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DNA Evidence Uncompromised by Active Oxygen

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Currently, forensic sciences can make use of the potential of instrumental analysis techniques to obtain information from the smallest, even invisible, samples. However, as laboratory techniques improve, so too should the procedures applied in the search for and initial testing of clues in order to be equally effective. This requires continuous revision so that those procedures may resolve the problems that samples present. As far as bloodstains are concerned, there are methods available that are recognized as being both highly sensitive and effective. Nevertheless, the marketing of new cleaning products, those that contain active oxygen, has raised doubts about the ability of those procedures to detect blood. It has been shown that stains washed with these detergents (and still visible) invalidated both the presumptive test (reduced phenolphthalein, luminol, and Bluestar) and that applied for determining human hemoglobin. These findings have caused considerable concern both within the forensic and scientific community, and among the general public, so obliging us to seek solutions. In this work, the effect of these new cleaning products on DNA analyses is studied. The results, encouraging ones, show that these detergents, despite invalidating all other tests, do not hinder the extraction, or the subsequent analysis, of DNA.
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Research Article
TheScientificWorldJOURNAL (2010) 10, 387392
ISSN 1537-744X; DOI 10.1100/tsw.2010.47
*Corresponding author.
©2010 with author.
Published by TheScientificWorld; www.thescientificworld.com
387
DNA Evidence Uncompromised by Active
Oxygen
Ana Castelló, Francesc Francés, and Fernando Verdú*
Department of Legal and Forensic Medicine, Faculty of Medicine, University of
Valencia, Spain
E-mail: Ana.Castello@uv.es; Francesc.Frances@uv.es; Fernando.Verdu@uv.es
Received November 20, 2009; Revised February 24, 2010; Accepted February 26, 2010; Published March 5, 2010
Currently, forensic sciences can make use of the potential of instrumental analysis
techniques to obtain information from the smallest, even invisible, samples. However, as
laboratory techniques improve, so too should the procedures applied in the search for
and initial testing of clues in order to be equally effective. This requires continuous
revision so that those procedures may resolve the problems that samples present. As far
as bloodstains are concerned, there are methods available that are recognized as being
both highly sensitive and effective. Nevertheless, the marketing of new cleaning
products, those that contain active oxygen, has raised doubts about the ability of those
procedures to detect blood. It has been shown that stains washed with these detergents
(and still visible) invalidated both the presumptive test (reduced phenolphthalein,
luminol, and Bluestar®) and that applied for determining human hemoglobin. These
findings have caused considerable concern both within the forensic and scientific
community, and among the general public, so obliging us to seek solutions. In this work,
the effect of these new cleaning products on DNA analyses is studied. The results,
encouraging ones, show that these detergents, despite invalidating all other tests, do not
hinder the extraction, or the subsequent analysis, of DNA.
KEYWORDS: forensic sciences, bloodstains investigation, presumptive test, hemoglobin test,
forensic genetics
INTRODUCTION
Impressive advances in instrumental analysis methods allow the smallest and even invisible samples to be
analyzed. This fact, favorable in any field of science, is even more so in the field of criminal
investigation, where it is commonplace to work with very little and often contaminated evidence.
However, to make use of the potential that laboratory analysis provides, it is essential to have the
sufficiently sensitive means available for searching for clues and evidence so as to obtain the initial basic
information on the sample type being worked on. These means are called the presumptive and
confirmatory tests, respectively.
With regard to bloodstains, effective search and presumptive procedures[1,2] have been available for
many years. Those using reduced phenolphthalein, luminol, and Bluestar®[3] are currently the most
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widely employed. These have been thoroughly studied and the possibilities of obtaining false-negative
results are known[4,5,6,7,8,9].
Recently, however, a new problem has arisen: cleaning products that contain active oxygen have
appeared on the market. These are detergents that do not contain hypochlorites, but are made with sodium
percarbonate[10,11]. Tests undertaken on bloodstains washed with these products have provided
worrying results (Castelló et al. 2009). After washing, although the stain remains visible, all the
presumptive tests as well as the human hemoglobin test have given a negative result, i.e., apparently the
stain is not a blood stain.
The article announcing these results caused deep concern among the scientific community. Headlines
such as “Why hair bleach is a murderer’s best friend”[12] or “New detergent washes away stains of
murder: study”[13], express the concern felt among forensic scientists and underlines the need to find a
solution to this problem.
This work attempted to extract and analyze DNA from stains washed with active oxygen that had
given negative results in the presumptive and human hemoglobin tests. The aim was to determine whether
these cleaning products are also capable of hindering genetic analysis. For this purpose, the following
work plan was employed.
