting 100ml TBE stock solution in 900ml of distilled water.
An agarose gel was made by mixing 2g of agarose powder (Life
Technologies) with 100ml of TBE buffer and weighed on a scale. The
mixture was then microwaved for 90 seconds and re-weighed. The
solution was made up to its original weight with distilled water. The
solution was cooled to 70ºC and 5µL of SafeView (NBS Biologicals) was
added, once the temperature was below 60ºC it was put into a tray and
left to set in the electrophoresis chamber. Once set the tray was turned
90º in the electrophoresis chamber and submerged in TBE buffer.
Each sample was prepared by combining 4µL of loading dye with 7µL of
extracted sample and mixed by withdrawing and ejecting the solution with
a pipette. The prepared samples are then loaded into separate wells
within the gel and recorded. The wells at either end of the gel are loaded
with a 1000bp ladder. The electrophoresis was turned on for one hour at
100V. Once completed the gel was viewed under UV light in order to
visualise any bands that were present.

27
3.5.14 Polymerase Chain Reaction (PCR)
Each sample was prepared individually by placing in a microcentrifuge
tube 1µL of the DNA extract, 2µL of both forward and backward horse
primers, 12.5µL redmix HotStart solution and 7.5µL denuclease water.
These were placed in a Bio-Rad thermal cycler which was programmed
to have an initial incubation time of 10 minutes at 95ºC, followed by 40
cycles of Denaturing at 95ºC for 15 seconds, annealing at 54ºC for 15
seconds and extension at 72ºC for 60 seconds. Once complete samples
were held at 72ºC for 10 minutes and cooled to 4ºC. The samples were
then stored in a freezer.
3.5.15 NuSieve® GTG® Agarose Gel Electrophoresis
A 4% Nuseive agarose gel was made by sprinkling 4g NuSieve® agarose
powder (Lonza) into 100ml of TBE buffer and left for 15 minutes for the
powder to soak in TBE buffer. This solution was weighed and covered
with plastic wrap and pierced. This was placed into a microwave oven on
medium power for two minutes. The solution was removed and gently
mixed to re-suspend unreacted powder. This was placed in the
microwave oven for a further minute on high and once boiling was left for
one minute. The solution was re-weighed and made up to its initial weight
with boiling distilled water, 5µL of SafeView was added and the solution
left to cool to between 50ºC and 60ºC. The solution was then casted in a
tray with a comb.
Each sample was prepared by combining 4µL loading dye and 7µL PCR
product. These were loaded into the wells and the sample position
recorded. The chamber was then turned on and set to 120V for 90
minutes. Images of the gel were taken at time intervals 30 minutes, 60
minutes, 75 minutes and 90 minutes these can be seen in Appendix 6.

28
4 Results and Discussion
4.1 Presumptive Testing results
Results from all presumptive tests were documented and tabulated.
Positive reactions were reported with how many seconds it took for the
reaction to take place, for example ‘20’ which would correspond to 20
seconds. On the other hand, if there was no reaction it was reported as
“-“. Each sample group: A (non-biological washing powder, bleach and
sodium percarbonate); B (biological washing powder, bleach and sodium
percarbonate); C (non-biological washing powder, stain removing spray
and sodium percarbonate) and D (biological washing powder, stain
removing spray and sodium percarbonate) will be discussed separately
and the results compared. It was expected that all results would be
negative due to the presence of sodium percarbonate which would have
oxidised any haemoglobin present on the fabric before these tests were
carried out.
Mean times for each set of results have been calculated and are
tabulated in this section. A full set of raw data can be found in Appendix 1
and images of test results and can be seen in Appendix 2.

29
4.1.1 Test Group A – Non-biological powder and Bleach
As can be seen in Table 2 below test methods 1 and 2 gave positive
presumptive test results for blood when KM reagent was applied but the
use of filter paper directly (test method 3) acquired negative results. This
may be a result of the use of a spatula although some negative results
were observed for wool and polyester using this method which may
indicate this may be due to the scraping method itself. It can also be seen
that there was a difference between times of reaction between the use of
6% H2O2 and 3% H2O2 in which 3% H2O2 reacted faster than 6% H2O2.
Table 2 - Mean reaction time for KM presumptive tests on Group A
using different testing methods
Material
Mean Reaction Time (s)
Test Method 1 –
3% H2O2 and
spatula
Test Method 2 –
6% H2O2 and
spatula
Test Method 3 –
6% H2O2 and
Filter paper
Wool (AW) 28* 44 -**
Cotton (AC) 23 40 -**
Denim (AD) 34 43 -**
Polyester (AP) 35* 42 -**
Colour Catcher
(AC)
- 20 -**
*1 out of 3 from both wool and polyester resulted in a negative.
**Positive reactions did take place but occurred after one-minute
Table 3 displays results for LMG tests which across the board shows
negative results bar a few outliers that were observed with 3% H2O2.
Table 3 - Mean reaction times for LMG presumptive tests on Group A
Material
Test Method 1 –
3% H2O2 and
spatula
Test Method 2 –
6% H2O2 and
spatula
Test Method 3 –
6% H2O2 and
Filter paper
Wool (AW) - - -
Cotton (AC) -* - -
Denim (AD) - - -
Polyester (AP) - - -
Colour Catcher
(ACC)
- - -
* 2 out of three repeat repeats gave a single dot appearing near the end
of reaction time window.

