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Evaluation of Six Presumptive Tests for Blood, Their Specificity, Sensitivity, and Effect on High Molecular-Weight DNA

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Luminol, leuchomalachite green, phenolphthalein, Hemastix, Hemident, and Bluestar are all used as presumptive tests for blood. In this study, the tests were subjected to dilute blood (from 1:10,000 to 1:10,000,000), many common household substance, and chemicals. Samples were tested for DNA to determine whether the presumptive tests damaged or destroyed DNA. The DNA loci tested were D2S1338 and D19S433. Leuchomalachite green had a sensitivity of 1:10,000, while the remaining tests were able to detect blood to a dilution of 1:100,000. Substances tested include saliva, semen, potato, tomato, tomato sauce, tomato sauce with meat, red onion, red kidney bean, horseradish, 0.1 M ascorbic acid, 5% bleach, 10% cupric sulfate, 10% ferric sulfate, and 10% nickel chloride. Of all the substances tested, not one of the household items reacted with every test; however, the chemicals did. DNA was recovered and amplified from luminol, phenolphthalein, Hemastix, and Bluestar, but not from leuchomalachite green or Hemident.
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TECHNICAL NOTE
Shanan S. Tobe,
1
M.Sc.; Nigel Watson,
1
Ph.D.; and Niamh Nic Dae
´id,
1
Ph.D.
Evaluation of Six Presumptive Tests for Blood,
Their Specificity, Sensitivity, and Effect on High
Molecular-Weight DNA
ABSTRACT: Luminol, leuchomalachite green, phenolphthalein, Hemastix
s
, Hemident
TM
, and Bluestar
r
are all used as presumptive tests for
blood. In this study, the tests were subjected to dilute blood (from 1:10,000 to 1:10,000,000), many common household substance, and chemicals.
Samples were tested for DNA to determine whether the presumptive tests damaged or destroyed DNA. The DNA loci tested were D2S1338 and
D19S433. Leuchomalachite green had a sensitivity of 1:10,000, while the remaining tests were able to detect blood to a dilution of 1:100,000.
Substances tested include saliva, semen, potato, tomato, tomato sauce, tomato sauce with meat, red onion, red kidney bean, horseradish, 0.1 M
ascorbic acid, 5% bleach, 10% cupric sulfate, 10% ferric sulfate, and 10% nickel chloride. Of all the substances tested, not one of the household
items reacted with every test; however, the chemicals did. DNA was recovered and amplified from luminol, phenolphthalein, Hemastix
s
, and
Bluestarr, but not from leuchomalachite green or Hemident
TM
.
KEYWORDS: forensic science, luminol, leuchomalachite green, phenolphthalein, Hemastix
s
, Hemident
TM
, Bluestar
r
, presumptive tests,
sensitivity, specificity, DNA recovery
Blood is the most common and perhaps the most important
form of evidence in the world of criminal justice today (1). Blood
evidence associated with a crime can provide essential informa-
tion that may help solve a case, collaborate witness testimony,
define a scene of crime, link a suspect and scene, or simply point
the investigation in a new direction (1,2). Therefore, it is import-
ant to identify any stain that could potentially be blood at a crime
scene. Obvious bloodstains should never be contaminated with
any reagent (3). When encountered with a potential bloodstain
that cannot be identified immediately, several questions enter the
mind of an investigator or forensic scientist. These include ‘‘What
is that stain?’’; ‘‘Could it be blood?’’; or if a stain is expected or
suspected, and is absent, ‘‘Was there blood here at one time?’’ Cox
(4) describes the attributes that a good presumptive test for blood
should have: it should be sensitive, specific, quick, simple, and
safe.
More recently, these questions have expanded to include
‘Whose blood is this?’’; ‘‘Can it be excluded from a control or
known sample?’’; and ‘‘Is there enough genetic material here to
obtain a complete DNA profile?’’ It is therefore obvious that
within a forensic context, the most important components of blood
are those that can be used for blood identification and to indi-
vidualize it (5).
In order for these presumptive tests for blood to function prop-
erly, they must detect a component of blood, which ideally should
not be commonly found in the everyday environment. Therefore,
most presumptive tests for blood rely on the peroxidase-like ac-
tivity of hemoglobin. This is a component of blood that is not
commonly found in the everyday environment, although there are
other substances found in fruits and vegetables that perform a
similar function.
In the past half-century, several studies have been conducted on
the sensitivity and specificity of presumptive tests for blood, and
their effect on subsequent DNA analysis.
