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Active oxygen doctors the evidence

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Investigation at the scene of a crime begins with the search for clues. In the case of bloodstains, the most frequently used reagents are luminol and reduced phenolphthalein (or phenolphthalin that is also known as the Kastle-Meyer colour test). The limitations of these reagents have been studied and are well known. Household cleaning products have evolved with the times, and new products with active oxygen are currently widely used, as they are considered to be highly efficient at removing all kinds of stains on a wide range of surfaces. In this study, we investigated the possible effects of these new cleaning products on latent bloodstains that may be left at a scene of a crime. To do so, various fabrics were stained with blood and then washed using cleaning agents containing active oxygen. The results of reduced phenolphthalein, luminol and human haemoglobin tests on the washed fabrics were negative. The conclusion is that these new products alter blood to such an extent that it can no longer be detected by currently accepted methods employed in criminal investigations. This inability to locate bloodstains means that highly important evidence (e.g. a DNA profile) may be lost. Consequently, it is important that investigators are aware of this problem so as to compensate for it.
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SHORT COMMUNICATION
Active oxygen doctors the evidence
Ana Castelló &Francesc Francès &Dolores Corella &
Fernando Verdú
Received: 26 June 2008 /Revised: 25 September 2008 /Accepted: 29 September 2008 / Published online: 21 October 2008
#Springer-Verlag 2008
Abstract Investigation at the scene of a crime begins with
the search for clues. In the case of bloodstains, the most
frequently used reagents are luminol and reduced phenol-
phthalein (or phenolphthalin that is also known as the
KastleMeyer colour test). The limitations of these reagents
have been studied and are well known. Household cleaning
products have evolved with the times, and new products
with active oxygen are currently widely used, as they are
considered to be highly efficient at removing all kinds of
stains on a wide range of surfaces. In this study, we
investigated the possible effects of these new cleaning
products on latent bloodstains that may be left at a scene of
a crime. To do so, various fabrics were stained with blood
and then washed using cleaning agents containing active
oxygen. The results of reduced phenolphthalein, luminol
and human haemoglobin tests on the washed fabrics were
negative. The conclusion is that these new products alter
blood to such an extent that it can no longer be detected by
currently accepted methods employed in criminal inves-
tigations. This inability to locate bloodstains means that
highly important evidence (e.g. a DNA profile) may be lost.
Consequently, it is important that investigators are aware of
this problem so as to compensate for it.
Keywords Crime scene investigation .Bloodstains .
Presumptive test .Haemoglobin test
Introduction
Investigations at a crime scene are mainly aimed to
discover clues that can be studied in the laboratory in order
to provide additional data for solving a case. Logically, the
perpetratorsintention is to prevent investigators from
finding clues, so they will strive to eliminate all traces
insofar as possible. The scene of the crime, or clothing worn
by the perpetrator, may often be cleaned to eliminate any
traces that could constitute evidence. DNA tests, however,
can be used to establish a genetic profile using traces of bio-
logical samples such as blood. The use of research tech-
niques capable of discovering latent clues, i.e. those that may
actually exist but cannot be seen, is, therefore, essential.
The presumptive test has proved to be highly effective in
detecting latent bloodstains due to its high sensitivity (Cox
1991; Higaki and Philp 1976). Currently, the reagents most
commonly used are reduced phenolphthalein and luminol.
Various researchers have assessed the effect of washing blood
stains on the sensitivity and specificity of these presumptive
tests (Cox 1990; Creamer et al. 2003; Quickenden and
Creamer 2004;Heuseretal.2006). The results showed that
of all the indicator reagents, luminol is the most resistant to
the possible influence of contaminants. Studies undertaken to
Naturwissenschaften (2009) 96:303307
DOI 10.1007/s00114-008-0466-9
A. Castelló :F. Francès :F. Verdú (*)
Facultad de Medicina, U. D. Medicina Legal,
University of Valencia EG,
Av/ Blasco Ibañez, n°15,
46010 Valencia, Spain
e-mail: Fernando.Verdu@uv.es
A. Castelló
e-mail: Ana.Castello@uv.es
F. Francès
e-mail: Francesc.Frances@uv.es
D. Corella
Facultad de Medicina, U. D. Preventive Medicine/CIBER
Fisiopatologia de la Obesidad y Nutrición,
Instituto de Salud Carlos III,
University of Valencia EG,
Av/ Blasco Ibañez, n°15,
46010 Valencia, Spain
e-mail: Dolores.Corella@uv.es
determine the effect of detergents of different characteristics
indicate that luminol does not react positively with the
products. Nevertheless, it has also been shown that on blood
stains treated with detergents, the intensity of the chemilu-
minescence is increased in comparison to that of standard
haemoglobin. This can be due to the fact that the detergents
make the plasma membrane of the erythrocytes more
permeable, exposing the Fe
3+
inside the cell and speeding
up the entire reaction (Heuser et al. 2006). That is to say,
that the effect of the detergent on blood results in a more
intense positive and not in a negative. We previously
reported that the luminol test was positive on blood stains
formed on cotton fabric even after being washed ten times
in a washing machine using a standard detergent.