30
4.1.2 Test Group B – Bio and Bleach
Negative presumptive tests were expected to be obtained with this group
however it shared the same characteristics as Test Group A as both
Testing Method 1 and 2 obtained positive reactions for KM and Testing
Method 3 had no positive reactions which can be seen in Table 4.
Table 4 Mean reaction times for KM presumptive tests for Group B
Material
Test Method 1 –
3% H2O2 and
spatula
Test Method 2 –
6% H2O2 and
spatula
Test Method 3 –
6% H2O2 and
Filter paper
Wool (BW) 36 35 -
Cotton (BC) 40 40 -
Denim (BD) 46* 43 -
Polyester (BP) 46 42 -
Colour Catcher
(BCC)
- 27 -
*Reaction for Denim at 3% resulted in a single positive within the
allocated minute, one above it and the last repeat as a no reaction the
details of which can be found in appendix 1.
Table 5 shows that a full set of negative results were gathered from
samples when tested with LMG. This may be due to LMG being less
sensitive than tests carried out using KM or that KM is reacting with other
chemicals in the materials.
Table 5 Mean Reaction times for LMG presumptive tests for Group B
Material
Test Method 1 –
3% H2O2 and
spatula
Test Method 2 –
6% H2O2 and
spatula
Test Method 3 –
6% H2O2 and
Filter paper
Wool (BW) - - -
Cotton (BC) - - -
Denim (BD) - - -
Polyester (BP) - - -
Colour Catcher
(BCC)
- - -

31
4.1.3 Test Group C – Non-bio and stain remover spray
It was expected that this group was more likely to give a positive reaction
due to the lack of damaging chemicals that are found in bleach and lack
of enzyme activity on proteins. Table 6 shows the same pattern that has
emerged from Test Groups A and B as Testing methods 1 and 2 has
given positive reactions for the presence of blood but negative results
were obtained from Testing Method 3 for KM.
Table 6 Mean reaction times for KM tests for test group C using
different testing methods
Material
Test Method 1 –
3% H2O2 and
spatula
Test Method 2 –
6% H2O2 and
spatula
Test Method 3 –
6% H2O2 and
Filter paper
Wool (CW) 35 37 -
Cotton (CC) 50 39 -
Denim (CD) 44 47 -
Polyester (CP) 51 35 -
Colour Catcher
(CCC)
- 35 -
Table 7 shows that LMG results were mainly negative, however a few
outliers can be observed in Testing Method 1 but are discounted as only
one out of three repeats resulted like this.
Table 7 Mean Reaction times for LMG presumptive tests for Group C
Material
Test Method 1 –
3% H2O2 and
spatula
Test Method 2 –
6% H2O2 and
spatula
Test Method 3 –
6% H2O2 and
Filter paper
Wool (CW) - - -
Cotton (CC) -* - -
Denim (CD) -* - -
Polyester (CP) -** - -
Colour Catcher
(CCC)
- - -
*Cotton and denim both had one out three repeats result was a positive
reaction
**For polyester a single dot was present

32
4.1.4 Test Group D – Bio and Stain remover spray
This group used biological washing powder and stain removing spray.
Table 8 shows that positive results were obtained in testing methods 1
and 2 and negatives for test method 3 for KM. Although positive reactions
were observed for both denim and polyester when using 3% H2O2, 2 out
of 3 results took longer to be react and were discounted as the reaction
occurred out with the 1 minute reaction time cut off.
Table 8 Mean reaction times for KM tests for test group D using
different testing methods
Material
Test Method 1 –
3% H2O2 and
spatula
Test Method 2 –
6% H2O2 and
spatula
Test Method 3 –
6% H2O2 and
Filter paper
Wool (DW) 54 38 -
Cotton (DC) 46 32 -
Denim (DD) -* 38 -
Polyester (DP) -* 46 -
Colour Catcher
(DCC)
29 35 -
*For denim and polyester there was a single positive reaction, the other
two repeats did not react until after the allocated minute.
LMG results which can be found in Table 9 show that the use of 3% H2O2
resulted in 2 positive results for LMG, as 2 out of 3 samples reacted this
way.
Table 9 Mean Reaction times for LMG presumptive tests for Group D
Material
Test Method 1 –
3% H2O2 and
spatula
Test Method 2 –
6% H2O2 and
spatula
Test Method 3 –
6% H2O2 and
Filter paper
Wool (DW) -* - -
Cotton (DC) 24** - -
Denim (DD) 26** - -
Polyester (DP) -*** - -
Colour Catcher
(DCC)
- - -
*Wool had a single dot of colour the other 2 reactions were negative.
**Cotton and Denim had 1 negative and 2 positively reacted repeats
***Polyester had a single positive reaction out of the 3 repeats