In the past 50 years, there have been many tests conducted on
the sensitivity of presumptive blood tests (4,6–15). The findings of
these studies are in great contradiction with each other. Sensitiv-
ities for luminol range from 1:200 (11) to 1:100,000,000 (6); from
1:200 (11) to 1:100,000 for leuchomalachite green (LMG) (8); and
from 1:2,000 (12,13) to 1:10,000,000 for phenolphthalein (9). The
various differences in the sensitivities reported by different re-
searchers of presumptive blood tests are probably caused by dif-
ferences in reagent concentrations, methods of preparation of
samples, reagents and results, and in the type of material contain-
ing the blood (4). Grodsky et al. (8) also add that dried bloodstains
are not comparable with the same amount of blood dissolved in a
solution. They further add that many of the discrepancies observed
are probably due to the presumptive test reagents being added
directly to a dilute blood solution, thereby also diluting the re-
agent, while in other cases the dilute blood solutions are dried first
and then tested with full-strength reagents (8).
In the past half century, many tests have been conducted on the
specificity of presumptive blood tests. These tests for specificity
include changing substrates, adding material and chemicals to the
bloodstains, and testing to see whether the reagents will react with
substances other than blood (4,7,11,13,14,16–21). Grodsky et al.
(8) believe that studies involving the various presumptive blood
tests indicate that there is a degree of interference with some of
them that effectively prevents their effective use as a test for the
1
Department of Pure and Applied Chemistry, Centre for Forensic Science,
Strathclyde University, 204 George Street, Glasgow G1 1XW, U.K.
Received 5 Feb. 2006; and in revised form 12 July 2006 and 2 Aug. 2006;
accepted 1 Sept. 2006; published 8 Dec. 2006.
Copyright r2006 by American Academy of Forensic Sciences
102
J Forensic Sci, January 2007, Vol. 52, No. 1
doi:10.1111/j.1556-4029.2006.00324.x
Available online at: www.blackwell-synergy.com
presumptive identification of blood. Therefore, this must be ad-
dressed and examined with experimentation.
The ideal presumptive blood test is one that is specific to blood
(more specifically to human blood), has a high sensitivity, will
meet the Frye standard, and will not damage underlying DNA so
that a full DNA profile can be obtained after the reagent’s use
(5,22). New reagents will be tested with the ones most commonly
used by police and forensic scientists throughout the world: Kas-
tle–Meyer (KM), leuchomalachite green, and luminol (23,24).
The ease of transport, ease of use, working life, and storage will be
determined and discussed for the three new reagents.
Current literature reports differing sensitivities for the various
blood detection tests, often conflicting in their results. Therefore,
the sensitivity limits of the reagents will be tested and the limits
will be determined.
The specificity of the new reagents will be tested with sub-
stances commonly known to interfere with traditional reagents, or
those that could be mistaken for blood spatter in some situations.
DNA will be collected and PCR performed to determine
whether the reagents have limits less than, equal to, or exceeding
that of current DNA detection techniques.
Materials and Methods
Samples
Blood samples were taken from an anonymous donor. All
equipment used to extract, store, apply, and manipulate the blood
for the experiments was sterile. The equipment was either open
from sterile packaging or autoclaved at 1201C for 20 min.
Blood from the donor was used for all experiments and for
positive controls. The blood was extracted by creating a small
lancet wound in the finger of the donor and was not subject to any
form of anticoagulants or other contaminants.
Reagents
Luminol (3-aminophthalhyrazidem), LMG, and phenolphtha-
lein KM were prepared according to Strathclyde University,
Centre For Forensic Science guidelines. Hemastix
s
(instructions
included with reagent), Hemident
TM
(MacPhails
TM
Reagent;
instructions included with reagent), and Bluestar
r
(instructions
included with reagent) were from commercially available kits
provided by WA Products (Essex, U.K.) (product codes: B23014,
B23013, B23014). All reagents were used according to the manu-
facturer’s guidelines.
Positive controls were taken by applying the reagent to a blood-
stained piece of filter paper. Negative controls were performed
by applying the reagents to a fresh piece of filter paper with no
trace of blood. The positive control was retained for further DNA
testing.
Sensitivity Testing
Autoclaved bottles (1251C for 20 min) and distilled H
2
O were
used. Water was measured using a graduated cylinder and blood
was added using a Gilson pipette. Differing low concentrations of
blood were achieved by making a stock solution of blood and
distilled water. Solutions of 1:10,000; 1:100,000; 1:1,000,000;
1:5,000,000; and 1:10,000,000 were prepared.
A set of 25 1 cm 1 cm pieces of filter paper were placed in
each of the diluted blood solutions for each of the presumptive
reagents tested. The pieces of filter paper were then removed and
allowed to dry for 72 h. Each of the pieces of filter paper was then
tested with its corresponding reagent to see whether the blood
present was detectable. The reagents were added directly to the
1cm
2
pieces of filter paper. The time taken for the reagent to
register a positive result was determined and recorded. Tests were
considered negative if reagents failed to react within 4 min of ex-
posure to the blood-stained filter paper. The treated pieces of filter
paper that had not reacted with any reagents were retained for
subsequent DNA analysis.
Specificity Testing
Substances found to give false positives previously as reported
by other authors, or substances which could be mistaken for
blood, were tested. The tests were also carried out on saliva and
semen.