MATERIALS AND METHODS
Material
A piece of cotton cloth was used as backing for bloodstains.
Reagents
For the presumptive test, Phenolphthalein Dischaps™ (Sirchie, http://sirchie.com/SearchResult.aspx),
luminol (3-aminophthalhydrazide) (Merck), sodium perborate (Panreac), sodium carbonate (Panreac),
Bluestar, and distilled water were used.
The hemoglobin test was made using the Hexagon OBTI® test[14].
Finally, for DNA analysis, the necessary reagents were the following: Tris hydroxymethyl
aminomethane (Panreac), hydrochloric acid (Panreac), ethylenediaminetetraacetic acid (Panreac), DL-
dithiothreitol solution (Fluka), sodium chloride (Panreac), sodium dodecyl sulfate (Panreac), proteinase K
(Sigma-Aldrich), phenolchloroformisoamyl alcohol (Sigma), agarose MS-8 (Promadisa), and 50xTAE
(National Diagnostics).
Sample Preparation[15]
Bloodstains were prepared using newly extracted blood samples without preservatives and the stains were
made on the backing with five drops of blood. Having made the stains, they were left to dry at room
temperature without any protection whatsoever. Drying time was initially set at 1 day. Subsequently, at
different times (1, 5, 10, 20, and 30 days), the following procedure was undertaken:
Four stains were cut out of the backing using a sterile scalpel. One of these was kept in the
laboratory for subsequent use as a control. Another was washed using a product containing active
oxygen (Neutrex™) whose composition, as appears on the label, is as follows: nonionic and
anionic tensoactive agents, polycarboxylates, zeolites (concentration less than 5%), and sodium
percarbonate (in a proportion greater than 30%). The pH of the product dissolved in water is 10.
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When washing, the manufacturer’s instructions were followed: the fabric was left in hot water (at
40ºC) containing the cleaning product for 2 h. Then it was rinsed in running water and left to dry
over 24 h.
The two remaining stains were used for control assays. One of them was washed in hot water (at
40ºC) without using detergent in order to determine the possible influence of water temperature
on the results of the tests. The other was washed 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.
To confirm the results, the entire process was repeated five times for each of the periods (1, 5, 10, 20,
and 30 days) using different samples.
Presumptive Test and Human Hemoglobin Test
The presumptive tests (reduced phenolphthalein, luminol, and Bluestar) and the human hemoglobin test
were undertaken on the dry stains, following the procedures described in the bibliography[15]. Having
checked that all the results were negative, the stains were then processed for DNA extraction.
DNA Extraction
A square of approximately 3 3 mm was cut from the stains using a sterile scalpel and inserted into
appropriately marked microtubes, and 300 µl of extraction buffer (10 mM TrisHCl, pH 8, 10 mM
EDTA, pH 8, 2% SDS, 100 mM NaCl), 12 µl of 39 mM DTT, and 7.5 µl of proteinase K (10
mg/ml)[16,17] were added.
The tubes were incubated at 56ºC for 24 h. After incubation, DNA was extracted employing the
phenolchloroform method. The process was as follows:
First, 300 µl of phenolchloroform was added to each sample, and after agitating, was centrifuged
at 13,000 rpm for 3 min. Then the liquid phase was transferred to a Microcon 100 filter
(www.millipore.com ref 42413), where 100 µl of distilled water (filter buffer) had previously
been added, and centrifuged at 13,000 rpm for 20 min.
After removing the liquid, leaving only the filter in the tube, 200 µl of sterile distilled water were
added that is the washing process and once again the sample was centrifuged at 13,000 rpm
for 20 min.
To recover the DNA, it was resuspended with 100 µl of distilled water and, as the last phase
before inserting the filter downwards in new tubes, it was centrifuged at 13,000 rpm for 5 min.
DNA Quantification
Before proceeding to amplification, checks were undertaken to determine whether DNA had been
extracted, and in what quantity and quality.
For this purpose, the extraction product was submitted to two quantification processes. At the first, a
horizontal electrophoresis in agarose gel of low electroendosmosis was used, at 0.8%. For each sample,
1:1, 1:5, and 1:25 dilutions were prepared. In addition, a control of 10 ng/pl concentration was added.
The results of the electrophoresis were observed under an ultraviolet lamp after having stained them
with ethidium bromide.
A second quantification was made using pectrophotometry. For this, a NanoPhotometer (IMPLEN
GmBH) was selected.