33
4.1.5 Controls
The controls were all tested neat on filter paper. The results of which can
be seen in Table 10. Control tests were carried out using both 3% and
6% H2O2 but gave the same reactions, however 3% H2O2 tended to be
less intense. As the reactions were similar they have been reported in the
same table below.
Table 10 Results of presumptive testing on control samples
Substance KM LMG
Biological washing
powder
- +
Light blue
Non-biological
washing powder
- +
Light blue
Stain removing spray +
light pink at 60
seconds
+
Deep blue
Bleach Yellow colouring on
contact
Foam on addition of
H2O2
Blue on contact with
reagent
Foam on addition of
H2O2
Vanish - +
Light blue
Blood +
Bright Pink
+
Bright Blue
No blood - -
As can be seen above most false positive reactions took place in the
presence of LMG reagent, however when compared to tests carried out
on washed fabrics results for KM reactions gave more positive reactions.
This may be due to the higher chance of false negatives occurring with
the use of KM as there are more chemicals that have an effect on results
than LMG due to the sensitivity of the chemical.

34
4.2 DNA Analysis
Results have been obtained from UV quantitation and final NuSieve®
Gel. The samples were quantitated with UV quant and calculations
carried out. For full results of calculations these can be found in Appendix
4 as well as the raw absorption data which can be found in Appendix 3.
4.2.1 UV Quantification
During this process each sample was put into a UV spectrophotometer
and an absorbance found for each individual sample. It was suspected
before carrying out analysis that there would be very low quantities and
low purity of DNA. A full set of absorptions obtained can be found in
Appendix 3.
The results from this can be found in Tables 11 – 15. No direct correlation
can be found between absorption and washing group. When looking at
the controls which can be found in Table 11 shows that there was a very
low concentration of DNA present in the negative sample in which there
should be none which may show some contamination had occurred.
Table 11 Results of UV quantification Calculations for Amount of
DNA and Range of Purity in control samples
Control Samples
Sample Amount present (µg) Range of purity (%)
Positive 0.73 0.39-0.4
Negative 0.23 0.7-0.8
Tables 12-15 show no correlations detected between fabric types or the
method in which they were washed. It would have been expected that the
samples washed with bleach would contain a lower quantity of DNA than
those treated with a stain removing stain. The amount of DNA found
between Group A (Table 11) which used non-biological powder and
Group B (Table 12) which used biological powder shows that the samples
washed in biological washing powder resulted in a lower DNA yield when
compared to non-biological washing powder.

35
DNA and Range of Purity for test group A
Test Group A - Non-Biological washing powder and Bleach
Wool 0.55 0.5-0.6
Cotton 0.55 1.9-2
Denim 0.48 2-2.2
Polyester 0.8 1.1-1.2
Colour catcher 0.57 0.9-1
DNA and Range of Purity in test group B
Test Group B - Biological washing powder and Bleach
Wool 0.46 0.62-0.68
Cotton 0.38 0.42-0.46
Denim 0.57 0.5-0.6
Polyester 0.34 0.3-0.34
Colour catcher 0.54 0.9-1
DNA and Range of Purity for test group C
Test Group C - Non-Biological washing powder and spray
Wool 0.4 1.6-1.7
Cotton 0.36 0.75-0.83
Denim 0.51 0.04-0.042
Polyester 0.54 1.3-1.5
Colour catcher 0.67 1.3-1.5
DNA and Range of Purity for test Group D
Test Group D - Biological washing powder and Spray
Wool 0.7 2.2-2.4
Cotton 0.69 0.9-1
Denim 0.43 0.7-0.8
Polyester 0.45 0.25-0.28
Colour catcher 0.41 0.44-0.48

36
Results obtained from Group C (Table 14) and Group D (Table 15) show
no correlation however between biological and non-biological washing
powder with only 3 out of the 5 fabrics showing a higher quantity of DNA
in non-biological washing powder.
When comparing the two groups it would be expected that blood treated
with bleach would obtain lower quantities of DNA however this cannot be
seen in the results.
4.2.2 Agarose Yield Gel
This was another precursor to identify if DNA was present in the samples.
As can be seen in Figure 7 there were no bands present which would
indicate that no DNA was present within the samples. It could be possible
that the DNA concentration in the sample was so low that it was unable to
be identified in the gel which correlates with results from UV
quantification. As can be seen in Figure 7, sample BP was run twice as it
was not detected in the gel 1 (left hand image) and so was run again in
gel 2 (right hand image).
The photographs in Figure 7 (above) run as follows. The wells on the left hand
gel run in the order from left to right: Ladder, space, BC, BD, BP, BW, BCC, AC,
AD, AP, AW, ACC, space, -ve, space and ladder. The samples in the wells in
the right hand image, from left to right, run in the order of ladder, BP, CCC, CP,
CW, CC, CD, DCC, DP, DW, DC, DD.
Figure 7 Yield gels obtained from Bio-Rad UV imager