The six different reagents were tested against saliva, semen,
potato, tomato, tomato sauce, tomato sauce with meat, red onion,
red kidney bean, horseradish, 0.1 M ascorbic acid, 5% bleach,
10% cupric sulfate, 10% ferric sulfate, and 10% nickel chloride.
For each of the presumptive reagents tested, a large piece of
filter paper (approximately 100 cm
2
) was exposed to each of the
substances being tested in 25 separate sample stains. These were
allowed to dry for a minimum of 18 h. Each of the pieces of filter
paper, and subsequent stains, were then tested with their corre-
sponding reagent to see whether the substance caused a reaction.
The time taken for the reagent to register a positive result was
determined and recorded. Tests were considered positive if there
was any color change, and were considered negative if there was
no observable color change within 4 min of exposure to the
stained filter paper.
DNA Testing
The Chelex method of DNA purification and recovery was
used. The protocol consisted of sterile distilled water, 20% Chelex
suspension, and extraction buffer. For each sample, 0.5 mL Sterile
H
2
O was pipetted to a 0.5 mL Eppindorf tube. A small 3 mm
2
section of the positive control (which had been exposed to the re-
agents) was added to the tube. For the sensitivity testing, the entire
1cm 1 cm section of the filter paper was added to the tube. For
the controls, a 3 mm
2
section of bloodstain was placed in the tube.
All the samples were incubated at room temperature for 25 min
with occasional inverting. They were then centrifuged at maxi-
mum for 2 min. Each tube had 0.35 mL of the supernatant re-
moved and then the pellet was resuspended. Fifty microliters of
20% Chelex was added to each tube and they were then incubated
at 561C for 30 min. Samples were then vortexed for 10 sec, boiled
for 10 min, and then vortexed for a further 10 sec. Samples were
then centrifuged at maximum for 2 min. The supernatant was re-
moved and retained in a separate Eppendorf tube and the pellet
was discarded. The retained supernatant was stored frozen.
A full commercial DNA profiling kit will not be used as the
amount of information that a full 10 or 14 loci (using SGMPlus
TM
or IdentifilerPlus
TM
, Applied Biosystems, Foster City, CA) profile
would provide is not needed in this study. Instead, two STR loci
from a well-used commercial kit, SGMPlus
TM
, will be amplified.
The STR loci to be used are D19S433 and D2S1338, the smallest
and largest loci, respectively (25). This will allow for both ends of
the spectrum to be amplified, as larger products are more likely to
drop out in degraded DNA than smaller loci are. Therefore, if only
D19S433 amplifies and D2S1338 drops out, it would mean that
partial amplification could likely be obtained from a commercial
kit. If both D19S433 and D2S1338 amplify, then this should
TOBE ET AL. .AN EVALUATION OF SIX PRESUMPTIVE TESTS FOR BLOOD 103
indicate that a commercial STR typing kit would be able to obtain
a full profile off the samples.
As the exact primer sequence used by Applied Biosystems is
not known, different primers were used. The primer sequences
were obtained from UniSTS (26), which is a comprehensive da-
tabase of sequence-tagged sites (STSs) defined by PCR primer
pairs and are associated with additional information such as ge-
nomic position, genes, and sequences (26). The primer informa-
tion for the two loci was given as:
D19S433
Forward: 50-HEX-CCTGGGCAACAGAATAAGAT-30
Reverse: 50-TAGGTTTTTAAGGAACAGGTGG-30
D2S1338
Forward: 50-HEX-CCAGTGGATTTGGAAACAGA-30
Reverse: 50-ACCTAGCATGGTACCTGCAG-30.
Primer sets for D19S433 and D2S1338 were each run in a sep-
arate PCR of 25 mL total; 2 mL of each primer was used and 5 mL
of template DNA. PCR was performed on a Perkin Elmer Gene-
Amp PCR System 2400 (Boston, MA). Thirty two cycles of 941C
for 30 sec; 551C for 30 sec; 721C for 1 min and 30 sec; and a final
extension of 45 min at 721C were performed.
Eleven experimental samples were run. Six correspond to all of
the positive controls, 3 were from the dilution sets 1:10,000,
1:100,000, and 1:1,000,000, a positive control from the blood
donor, and a negative control.
An ABI PRISM
s
310 Genetic Analyzer (Applied Biosystems)
was used to analyze all samples.
Statistical Tests
The test used to compare the different reagents was the w
2
test
for consistency in a 2 Ktable.
Results and Discussion
The positive control of luminol reacted instantly with blood,
giving a blue luminescence appearing at the site of the deposition;
this persisted for over 1 min. The negative control did not react on
addition of the reagent. Grodsky et al. (8) believe that luminol’s
only serious disadvantage, other than interference, is its require-
ment of near or complete darkness in order to perceive the chem-
iluminescence.
The LMG-positive control reacted within a few seconds of ap-
plication of the H
2
O
2
, with a blue/green color appearing at the site
of blood deposition. The negative control did not react on addition
of the H
2
O
2
but if left out will develop a green ring around where
reagents were deposited.