Moreover, DNA could be extracted from samples washed
three times (Castelló et al. 2002).
Cleaning products containing hypochlorites interfere
with blood test (Quickenden and Creamer 2001; Creamer
et al. 2005). A surface treated with bleach is known to
produce a positive test result even when there is no blood.
This form of interference has been thoroughly studied and
is easily recognised by experts investigating the scene of
the crime. One possible and quite effective solutionthe
use of amines to eliminate the chlorine as chloramineshas
recently been put forward to counteract the effect of bleach
on luminol (Kent et al. 2003; King and Miskelly 2005).
Although the problem created by chlorine bleach is on
the way to be resolved, other new problems are appearing.
Cleaning products have also evolved with the times, and
products containing active oxygen are widely used nowadays.
These are new formulations that contain no chlorine bleach,
yet are quite efficient at removing all types of stains. They are
used to clean clothes and many other types of surfaces.
1
In view of this new way of removing stains, it is
essential to evaluate their possible effect on presumptive
test results. The aim of this study was to determine whether
products containing active oxygen have any effect on the
presumptive testing methods used in the search for latent
bloodstains. Their possible interference on the results of
human haemoglobin analysis using forensic kits was also
evaluated.
Materials and methods
Materials
Backings for bloodstains: soft cotton cloth, jeans
fabric, towelling
Reagents: PHENOLPHTHALEIN DISCHAPS
(Sirchie Cat. No. DCB100), luminol (3-aminophthal-
hydrazide; Merck), sodium perborate (Panreac), sodium
carbonate (Panreac), distilled water, Hexagon OBTI® test
Light sources: Bluemax(390550 nm), UV
Methods
Sample preparation
Bloodstains were prepared using:
(a) newly extracted blood samples with no preservatives
(b) newly extracted blood samples to which EDTA was
added
Stains were made on each of the backings (cotton, jeans
fabric, towels) using each sample (a and b). Each stain was
made with five drops of blood.
Having made the stains, they were left to dry at room
temperature without any protection whatsoever. Prior to
analysis, they were left on the laboratory desk subject to
surrounding conditions. At different times, 1, 5, 10, 20, 30
and 40 days, the following procedure was undertaken: Four
stains from each group (a and b) were cut out from each of
the backings (cotton, jeans fabric, towels) using a sterile
scalpel. One of these was kept in the laboratory for
subsequent use as a control. The other three were washed
(in separate containers) using a product containing active
oxygen (Neutrex). The composition of the productas
appears on the labelis as follows
2
: non-ionic and anionic
tensoactive agents, polycarboxilates and zeolites (concen-
tration less than 5%) and sodium percarbonate (in a
proportion greater than 30%). The pH of the product
dissolved in water is 10. When washing, the manufacturers
instructions were followed: The fabric was left in hot water
(at 40°C) with the cleaning product for 2 h. Then, it was
rinsed in running water and left to dry. Drying time was
established at 1 day.
Once dry, all the stains were observed with visible and
UV lights before applying the reagents. Afterwards,
together with those that had been kept as controls, they
were subjected to the presumptive test and the haemoglobin
determination test following the procedures detailed below.
In order to confirm the results, the entire process was
repeated five times for each of the periods and supports (1,
5, 10, 20, 30 and 40 days) using different samples.
1
http://www.henkel.es/prensa/ppal/notadetalle.asp?idnota=230
(accessed on 24 June 2008). (http://www.alsnetbiz.com/homeimprove
ment/oxybleach.html accessed on 24 June 2008).
2
Other commercial makes of products containing active oxygen were
found to have the same composition as that used in the study, but also
all of them contain protease. The proteases have a destructor effect of
the proteins. Therefore, they will operate on the haemoglobin and will
join their effects with that of the active oxygen, and it is expected that
all the products present similar results.