Castelló et al.: DNA Evidence Uncompromised by Active Oxygen
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PCR Amplification
Multiplex PCR with the AmpflSTR Profiler PCR amplification kit (Applied Biosystems) was performed
at a final volume of 10 µl, composed of 6.6 µl of master mix (mixed 4.2 µl of PCR mix, 2.2 µl of primer
set, and 0.2 µl of AmpliTaq Gold polymerase per tube) and 0.7 ng of sample DNA on a GeneAmp PCR
System 9700 thermal cycler (Applied Biosystems), according to the manual.
Electrophoresis
Amplified PCR products were run on an ABI PRISM 310 Genetic Analyzer and analyzed using
GeneScan analysis software version 3.1 and Genotyper software version 2.5. Alleles were designated by
comparison with the allelic ladder marker contained in the kit, according to the Amp-FlSTR User’s
Manual.
In order to confirm the results, the entire process was repeated three times for each of the periods (1,
5, 10, 20, and 30 days) using different samples.
RESULTS
Presumptive Test and Human Hemoglobin Test
The presumptive test and human hemoglobin test were negative for the stains analyzed.
DNA Analysis
We were able to extract DNA from all the samples analyzed, and of a sufficient quantity and quality to
obtain a genetic profile, coinciding with that of the control samples.
Fig. 1 shows the presence of the DNA extracted from the problem samples. From the image, a certain
degradation can be observed that is due to the stains being treated with active oxygen, which is not
observed in the controls. However, this effect has not hindered their subsequent amplification.
FIGURE 1.
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The results on the quantity of DNA extracted from NanoPhotometer were coincident with those from
the electrophoresis in gel. Nevertheless, it is not possible, through spectrophotometry, to observe the
degradation of the DNA that is observable in the in-gel development. Table 1 shows the average
quantities of DNA obtained from problem and control samples in each period (1, 5, 10, 20, and 30 days).
TABLE 1
Average Quantities (Concentration) of DNA Obtained from Problem and Control
Samples in Each Period
Time
(Days)
DNA Concentrations (ng/µl)
Control Sample
Control Assay 1
Control Assay 2
Problem
1
41
35
10
7
5
37
33
12
5
10
40
34
11
6
20
36
30
11
7
30
35
36
10
7
Control sample: Bloodstains not washed.
Control assay 1: Bloodstains washed in hot water (at 40ºC) without using detergent.
Control assay 2: Bloodstains washed with active oxygen and cold water.
Problem: Bloodstains washed with active oxygen and hot water.
DISCUSSION
The results reaffirm the deleterious effect of new detergents on the blood search, presumptive, and
confirmation procedures. However, although these initial tests are invalidated, DNA analysis is not
impeded, as it remains positive in all the cases studied.
Consequently, faced with a suspicious stain, one has to think of the possibility that it has been washed
with an active oxygen product. Then, even though everything indicates that it is not blood, it would be
recommendable to try a DNA analysis.
Obviously invisible or latent stains cannot be identified when these products have been used. The
evidence will be lost and this poses a problem that is yet to be solved. Perhaps in the future it will be.
In conclusion: active oxygen cannot doctor the evidence, at least, not completely.
ACKNOWLEDGMENTS
We thank the staff of the Laboratory of Forensic Identification (University of Granada, Spain) and to the
Genetic and Molecular Epidemiology Unit (University of Valencia, Spain) for their invaluable
collaboration.
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This article should be cited as follows:
Castelló, A., Francés, F., and Verdú, F. (2010) DNA evidence uncompromised by active oxygen. TheScientificWorldJOURNAL
10, 387392. DOI 10.1100/tsw.2010.47.
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Chapter
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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A wide range of domestic and industrial substances that might be mistaken for haemoglobin in the forensic luminol test for blood were examined. The substances studied were in the categories of vegetable or fruit pulps and juices; domestic and commercial oils; cleaning agents; an insecticide; and various glues, paints and varnishes. A significant number of substances in each category gave luminescence intensities that were comparable with the intensities of undiluted haemoglobin, when sprayed with the standard forensic solution containing aqueous alkaline luminol and sodium perborate. In these cases the substance could be easily mistaken for blood when the luminol test is used, but in the remaining cases the luminescence intensity was so weak that it is unlikely that a false-positive test would be obtained. In a few cases the brightly emitting substance could be distinguished from blood by a small but detectable shift of the peak emission wavelength. The results indicated that particular care should be taken to avoid interferences when a crime scene is contaminated with parsnip, turnip or horseradish, and when surfaces coated with enamel paint are involved. To a lesser extent, some care should be taken when surfaces covered with terracotta or ceramic tiles, polyurethane varnishes or jute and sisal matting are involved.
Critical review of presumptive tests for blood stains Available http://www.fbi.gov/hq/lab/fsc/backissu/july1999/ponce
  • A Castelló
  • F A Verdú
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