The phenolphthalein KM positive control reacted within a few
seconds of application of the H
2
O
2
, with a pink color appearing at
the site of blood deposition. The negative control did not react on
addition of the H
2
O
2
; however, there was a reaction after several
minutes (greater than the 4 min timed) with a pink color devel-
oping around the edges of the area of reagent deposition.
The Hemastix
s
reagent strips-positive control reacted instantly
on application to the blood by turning dark gray/green; the site
where the reagent strip touched the filter paper also turned dark
green/blue where there was blood. The negative control did not
react on addition of the H
2
O and there was no reaction on the filter
paper; however, there was a reaction after several minutes (greater
than the 4 min timed), with the reagent pad turning light green.
The ease of transport and use of the Hemastix
s
reagent strips is
excellent. The strips are easily stored and transported and there is
no risk of chemical spills or solution breakdown or contamination,
all that is required is some distilled water (tap water would most
likely also be fine) and a desiccant (provided with the strips) with
the reagent strips. They are easy to use, and easy to transport.
There is a range of color reactions to compare with on the con-
tainer, for accurate reading of the strips. According to the manu-
facturer, storage is provided in the container and has a life of 6
months from initial opening, and about a year if unopened.
The Hemident
TM
(MacPhail’s reagent)-positive control reacted
within a few seconds of application of the H
2
O
2
, with a dark green/
blue color appearing at the site of blood deposition. The negative
control did not react on addition of the H
2
O
2
but if left out will
develop a green ring around where reagents were deposited.
If used according to the manufacturer’s guidelines, the Hemi-
dent
TM
test is easy to transport and use. The ampoules are not
easily broken, and the case provides a convenient disposal vessel.
There are no instructions for any special storage conditions, or any
expiry date indicated.
The Bluestar
r
-positive control reacted instantly, with a blue
luminescence appearing at the site of blood deposition; this dis-
sipated within 30 sec. The negative control did not react on addi-
tion of the reagent.
The reagent was easy to prepare from the two tablets, which
were mixed directly together into a spray bottle with water (tap
water can be used). The tablets can be brought to a scene separ-
ately, so there is no risk of leaking bottles of reagents. Bluestar
r
was extremely easy to use and was effective by just spraying over
an entire area for full coverage. The working life of the solution is
a problem as it is quite short once the solution is mixed and may
only be reactive for a few hours. The tablets come in two separate
foil-wrapped packages, but a warning is given that the product is
stored under pressure and it should not be stored in the home or
car without proper precautions according to the manufacturer.
There is no expiry date given with the tablets, indicating that they
are stable if stored with the proper precautions. The only problem,
much like luminol, is that the product requires complete or near-
complete darkness to be effective.
Sensitivity
The approximate numbers of erythrocytes, leukocytes, and
hemoglobin molecules, as given by Marieb (27), were calculated
for each of the five dilution factors and are shown in Table 1.
Table 2 illustrates the results obtained for the sensitivity portion of
the experiment.
The luminol reagent reacted instantly, with both the 1:10,000
and 1:100,000 dilution factors producing a blue luminescence.
The luminescence lasted for close to a minute. However, both di-
lution factors were much less intense than the positive control of
whole blood. The reaction with the 1:100,000 dilution factor was
extremely faint. There was no reaction with dilutions of
1:1,000,000, 1:5,000,000, or 1:10,000,000 within the 4 min of
timed experimentation.
LMG reacted at a dilution factor of 1:10,000. All samples ex-
cept one showed a positive reaction within 1 min. The samples
turned a green color within 1 sec of the application of the LMG
reagent and the H
2
O
2
. A single sample did not show a positive
reaction within the 4 min of timed experimentation. The LMG
reagent did not show a positive reaction at dilution factors of
1:100,000, 1:1,000,000, 1:5,000,000, or 1:10,000,000 within the
4 min of timed experimentation.
104 JOURNAL OF FORENSIC SCIENCES
The phenolphthalein reagent registered a positive reaction for
all samples at a dilution factor of 1:10,000. The samples turned
pink after 45 sec of the introduction of the reagent and H
2
O
2
.Ata
dilution factor of 1:100,000, three of 25 samples showed a positive
reaction: two of them at 1 min and 30 sec, and the third at 2 min
and 30 sec. The phenolphthalein reagent did not show any reaction
with dilution factors of 1:1,000,000, 1:5,000,000, or 1:10,000,000
within the 4 min of timed experimentation.
The Hemastix
s
reagent strips reacted with the 1:10,000 dilu-
tion by first causing a color reaction with the filter paper. The filter
paper changed to a green color where the Hemastix
s
was pressed
within a few seconds. The actual Hemastix
s
took between 30 and
60 sec to register a reaction. Eighteen Hemastix
s
were positive
for 125 erythrocytes; the remaining seven registered positive for
180 erythrocytes.