37
4.2.3 NuSieve® GTG® Agarose Gel
As can be seen in Figure 8 DNA was obtained from 5 out of 10 samples.
As these tests groups were treated with bleach it was unlikely to get any
DNA from the material, however it was obtained from half of the samples.
The gels in which stain removing spray was used managed to have a
DNA profile from all but one sample amplified which can be seen in
Figure 9.
v
The image on the left was the original image and the image on the left was the
inverse of this image. From left to right the wells go in the order of Ladder,
space, BCC, BD, BP, BC, BW, ACC, AP, AD, AC, AW, space, +ve, space and
ladder
The image on the left was the original image and the image on the left was the
inverse of this image. From left to right the wells go in the order of Ladder,
space, DCC, DP, DD, DC, DW, CCC, CP, CD, CC, CW, space, -ve, space and
ladder.
Figure 8 NuSieve® Gel Image from Groups A, B and positive
control.
Figure 9 NuSieve® Gel Image from Group C, D and negative control

38
Figure 11 Control reaction of LMG on neat bleach
5 Discussion
5.1 Interferences from products on presumptive tests
When looking at the results obtained from both control tests and those
done on the samples there is little to compare. In controls it was LMG that
that reacted mostly with the chemicals present but during testing on
samples KM reacted with the samples more than LMG. Bleach has
shown to react with both KM and LMG before the addition of H2O2 and
when added started to fizz. Figure 10 shows the reaction KM with bleach
before H2O2 is added the bleach went yellow in colour and upon the
addition of H2O2 started to fizz and turn a light shade of pink however this
is not as noticeable in the image.
Picture on left is reaction with reagent only and
Picture on the right is after 6% H2O2 is applied
Figure 11 below is the reaction of LMG with neat bleach the regent goes
blue in the presence of bleach and fizzes on the addition of H2O2. This
reaction may take place due to oxidising agents within the bleach.
Reactions are the same with the addition of 3% H2O2 but less foam is
produced.
Picture on left is reaction with reagent only and
Picture on the right is after 6% H2O2 is applied
Figure 10 Control reaction of KM on neat bleach

39
5.2 Comparison of hydrogen peroxide concentration on testing
During this experiment two different concentrations of H2O2 were used.
To compare the effects of different concentrations, 6% H2O2 and 3%
H2O2, results from Testing Method 1 and 2 were used. Both methods
used the same scraping technique and as testing conditions were the
same could be compared. The results of these were put into a graph
which can be seen in Table 16.
Table 16 Graph comparing 3% and 6% H2O2 used when coupled with
KM presumptive test on samples
As can be seen above results were consistently obtained with the use of
6% H2O2 whereas 3% H2O2 did not consistently give results. It may also
be seen that 3% H2O2 results in most cases a longer reaction time than
that of 6% H2O2 this could be due to the lower concentration of hydrogen
peroxide present to oxidise haemoglobin. Although it cannot be seen in
Table 16 more detail can be found in Appendix 1. The results obtained
using 3% H2O2 were much more sporadic in that it did not react all the
time as well as having large variations in timings. For example, the
polyester sample from group D (DP) reaction time ranged from 59 to 80
seconds but when tested with the 6% H2O2 repeated reacted within 42 to
52 seconds.
0
10
20
30
40
50
60
AW AC AD AP ACC BW BC BD BP BCC CW CC CD CP CCC DW DC DD DP DCC
Time(s)
Sample
Comparison of the use of 3% and 6% hydrogen peroxide on reaction
times for Kastle-Meyer presumptive test
3% Hydrogen peroxide 6% hydrogen peroxide

40
Due to the large range of reaction times observed when using 3% H2O2
the obtained results do not come across as reliable. However, the above
statement only refers to that of KM testing. LMG testing on the other hand
shows different results. LMG results, which can be found in Appendix 1,
only had a few positive reactions which occurred in the presence of 3%
H2O2, as a reaction did take place a chemical must have been present to
have caused this, however this was not consistent throughout and when
tests employed 6% H2O2 all results were negative.
Overall, 6% H2O2 has proved to result in faster reaction times in most
cases and more consistent timings making the use of this more reliable in
comparison to 3% H2O2. This is also seen with LMG testing as random
positive results were obtained with 3% H2O2 and not consistent, yet with
the 6% H2O2 all results came back negative.
5.3 Correlation between washing groups and presumptive tests
When investigating the effects of the washing process on blood stains
there are two comparisons that can be made. Firstly, a comparison of
bleach and stain removing spray and secondly the use of biological or
non-biological washing powder. However, the presence of sodium
percarbonate during the washing cycle must be taken into consideration
as this should result in negative presumptive test results.
5.3.1 Comparison of pre-treatments
To compare the effects of bleach and stain removal spray on presumptive
tests the results of Group A and Group C were compared as well as
those of Group B and Group D. In doing this the type of washing powder
used was kept constant allowing a direct comparison between bleach and
stain removal spray to be made. The mean times calculated from the
reactions in both testing methods 1 and 2 were used and are displayed in
chart form and from here a comparison could be made.