304 Naturwissenschaften (2009) 96:303307
Presumptive tests
Reduced phenolphthalein
A pre-prepared reagent contained in an ampoule is used. As
the manufacturer
3
claims, this test is considered by many
field technicians to be one of the most reliable presumptive
tests for the presence of blood. Its sensitivity is 1:100,000.
The reagent was used following the manufacturers instruc-
tions: First, prints are obtained by moistening filter paper
with distilled water and pressing it onto the stain. The test is
then performed on the filter paper, rather than the original
stained substrate. The ampoules are then crushed by
applying pressure to the middle of the tube. After shaking
the tube to mix the reagents, they are applied to the sample.
If there is no positive reaction, the process is repeated,
applying the reagent directly onto the bloodstain. The same
procedure is applied to the control samples.
Luminol
Luminol was prepared in accordance with the Grodsky
formula (Grodsky et al. 1951). The reagent was then
sprayed onto the stain. This was performed in a darkroom.
To ensure that the reagents were correctly prepared, tests
were performed on the stains kept as controls. A small
portion of the stain was cut out using a scalpel, and the
reagent was applied. The test is considered positive when
luminescence is observed a few seconds after having
applied the reagent.
The same procedure was applied on the control samples.
Human haemoglobin test
The Hexagon OBTI® test kit was used. This testalso
widely used in forensic laboratoriesis an immunochro-
matographic assay that works with immobilised monoclo-
nal antibodies and a coloured particular reagent.
4
The stain
was steeped in distilled water. The test was then performed
following the manufacturers instructions
5
.
The same procedure was applied on the control samples.
Additional control assays
With the aim of evaluating more accurately the possible
effect of the product with active oxygen on the develop-
ment of the tests (presumptive and haemoglobin), the
following control assays were undertaken:
Assay 1. Washing of stains in hot water (at 40°C), without
using detergent, in order to determine the
possible influence of water temperature on the
results of the tests
Assay 2. Washing of stains with active oxygen and cold
water with the aim of determining whether
differences occur with the results obtained when
using the product with hot water
For each of the control assays, ten stains of between 1
and 10 days old were used.
Results
For the samples
The results obtained are shown in Table 1. A positive test is
represented as +; a negative test as . A presumptive
test is regarded as positive when the coloration (in the case
of the reduced phenolphthalein) or luminescence (luminol)
is observed a few seconds after applying the reagent. A
slower reaction may be due to the oxidation of the reagent
by agents other than the peroxidases of the sample.
The human haemoglobin test is regarded as positive
whenas stated in the manufacturers instructionsthe
blue test line (indicating the positive result of the sample) is
formed and observed in the first 5 min following the
beginning of the test. Negative results are confirmed at
10 min.
After washing, the stainsboth problem and control
were still visible with the naked eye, although slightly
faded. Observation using visible light lamps and UV did
not improve the perception of the stains.
The results of the test were the same for all the stains
being washed: The presumptive tests (reduced phenol-
3
http://sirchie.com/SearchResult.aspx (accessed on 24 June 2008).
4
www.bluestar-forensic.com/pdf/en/hexagon_obti_verif_report.pdf
(accessed on 24 June 2008).
5
http://www.bluestar-forensic.com/gb/documentation_hexagon.php
(accessed on 24 June 2008).
Table 1 Results of phenolphthalin, luminol and human haemoglobin
tests on bloodstain samples (of different ages) after having been
washed with a product containing active oxygen
Test Sample Age of sample (days)
1510203040
Phenolphthalin Control + + + + + +
Problem −−− − − −
Luminol Control + + + + + +
Problem −−− − − −
Human Haemoglobin Control + + + + + +
Problem −−− − − −
+positive test, negative test
Naturwissenschaften (2009) 96:303307 305
phthalein and luminol) and the haemoglobin test gave
negative results.
Although the results were negative using the 1-day
samples, it was considered advisable to continue studying
the older stains (up to 40 days), because luminol, as
indicated in the bibliography (Proescher and Moody 1939;
James and Eckert 1999), is more effective on older than
fresher stains. However, no positive results were obtained
in any case. All control bloodstains gave positive results.
For additional control assays
The results for assay 1 (washing of stains with hot water (at
40°C) without using detergent) showed that the presump-
tive and the human haemoglobin tests were positive for all
the stains analysed.
In assay 2 (washing of stains using active oxygen and
cold water), it is important to point out that the product with
active oxygen does not dissolve easily in cold water. Only
by agitating the solution a partial dissolution can be
achieved. Consequently, it is to be expected that it is not
so effective as when used following the manufacturers
instructions (water at approximately 40°C).