The Hemastix
s
reagent strips reacted with the 1:100,000 dilu-
tion by first causing a color reaction with the filter paper. At 1 min,
one of the samples showed a color change on the filter paper of a
green color. The rest of the samples showed this same reaction at
between 1 min and 45 sec and 2 min. At 3 min and 45 sec, 17 of the
reagent strips were a very light shade of green, corresponding with
a trace 10 hemolyzed sample according to the instructions. Four of
the strips registered 125 erythrocytes at the same time. The re-
maining four strips registered a negative result at 4 min.
The Hemastix
s
reagent strips did not react with the
1:1,000,000, 1:5,000,000, and 1:10,000,000 dilutions. There was
no color change on the filter paper or on the reagent strips.
There were no previous literature values for the sensitivity of
Hemastix
s
although the package claims to be able to detect blood in
urine down to 10 erythrocytes, which equates to between a
1:100,000 and 1:1,000,000 dilution factor (Table 1). This is con-
sistent with the results obtained in this experiment, although the
strips should be read at 60 sec and a reaction was not observed on the
strips until between 3 and 4min after initial application to the stain.
The Hemident
TM
reagent reacted with most of the samples at
the 1:10,000 dilution. One sample reacted before the addition of
the H
2
O
2
with a color change to green; two other samples did not
register a reaction. The samples that did react showed a green/blue
color change at the edges of the filter paper, predominantly in the
corners, which occurred within 1 min of addition of the H
2
O
2
.
The Hemident
TM
reagent did not react with most of the samples
at the 1:100,000 dilution. Two of the samples showed a positive
result at 4min. The remainder did not show any reaction. There was
no reaction with the samples diluted to 1:1,000,000, 1:5,000,000, or
1:10,000,000 within the 4min of timed experimentation.
There were no previous literature values for the sensitivity of
Hemident
TM
, but the package claims a capability of identifying
one part per million of blood (28). The findings of this study do
not confirm this. Hemident
TM
is slightly more sensitive than leu-
chomalachite green, but does not even approach the sensitivity it
claims to have. Two samples showed a positive reaction at
1:100,000 dilution, which is 10 times more sensitive than leucho-
malachite green, but this was not consistent over all 25 samples,
and it is still one-tenth of the sensitivity claimed.
The Bluestar
r
reagent reacted instantly with the 1:10,000 with
a blue luminescent glow but faded within a few seconds. The
1:100,000 dilution showed slight reactivity, with five of the 25
samples showing a very faint positive, which faded in a few sec-
onds. However, both dilution factors were much less intense than
the positive control of whole blood. There was no reaction with
dilutions of 1:1,000,000, 1:5,000,000, or 1:10,000,000 within the
4 min of timed experimentation.
The Bluestar
r
reagent has no previous tested sensitivities, al-
though the company claims sensitivity to 1:1,000 dilution (29).
This was not found to be consistent with this study as lumines-
cence was detected at 1:100,000 dilution of blood in water, 100
times more sensitive than what is claimed by the company. This
luminescence was faint and short-lived, but was still detectable.
Specificity
Table 3 gives the specificity results for all reagents.
TABLE 1—Distribution of blood cells for the different dilution factors, calculated from the values given in Marieb (27).
Blood 1 mL 51mm
3
Erythrocytes Hemoglobin Leukocytes
Minimum Maximum Minimum Maximum Minimum Maximum
1:l 4,300,000 5,200,000 1.075E115 1.3E115 4000 11,000
10:l 43,000,000 52,000,000 1.075E116 1.3E116 40,000 110,000
1:10,000 430 520 1.075E111 1.3E111 0.4 1.1
1:100,000 43 52 10,750,000,000 13,000,000,000 0.04 0.11
1:1,000,000 4.3 5.2 1,075,000,000 1,300,000,000 0.004 0.011
1:5,000,000 0.86 1.04 215,000,000 260,000,000 0.0008 0.0022
1:10,000,000 0.43 0.52 107,500,000 130,000,000 0.0004 0.0011
TABLE 2—Sensitivity results for the six different reagents.
Dilution
Reagent
Luminol LMG KM Hemastix
s
Hemident
TM
Bluestar
r
1:10,000 1 1 1 1 1 1
1:100,000 1 NR 2 2 4 1
1:1,000,000 NR NR NR NR NR NR
1:5,000,000 NR NR NR NR NR NR
1:10,000,000 NR NR NR NR NR NR
The shortest reaction time is shown here.
A positive reaction was any sort of color change to the stain (or reagent strip in the case of Hemastix
s
); 0, color change before all reagents were added; 1, color
change within 1 min of all reagents being added; 2, color change within 1–2 min of all reagents being added; 3, color change within 2–3 min of all reagents being
added; 4, color change within 3–4 min of all reagents being added;
NR, indicates that there was no reaction within the 4 min of timed experimentation; KM, Kastle–Meyer; LMG, leuchomalachite green.