41
Table 17 shows a comparison between the two testing methods the first 5
columns are Group A which used bleach as a pre-treatment and the
following 5 is Group C which used a stain removing spray. Group A
shows to have faster reaction times than Group C, however when looking
at results obtained from 6% H2O2 show the opposite effect and doesn’t
have a specific pattern.
Table 17 Graph comparing times of presumptive testing between
bleach and stain removing spray
Table 17 shows reaction times are much slower for stains treated with
stain removing spray in comparison to bleach when looking at results
from Testing Method 1 but this is not the same when Testing Method 2 is
compared. It should also be noted that not all tests run using 3% H2O2
(Testing Method 1) reacted but 2 out of 3 which may mean that the mean
times are not fully representative.
AW AC AD AP ACC CW CC CD CP CCC
3% Hydrogen peroxide 28 23 34 35 0 35 50 44 51 0
6% hydrogen peroxide 44 40 43 42 20 37 39 47 35 35
0
10
20
30
40
50
60
Time(s)
Comparison of mean times between samples treated with
bleach and spray detergent in the presence of biological
washing powder

42
In contrast Table 18 below shows the presence of bleach in Group B and
stain removing spray in Group D samples washed in biological washing
powder. 3% H2O2 resulted in slower reaction times than 6% H2O2. It may
also be observed that reaction times for samples were spray was used
are longer than that of bleach.
Table 18 Graph comparing times of presumptive testing between
bleach and stain remover
Overall bleach shows to result in faster reaction times than stain remover
spray. This could be due to the oxidising ability of the sodium hypochlorite
in the bleach which would accelerate the rate in which oxidation of
haemoglobin takes place once H2O2 was added in comparison to the
spray which contains no chemicals which would affect the haemoglobin
and in turn affect test results. It can also be stated that here only a
comparison of KM results can be made this is due to the sporadic results
of LMG as there was not consistent positive results that were
comparable.
BW BC BD BP BCC DW DC DD DP DCC
0
10
20
30
40
50
60
Time(s)
Comparison of mean times between samples treated with
bleach and spray detergent in the presence of non - biological
washing powder

43
5.3.2 Comparison of washing powder type
Table 19 and 20 are used to compare any difference in reaction times
when the sample was washed with non-biological washing powder or
biological washing powder when pre-treated with the same treatment.
Table 19 shows reaction times between bio and non-bio washing
powders in the presence of bleach. It can be observed that the use of 6%
H2O2 caused reaction times for bio washing powders to be faster in
comparison to that of non-bio washing powder reaction times. In contrast
the opposite effect is seen from results obtained when 3% H2O2 was used
but this may be down to a lower amount of oxidation taking place.
As 6% H2O2 has proven to be more reliable in that results are more
consistent these results may be said to be a better comparison. Overall
bio washing powders have faster reaction times bar that of the colour
catcher. It is possible that the enzymes in the washing powder affect
some materials more than others and this should be taken into account.
Table 19 Comparison of washing powder type on reaction time for
KM testing in the presence of bleach
AW AC AD AP ACC BW BC BD BP BCC
0
10
20
30
40
50
60
Time(s)
Comparison of the use of non-biological and biological
washinh powders in the presence of bleach

44
Table 20 in comparison was treated with stain removing spray and it can
be seen that reaction times are slower in comparison. When looking at
results obtained from 3% H2O2 the same pattern can be seen that per
each fabric biological washing powder reacts faster, this could be due to
the lack of enzyme activity on the proteins within the blood. However,
when 6% H2O2 is used there is no obvious pattern between them.
Table 20 Graph comparing washing powder type on reaction times
for KM in the presence of stain removing spray
Overall when comparing Table 19 and Table 20 reaction times are visibly
slower when stain removing spray is used. This may be due to the
presence of bleach causing the proteins to denature making haemoglobin
more accessible as well as the possibility that residue from the bleach
was left on the material after washing which could cause a faster false
reaction for blood that stain removing spray alone.
Results from testing method 3 are unable to be compared as there were
negative results obtained across the board. This method however is used
when working in a forensic laboratory and therefore more reliable so it
could be said that the results obtained are not accurate.
CW CC CD CP CCC DW DC DD DP DCC
0
10
20
30
40
50
60
Time(s)
Comparison non-biological and biological washing powder on
reaction times in the presence of stain removing spray