The results obtained were as follows: The reduced
phenolphthalein test gave negative results for all the stains
analysed. The luminol test gave negative results on all the
stains analysed. Having said that, a slight luminescence was
observed in several samples (approximately 25%) several
minutes after applying the reagent. As this late response
cannot be interpreted as a positive test, the results were
considered negative.
The human haemoglobin test gave negative results for
all the stains analysed. However, it should be noted that for
some of the samples (approximately 20%), the formation of
a slight blue test line (indicating the positive result of the
sample) was observed more than 10 min after the beginning
of the test. Nevertheless, the test had to be considered
negative.
Discussion
The results show that regardless of the type of blood used
to generate stains, the age or backing on which they were
placed (soft cotton cloth, jeans fabric, towelling) or the
washing conditions employed (hot or cold water), washing
fabric with products containing active oxygen prevents
positive results from being found using presumptive and
human haemoglobin tests.
Under the conditions employed in this study, the stains
both problem and controlwere still visible after washing
with the naked eye (although slightly faded). It is,
nevertheless, important to note that when there is a lower
blood concentration in the samples or the material on which
the stain is formed is less porous or coloured, it is quite
possible that the stains will become invisible after washing. In
this case, presumptive tests are the only way to find evidence
and start investigations that may lead to a genetic profile.
It is not the aim of this work to determine the reason for
this interference. However, a final test was undertaken to
account for its origin, which was as follows: It has been
proved that sodium percarbonate, dissolved in water, gives
off an appreciable amount of hydrogen peroxide (Jones
1999). A blood stain was inserted into an Eppendorf test
tube, and hydrogen peroxide was added. It was left in
contact with the reagent over 2 h, then the presumptive tests
and the human haemoglobin test were undertaken. The
results were negative for all of them. It is, therefore,
possible that the inhibiting effect is due to the action of
hydrogen peroxide as the ability of haem to breakdown
hydrogen peroxide has been exhausted by the considerable
amount of hydrogen peroxide produced by the active
oxygen cleaner. Nevertheless, this can only be considered
as a setting-out point for trying to find a valid answer.
Meanwhile, from the results presented in this work, it
may be concluded that these new products alter human
blood to such an extent that it can no longer be detected by
currently accepted methods employed in criminal investi-
gation. The inability to to identify or locate bloodstains
means no DNA can be extracted, and, therefore, important
evidence may be lost. Consequently, it is important for
investigators to be aware of this problem and to seek for a
solution.
Acknowledgements We are grateful to the referees and the editor
for their comments that have helped to improve this work.
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... Only a few studies have explored substances that can suppress the luminol reaction and generate a false--negative result. For instance, recent research has demonstrated that cleaning products that contain sodium percarbonate can remove bloodstains so that presumptive and confirmatory tests are unable to detect the presence of blood 18,19 . While the occurrence of these false--negatives might be rare, the potential exists for crucial DNA evidence to go undetected. ...
... This series of experiments was designed in order to investigate the persistence and detect--ability of blood when exposed to different conditions and washed in conjunction with sodium percarbonate. As noted by Castello 18 and substantiated by the experiments con--ducted in this study, sodium percarbonate is more effective than a detergent alone in remov--ing traces of blood in denim and carpet. However, in contrast to the findings presented by Castello 19 this study has demonstrated that the removal of blood may also be contingent on a number of variables such as the washing temperature, the type of detergent used, the drying time of blood and exposure of bloodstains to environmental conditions. ...
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O exame de corpo de delito pode constituir-se tarefa árdua para o perito criminal com ênfase em local de crime. A identificação desses vestígios muitas vezes inclui a presença de sangue. O objetivo desse estudo é testar a quimiluminescência do Bluestar® na identificação de manchas de sangue em tecido de algodão após as superfícies terem sido lavadas em diferentes períodos de tempo e com diferentes produtos de lavagem por uma máquina de lavar roupas automática. Sete corpos de prova foram preparados e em cada um deles foi dispensado 2 ml de sangue: um grupo controle, que não sofreu lavagem, e três duplas, divididas de acordo com os seguintes ciclos: algodão, lavagem ecológica e lavagem diária. Em cada dupla, um substrato foi lavado com água e sabão em pó e outro foi lavado com água, sabão em pó e alvejante em pó sem cloro. Os grupos ainda foram divididos de acordo com a quantidade de vezes que foram lavados na lavadora de roupas: os que foram lavados apenas uma vez (t1) e os que tiveram duas lavagens, no qual, além da primeira, tiveram uma segunda lavagem após 60 dias da primeira (t2). Conclui-se que o Bluestar® apresentou quimiluminescência em tecidos de algodão manchados de sangue em t1. Entretanto, em t2, em grupos que tiveram e os que não tiveram uma segunda lavagem, Bluestar® apresentou pouca quimiluminescência, podendo ser confundido com falso positivo, ou não apresentou reação quimiluminescente, como nos grupos onde foi usado o alvejante em pó sem cloro.