TOBE ET AL. .AN EVALUATION OF SIX PRESUMPTIVE TESTS FOR BLOOD 105
There was a reaction between the luminol reagent and 10%
cupric sulfate, 10% ferric sulfate, and 10% nickel chloride indi-
cated by a blue chemiluminescence. Both the 10% cupric sulfate
and 10% ferric sulfate showed an immediate reaction on addition
of the luminol. The 10% nickel chloride also showed a reaction on
addition of the luminol; however, the intensity of this reaction was
far less than for the ferric and cupric sulfates and it did not occur
instantly. The reaction took several seconds to become visible.
Luminol’s reaction with the metal salts was expected as Grod-
sky et al. (8) noted that the common substances that interfere with
luminol are copper-containing surfaces.
Contrary to the literature findings, this study found that luminol
only reacted with blood and the metal salts. Bleach gave no re-
action, but this could be because the bleach solution was only 5%
concentration, and that it was not tested right away but first al-
lowed to dry for at least 18 h. Kent et al. (20) noted that when
bleach-treated blood is left for several days, the interference by
bleach is diminished. The negative reaction observed may be due
to the storage time of the sample. Luminol was expected to react
with the potato and horseradish as it has been used to study vege-
table peroxidase reactions, such as the horseradish peroxidase re-
action (30), and Albrecht noted that fresh potato juice caused
luminescence (16). This could once again be due to the sub-
stances’ drying time before testing.
LMG showed a reaction with several of the substances tested.
However, the results of these reactions would not be mistaken for a
reaction with blood. All of the substances that reacted did so after
the addition of the LMG reagent but before the addition of the H
2
O
2
.
This agrees with the findings of Alvarez de Toledo and Valero, who
noted that many chemical oxidants may yield the reaction in the
absence of H
2
O
2
(31). Blood only reacts after the addition of the
hydrogen peroxide and then only at the site of blood deposition.
Therefore, none of the substances tested react in the same manner as
blood and could not be mistaken for a reaction with blood.
Several substances reacted with the phenolphthalein reagent.
Semen stains showed a very light pink color change at 45 sec,
which grew stronger as the timing approached 2 min. Seven potato
samples showed a slight pink color change within 2 min of intro-
duction of the reagents. Six tomato sauce samples showed a pink
color at 3 min and 45 sec. Red onion samples turned yellow after
the addition of the KM reagent, but before the addition of H
2
O
2
.
The horseradish samples all showed a very slight pink color
change at 3 min. The 0.1 M ascorbic acid samples turned yellow
after the addition of the phenolphthalein reagent, but did not show
any further color change on addition of the H
2
O
2
. Two of the 5%
bleach samples showed a slight pink color at 2 min, but the rest did
not show a color change within the 4 min of timed experimenta-
tion. On addition of the phenolphthalein reagent, the 10% cupric
sulfate samples turned blue. On addition of the H
2
O
2
, the samples
instantly turned brown and foamed. All 25 of the 10% cupric sul-
fate samples then developed an intense pink color around the
stain, 11 within 1 min and the remaining 14 within 2 min and
30 sec. The 10% ferric sulfate samples turned yellow/brown on
addition of the phenolphthalein reagent and the stains had dark
edges. The samples foamed on addition of H
2
O
2
. At 1 min and
30 sec, 13 of the samples showed a pink color developing around
the stain, and two additional samples showed this same color
change at 3 min. The remaining samples did not develop any color
around the stains in the 4 min of timed experimentation. The 10%
nickel chloride turned light green on addition of the phenolphtha-
lein reagent. This color deepened on addition of the H
2
O
2
. Eight
of the samples developed a pink color around the stain at 3 min.
The rest of the samples failed to react within the 4 min of timed
experimentation.
The reaction of the phenolphthalein reagent with other sub-
stances differs from Pinker (32), who did not find even one sub-
stance that would give a true positive reaction with
phenolphthalein. This does not correspond with the current find-
ings as semen caused a reaction at 45 sec, that grew stronger with
time. The potato stains reacted the same way, as did tomato sauce,
red kidney beans, horseradish, and 5% bleach, which all reacted at
some point within the 4 min of timed experimentation. This time
delay in reaction is much like the time delay observed for dilute
blood samples (1:100,000) and therefore, any of these stains could
be conceivably mistaken for very dilute blood.
The remaining substances reacted before all the reagents were
added, or did not form the pink color as an expected bloodstain
would. Both the red onion and the 0.1 M ascorbic acid samples
turned yellow on addition of the phenolphthalein reagents but be-
fore the H
2
O
2
was added. The metal salts also all reacted before
TABLE 3—Specificity results for the six different reagents.