45
5.4 Difference between use of spatula and filter paper
For this section Testing Method Groups 2 and 3 were looked at as they
both employed the use of 6% H2O2. Results varied massively for KM as
those tests carried out with a spatula resulted in all positive results
without any outliers but those done with the filter paper folded and
scraping gave all negative. There are several factors that may have led to
this set of results, firstly the use of a spatula may have been what caused
the reaction; however, when the spatula was wiped onto a damp filter
paper and then tested no reaction occurred.
Secondly, the amount of material scraped off the fabric during the
process. When using the spatula more force could be applied to remove
fibres from the fabric and due to the curved edge of the spatula more
fibres could remain on the surface rather than fall off, the filter paper on
the other hand was much softer and had to be folded to scrape the
material. It was also noticeable that the fibres did not easily attach to the
surface and were likely to fall off especially when unfolding the filter paper
so extra care had to be used.
It is possible that the amount of fibres present on the filter paper after
scraping could affect the results and the use of which reduces the chance
of a positive reaction. The sensitivity of the reagent although may suggest
otherwise as even if a few fibres on the filter paper used to scrape had
the presence of blood there may have been a reaction, but then again
sodium percarbonate was present and no reaction should have occurred.
However, when it comes to results obtained from LMG tests both the use
of spatula and filter paper resulted in negative reactions. It is widely
known that LMG results in less false positives than that of KM which
would further indicate that the spatula may have caused the reaction.
However, as the spatula was made of stainless steel there is a low
chance that the KM would react with the metal. The results obtained may
be due to a combination of both scraping method and quantity of fibres.

46
5.5 Sodium percarbonate and presumptive testing
Results obtained from Testing Method 3 were used to evaluate the effect
of sodium percarbonate on presumptive tests as there was no other
factors introduced that may affect the results that were produced. In
consideration to Testing Methods 1 and 2 a spatula was used during
testing which may have affected the results obtained.
Testing Method 3 for both KM and LMG resulted in negative reactions
being obtained suggesting that the materials had been in no contact with
blood however this was not the case. This method used quartered filter
paper to directly scrape materials, unfolded and the reagent added. The
unfolding of the filter paper resulted in some fibres being lost as they
could not adhere properly. This may have resulted in negative results due
to the low quantity of material present for the reagents to react with. On
the other hand, it is likely that the negative result was obtained due to the
exhaustion of haemoglobin caused by the sodium percarbonate present
in the detergents that the stains were washed in.
Test method 1 is the most reliable way to test and is often used in
forensic laboratories, from this it is possible that false negatives for the
presence of blood may be attained if used in a professional setting.
These results occur due to the exhaustion of haemoglobins ability to be
oxidised when H2O2 was added. Due to this care should be taken when
running tests if clothing is suspected to have blood on them after being
washed as it is possible that oxidising agents may be present which
would hinder results for both KM and LMG.

47
5.6 DNA analysis
The results from the NuSieve® gel were unexpected due to lack of results
obtained from the yield gel and the law quantity and purity found by UV
quantification. However, PCR was run with 40 cycles instead of 27
causing the DNA present to multiply at a much greater rate and due to
this bands could be seen in the gel. Control samples were run and a
strong band for the presence of DNA could be seen for the control and no
band was present for the negative control, results of which have been
tabulated in Table 21. The success of the controls shows that extraction
and PCR were run smoothly and that no contamination can be identified.
Table 21 NuSieve gel results for control samples
Control DNA present
Positive +
Negative -
The results of Test Group A and B have been put in table form and can
be seen in Table 22. These groups were both treated with bleach which
would mean there was a lower chance of obtaining DNA. The results
showed that the wool and cotton from Group A as well as the polyester
from Group B were able to give DNA. The colour catchers from both
groups also showed the presence of DNA. Although the items themselves
were not in contact with blood they had the ability to have DNA adhere to
them during the washing cycle through secondary transfer.
Table 22 NuSieve gel 1 results for groups A and B
Washing Group Fabric DNA present
A
Wool +
Cotton +
Denim -
Polyester -
Colour catcher +
B
Wool -
Cotton -
Denim -
Polyester +
Colour catcher +

48
The second gel was run to look at samples from Group C and D which
were treated with a stain removing spray, the chemicals in which should
not affect the ability to obtain DNA or cause any damage to it. Table 23
shows the tabulated results from this gel and all but one sample
contained DNA. In some instances, DNA cannot be amplified from
samples on a denim substrate as dyes present such as indigo blue act as
PCR inhibitors resulting in DNA not being visible in a product gel.
However, DNA was obtained from denim samples in these groups this
could be due to a non-inhibiting dye being used. DNA was also obtained
from both colour catchers which further validated that DNA is obtainable
not only from the item in question but may be obtained from other items
that were washed in their presence. No DNA was obtained from cotton in
Group D this may be due to a failure in extraction or a handling error.
Table 23 NuSieve® Gel 2 results for Group C and D
Washing Group Fabric DNA present
C
Wool +
Cotton +
Denim +
Polyester +
Colour catcher +
D
Wool +
Cotton -
Denim +
Polyester +
Colour catcher +
Looking at results there is no correlation noticeable between fabric type
and quantity of DNA obtained or the effect of the washing powder itself or
that of the oxygen bleach. The only thing that affects DNA is the presence
of bleach during the washing process, but even then some DNA may
remain and be able to be amplified. As colour catchers of all four groups
managed to have DNA extracted from them it shows that no matter how
the materials are cleaned DNA can still be undamaged. DNA would have
been carried through the water in the washing cycle and adhered to the
fabric where subsequently could be extracted and enhanced.