Chapter
This chapter tabulates a summary of the blood enhancement processes, confirmatory tests and their relative specificities to blood. Two generic types of processes that are most widely used for enhancement of marks in blood are the protein stains and the peroxidase reagents. Both types of blood enhancement processes are routinely used by fingermark development laboratories worldwide, with protein stains being more widely used when development of fingermarks is required and the area where they may be present is well defined. The most widely used of peroxidase reagents is Leuco Crystal Violet, which has been shown to be an effective treatment for marks in blood, albeit less sensitive than the protein stains. Other peroxidase reagents that are in use to a lesser extent include diaminobenzidine. In common with protein stains, there are also peroxidase reagents that give fluorescent or chemiluminescent reaction products. Luminol is a highly sensitive reagent for detection of blood.
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An introduction to the preparation and properties of hydrogen peroxide The activation of hydrogen peroxide using inorganic and organic species The application of hydrogen peroxide for the synthesis of fine chemicals The heterogeneous activation and application of hydrogen peroxide The environmental application of hydrogen peroxide Miscellaneous uses for hydrogen peroxide technology.
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The luminol chemiluminescence presumptive test for blood is based oil the mild peroxidase activity of hemoglobin in basic peroxide solution. However, this test is subject to interference by strong oxidants, certain transition metal ions, and true peroxidases. This paper reports methods for reducing the interference caused by hypochlorite-containing bleaches. Amines such as 1,2-diaminoethane react rapidly with hypochlorite without interfering significantly with the hemoglobin-catalyzed oxidation. Thus, addition of 0.1 mol/L 1,2-diaminoethane to a standard luminol-peroxide spray lead to almost complete inhibition of hypochlorite-induced chemiluminescence while satisfactory chemiluminescence was still observed from bloodstains. If time allows, an alternative method for reducing interference from hypochlorite bleach is to wait several days until the bloodstains have dried thoroughly, by which time the hypochlorite will have decomposed.
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The forensic luminol test has long been valued for its ability to detect trace amounts of blood that are invisible to the naked eye. This is the first quantitative study to determine the effect on the luminol test when an attempt is made to clean bloodstained tiles with a known interfering catalyst (bleach). Tiles covered with either wet or dry blood were tested, and either water or sodium hypochlorite solution (bleach) was used to clean the tiles. As expected, the chemiluminescence intensity produced when luminol was applied generally decreased with the number of times that a tile was cleaned with water, until the chemiluminescence was neither visible nor detectable. However, when the tiles were cleaned with bleach there was an initial drop in chemiluminescence intensity, followed by a rise to a consistently high value, visibly indistinguishable from that of blood. Examination of bleach drying time suggested that any interfering effect becomes negligible after 8 h.
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INTERPRETATION OF BLOODSTAIN EVIDENCE AT CRIME SCENES: Stuart H. James & William G. Eckert (2nd Edition) CRC Press, Boca Raton, 1999; 324pp; RRP US$69.95 hardcover; Australian distributor DA Books & Journals; ISBN 0 8493 8108 8.
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Sprays containing alkaline solutions of peroxide and luminol are used as presumptive screens for bloodstains at crime scenes. These sprays can be subject to interference from hypochlorite-based cleaning agents (bleaches), leading to false positive results. This paper reports the screening of amines for their ability to decrease the interference by bleach while not greatly affecting the reaction with blood. The addition of glycine (0.05molL(-1)) to the Grodsky formulation of luminol spray, together with an adjustment of the pH to 12, gave good discrimination between blood and bleach, and has the advantage that glycine is non-toxic compared to many other amines. The modified spray gave similar chemiluminescence intensity and duration as the unmodified Grodsky spray. However, it is recommended that this modification only be used when there is evidence that hypochlorite bleach may have been used at a scene. The amines triethylamine and sulfamate led to enhanced chemiluminescence in the presence of hypochlorite.