Substance
Reagent
Luminol LMG KM Hemastix
s
Hemident
TM
Bluestar
r
Saliva NR NR NR 1 (3) NR NR
Semen NR NR 1 (25) NR 0 (25, white) NR
Potato NR 0 (6, green) 3 (7) 1 (25) NR 1 (25)
Tomato NR NR NR 1 (23) NR 1 (25)
Tomato sauce NR NR 4 (6) NR NR NR
Tomato sauce w/meat NR NR NR 4 (6) NR 1 (25)
Red onion NR 0 (5, pink) 0 (25, yellow) 1 (21) 0 (6, pink) 1 (25)
Red kidney bean NR NR 2 (5) NR NR 1 (25)
Horseradish NR NR 4 (25) NR NR 1 (25)
1 M Ascorbic acid NR NR 0 (25, yellow) NR 1 (11) 1 (25)
Bleach solution 5% NR NR 3 (2) NR 1 (5) 1 (25)
10% Cupric sulfate 1 (25) 0 (25, blue) 0 (25, blue) 1 (25) 0 (25, blue) 1 (25)
10% Ferric sulfate 1 (25) 0 (25, orange) 0 (25, yellow) 1 (25) 0 (25, red/brown) 1 (25)
10% Nickel chloride 1 (25) 0 (25, blue) 0 (25, green) 1 (25) 0 (25, green) NR
The shortest reaction time is shown here. Numbers in parentheses indicate the number of samples that reacted out of 25 and the color change observed if different
from that of a reaction with blood.
A positive reaction was any sort of color change to the stain (or reagent strip in the case of Hemastix
s
); 0, a color change before all reagents were added; 1,
indicates a color change within 1 min of all reagents being added; 2, color change within 1–2min of all reagents being added; 3, color change within 2–3 min of all
reagents being added; 4, color change within 3–4 min of all reagents being added; NR, indicates that there was no reaction within the 4 min of timed experi-
mentation.
106 JOURNAL OF FORENSIC SCIENCES
the H
2
O
2
was added by turning blue (10% cupric sulfate), yellow/
brown (10% ferric sulfate), and light green (10% nickel chloride).
There was a reaction between the Hemastix
s
reagent strips and
saliva, potato, tomato, tomato sauce with meat, red onion, 10%
cupric sulfte, 10% ferric sulfate, and 10% nickel chloride. The
Hemastix
s
reagent strips reacted with three of the saliva samples
turning the paper green. One reacted within 1 min, and the re-
maining two samples reacted within 2 min. The actual reagent
strips did not show a reaction within the 4 min of timed experi-
mentation, nor did any of the remaining 22 samples. All of the
potato stains produces a color reaction by turning green within
15 sec, which darkened to blue as time progressed. There was no
reaction with the actual reagent strips within the 4 min of timed
experimentation. The tomato samples reacted within 1 min by
turning a very light green, which darkened as time passed. Two of
the stains did not react, and most of the reagent pads did not react
within the 4 min of timed experimentation. Two of the pads
showed a very slight green color change at 4 min. Six of the to-
mato sauce with meat samples reacted at 3 min. The six stains
turned green around the edges at 3 min. The rest of the sample
stains as well as all of the reagent pads did not react within the
4 min of timed experimentation. Twenty-one of the red onion
samples turned a very light green at 1 min with the color darken-
ing to dark green/blue as time progressed. Three of the reagent
pads show a green line running horizontally, the remaining pads
and 4 stains did not react within the 4 min of timed experimen-
tation. The 10% cupric sulfate samples showed an instant blue/
green color on the stain. The reagent pads appear as trace (spots of
green) non-hemolyzed and progress to a dark green/blue uniform
color. The 10% ferric sulfate samples all turned instantly green/
blue at the center of the stain, which progressed to brown and then
yellow along the outer margins of the stain. The reagent pads
showed a small trace (spots of green color) at 4 min. The 10%
nickel chloride sample stains all turned instantly green/blue on
application of the reagent. The reagent pads were all negative after
the 4 min of timed experimentation except for one, which showed
a green/blue color at the end of the strip.
The Hemastix
s
reagent strips were quite reactive with eight of
the substances tested. All of the substances showed a green color,
which may or may not have progressed to blue. This is the same
reaction observed on blood samples, except that when exposed to
blood the reagent strips also showed a reaction. This was not the
case for saliva, potato, and the tomato sauce with meat, which did
not react with the reagent pads.
The samples that did show a reaction on the actual reagent pads
were tomato, red onion, 10% cupric sulfate, 10% ferric sulfate,
and 10% nickel chloride. These samples showed the same reaction
as dilute blood samples would, and in the case of the 10% nickel
chloride samples, showed a color change indicative of whole
blood.