49
5.7 Connection between presumptive testing and DNA analysis
For this comparison I am going to use the results obtained for
presumptive Testing Method 3 which used filter paper to scrape and 6%
H2O2 as this method is employed in professional settings. As could be
seen all presumptive tests for both LMG and KM were negative although
blood was present on the fabric, this was due to the presence of sodium
percarbonate oxidising the haemoglobin before testing took place. The
subsequent DNA testing showed that this did not affect the ability for DNA
to remain on the fabric nor for it to be extracted and amplified.
As no visible stain could be seen after the fabrics were washed and a
negative presumptive result found it is possible that DNA analysis may
not be carried out. However, it is still possible to obtain a DNA profile.

50
6 Conclusions
Overall, it can be seen that testing method will have an impact on
presumptive testing results. The use 6% H2O2 has shown to be more
reliable than that of 3% H2O2 as more reproducible results have been
obtained and consistent throughout testing. The KM presumptive tests
resulted in more positive results than that of LMG this may due to the
reagent reacting with something else that was present, in this instance
the spatula that was used in Testing Methods 1 and 2 may have impacted
upon this. As Testing Method 3 used filter paper directly to the material
and gave all negative results the spatula used is the probable source of
error. The way in which the material washed seemed to have little to no
effect on test results with only small variations or patterns being
observed. All in all, when the final method was used all results were
negative, and as scraping with filter paper and 6% H2O2 is usually
employed in forensic laboratories and from this the results obtained would
be more reliable. From this it shows that the sodium percarbonate
present in the Vanish Oxi-Action does impact upon forensic presumptive
testing for blood.
DNA analysis however, remains un-effected by the presence of sodium
percarbonate and can still be extracted and amplified. The presence of
bleach however does affect DNA analysis and fewer profiles can be
found. Either way although negative presumptive tests were obtained,
DNA analysis was successful. This is evident in colour catchers as well
which have shown to have the ability to catch DNA that is transferred
when washed with items that have blood on them.
When a combination of active oxygen containing detergent and bleach is
used this may be an obstacle that forensic scientists may be faced with.
Nevertheless, sodium percarbonate only affects that of presumptive
testing and not DNA analysis.

51
Future Work
The effects of washing blood stains with sodium percarbonate on
confirmatory blood tests. Investigate if the oxidation of haemoglobin
would lead to takayama confirmatory providing a negative result.
The effects of washing temperature with a combination of oxygen bleach
concentrations on blood stain detection. In this experiment washing cycle
temperatures would vary for example at 30ºC, 60ºC and 90ºC and this
could be done with varying amounts of oxygen bleach instead of using a
single scoop use two or three.
Effects of washing on seminal stains and the consequential presumptive
testing. Does washing powder type or pre-treatments have an effect on
acid phosphatase reaction for seamen and if so, do temperature of
washing cycle and do several cycles reduce chance of identification.
Investigation into aging periods before clothing is washed with
oxygenated bleach detergents. Does leaving blood on material for
different periods of time before washing have an impact on reactions and
does leaving stained clothing too steep before treatment in active oxygen
containing detergent have a larger impact on the ability to obtain DNA.
Further investigation into colour catchers used in washing cycles. When
added to a washing cycle that does not contain active oxygen can
positive presumptive tests be obtained and if so does the washing cycle
have an effect.

52
Reference list
Akane, A. et al. 1994. Identification of the Heme Compound
Copurified with Deoxyribonucleic Acid (DNA) from Bloodstains, a
Major Inhibitor of Polymerase Chain Reaction (PCR) Amplification.
Journal of Forensic Sciences. 39(2): pp.362-372.
Brevnov, M. G., Pawar, H. S., Mundt, J., Calandro, L. M., Furtado, M.
R. and Shewale, J. G. 2009. Developmental Validation of the
PrepFiler™ Forensic DNA Extraction Kit for Extraction of Genomic
DNA from Biological Samples. Journal of Forensic Sciences. 54(3):
pp.599-607.
Brownlow, R. J., Dagnall, K. E. and Ames, C. E. 2012. A Comparison
of DNA Collection and Retrieval from Two Swab Types (Cotton and
Nylon Flocked Swab) when Processed Using Three QIAGEN
Extraction Methods. Journal of Forensic Sciences. 57(3): pp.713-717.
Buckleton, J. and Triggs, C. 2006. The effect of linkage on the
calculation of DNA match probabilities for siblings and half siblings.
Forensic Science International. 160(2–3): pp.193-199.
Castelló, A. Francés, F. Corella,D. and Verdú, F. 2009. Active oxygen
doctors the evidence. Naturwissenschaften. 96(2): pp.303-307.
Castelló, A., Francés, F. and Verdú, F. 2012. Chemistry in Crime
Investigation: Sodium Percarbonate Effects on Bloodstains Detection.
Journal of Forensic Sciences. 57(2): pp.500-502.
Cavenagh, J. 2007. White blood cells. Surgery (Oxford). 25(2): pp.61-
64.
Cox, M. 1990. Effect of Fabric Washing on the Presumptive
Identification of Bloodstains. Journal of Forensic Sciences. 35(6):
pp.1335-1341.
Cox, M. 1991. A Study of the Sensitivity and Specificity of Four
Presumptive Tests for Blood. Journal of Forensic Sciences. 36(5):
pp.1503-1511.
Dickson, C. F., Jacques, D. A., Clubb, R. T., Guss, M. J. and Gell, D.
A. 2015. The structure of haemoglobin bound to the haemoglobin
receptor IsdH from Staphylococcus aureus shows disruption of the
native α‐globin haem pocket. Acta Crystallographica Section D. 71(6):
pp.1295-1306.
Egan, J. M., Rickenbach, M., Mooney, K., E. Palenik, C. S.,
Golombeck, R. and Mueller, K. T. 2006. Bank Security Dye Packs:
Synthesis, Isolation, and Characterization of Chlorinated Products of
Bleached 1‐(methylamino)anthraquinone. Journal of Forensic
Sciences. 51(6): pp.1276-1283.

53
Forsdyke, D. R. and Mortimer, J. R. 2000. Chargaff's legacy. Gene.
261(1): pp.127-137.
Fregel, R., Almeida, M., Betancor, E., Suárez, N. M. and Pestano, J.
2011. Reliable nuclear and mitochondrial DNA quantification for low
copy number and degraded forensic samples. Forensic Science
International: Genetics Supplement Series. 3(1): pp.e303-e304.
French-Fuller, K. 2006. Gendered Invisibility, Respectable
Cleanliness: The Impact of the Washing Machine on Daily Living in
Post-1950 Santiago, Chile. Journal of Women's History. 18(4): pp.79-
100.
Gacko, M. and Głowiński, S. 1998. Activities of proteases in parietal
thrombus of aortic aneurysm. Clinica Chimica Acta. 271(2): pp.171-
177.
Garibyan, L. and Avashia, N. 2013. Polymerase chain reaction.
Journal of Investigative Dermatology. 133(3): pp.1-4.
Glaister, J. 1926. The Kastle-Meyer Test For The Detection Of Blood:
Considered From The Medico-Legal Aspect. The British Medical
Journal. 1(3406): pp.650-652.
Golebiewska, E. M. and Poole, A. W. 2014. Secrets of platelet
exocytosis – what do we really know about platelet secretion
mechanisms? British Journal of Haematology. 165(2): pp.204-216.
Gordon-Smith, T. 2013. Structure and function of red and white blood
cells. Medicine (United Kingdom). 41(4): pp.193-199.
Greenspoon, S. A., Scarpetta, M. A., Drayton, M. L. and Turek, S. A.
1998. QIAamp Spin Columns as a Method of DNA Isolation for
Forensic Casework. Journal of Forensic Sciences. 43(5): pp.1024-
1030.
Grosberg, A. Y. 2012. How two meters of DNA fit into a cell nucleus:
Polymer models with topological constraints and experimental data.
Polymer Science Series C. 54(1): pp.1-10.
Grubb, J. C., Horsman-Hall, K. M., Sykes, K. L. V., Schlisserman, R.
A., Covert, V. M., Rhee, H. N., Ban, J. D. and Greenspoon, S. A.
2010. Implementation and validation of the teleshake unit for DNA
IQ™ robotic extraction and development of a large volume DNA IQ™
method. Journal of Forensic Sciences. 55(3): pp.706-714.
Harris, K. A., Thacker, C. R., Ballard, D. and Court, S. D. 2006. The
effect of cleaning agents on the DNA analysis of blood stains
deposited on different substrates. International Congress Series.
1288: pp.589-591.
Hashmi, G. 2007. Red blood cell antigen phenotype by DNA analysis.
Transfusion. 47(1): pp.60S-63S.

Rebekah E. Findlay 1202708 Dissertation

Rebekah E. Findlay 1202708 Dissertation

Recommended

Recommended

More Related Content

What's hot

What's hot (18)

Similar to Rebekah E. Findlay 1202708 Dissertation

Similar to Rebekah E. Findlay 1202708 Dissertation (20)

Rebekah E. Findlay 1202708 Dissertation