The Hemident
TM
reagents did show a color reaction with
semen, red onion, 0.1 M ascorbic acid, 5% bleach, 10% cupric
sulfate, 10% ferric sulfate, and 10% nickel chloride. All of the
semen stains turned white on addition of the first reagent, but there
was no further reaction on addition of the H
2
O
2
. Six of the red
onion samples turned pink on addition of the first reagent but there
was no further reaction on addition of the H
2
O
2
during the 4 min
of timed experimentation. Two of the 0.1 M ascorbic acid samples
turned instantly positive with a blue/green color. Within 30 sec,
nine other samples a slightly positive, with a light blue/green color
developing and increasing with intensity as time progressed. The
remaining 14 0.1 M ascorbic acid samples did not react with the
reagents during the timed experiment. Five of the 5% bleach sam-
ples turned light blue/green along their margins 30 sec after ad-
dition of the H
2
O
2
; the remaining 20 samples did not react with
the reagents within the 4 min of timed experimentation. The 10%
cupric sulfate samples all turned slightly blue on addition of the
first reagent, and 12 developed an instant blue/green color around
their edges on addition of the H
2
O
2
. A further five samples
showed the same blue/green edges after 1 min and this color in-
tensified with time, but no other samples reacted. The 10% ferric
sulfate sample all turned brown/red on addition of the first reagent
and instantly turnedgrass green on addition of the H
2
O
2
. This
color darkened to blue/green over time. The 10% nickel chloride
samples all turned very light green after the addition of the first
reagent, but there was no further color change on addition of the
H
2
O
2
during the 4 min of timed experimentation.
100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250
3000
2000
1000
0
FIG. 1—Positive control DNA. Loci D2S1338 and D19S433 are shown with
peak size and height.
TABLE 4—DNA results for the various presumptive tests, positive and negative controls, and dilution series.
D2S1338 D19S433
Peak 1 (Height) Peak 2 (Height) Peak 1 (Height) Peak 2 (Height)
Positive control 170.29 (636) 178.32 (768) 207.58 (3373) 209.74 (3560)
Luminol 170.40 (1528) 178.34 (1596) 207.63 (4984) 209.81 (4510)
LMG N/R N/R N/R N/R
KM 170.39 (489) 178.34 (436) 207.57 (580) 209.84 (400)
Hemastix
s
170.38 (1934) 178.28 (1814) 207.52 (1873) 209.59 (1938)
Hemident
TM
N/R N/R N/R N/R
Bluestar
s
170.31 (3685) 178.27 (2652) 207.36 (4840) 209.46 (4300)
1:10,000 N/R N/R N/R N/R
1:100,000 N/R N/R N/R N/R
1:1,000,000 N/R N/R N/R N/R
Negative control N/R N/R N/R N/R
KM, Kastle–Meyer; LMG, leuchomalachite green; N/R, no result.
TOBE ET AL. .AN EVALUATION OF SIX PRESUMPTIVE TESTS FOR BLOOD 107
The Hemident
TM
reagent reacted with several of the substances
tested. Semen, red onion, 0.1 M ascorbic acid, 5% bleach, 10%
cupric sulfate, 10% ferric sulfate, and 10% nickel chloride all
showed a color reaction with one or both of the reagents. Semen,
red onion, 10% cupric sulfate, 10% ferric sulfate, and 10% nickel
chloride all reacted after the addition of the first reagent and would
therefore not be mistaken for a possible bloodstain.
The 0.1 M ascorbic acid and 5% bleach samples reacted with a
blue/green color on addition of the H
2
O
2
as would blood. The 5%
bleach samples showed a color change around the margins of the
stain, which is not indicative of blood, which reacts on top of the
actual stain. The 0.1 M ascorbic acid reacted as blood would for
11 of the 25 samples.
The Bluestar
r
reagent reacted with potato, tomato, tomato
sauce with meat, red onion, red kidney bean, horseradish, 0.1 M
ascorbic acid, 5% bleach, 10% cupric sulfate, and 10% ferric sul-
fate indicated by a blue chemiluminescence upon application.
DNA Analysis
Figure 1 shows the results for the positive control DNA, with
the D2 and D19 loci clearly visible. This demonstrates the func-
tionality of the primers and indicates that they would react with
any viable DNA obtained from samples exposed to the presump-
tive tests. These results can be seen in Table 4.
Luminol, phenolphthalein, Hemastix
s
, and Bluestar
r
all
achieved amplification at both loci tested, which corresponded
to the alleles found on the positive control. All four tests gave
amplification, although Bluestar
r
claimed that it destroyed DNA
(29). Phenolphthalein had a much reduced peak height compared
with the other three tests. This is consistent with Hochmeister
et al. (24), who found that phenolphthalein reduces the amount of
extractible high-molecular-weight DNA. LMG and Hemident
TM
did not achieve amplification.
No DNA results were obtained from any of the dilution series.
This could be because there is such a small amount of template
DNA that in order to achieve detectable amplification product, it
would need several more PCR cycles.
Statistical Interpretation
The results of the w
2
test for consistency can be seen for sen-
sitivity and specificity in Tables 5 and 6, respectively. The null
hypothesis was that the two samples originate from two popula-
tions with the same distributions.
The samples that come from populations with the same distri-
butions as each other do not necessarily react with the same sub-
stances or at the same rates. Therefore, what one test might react
with, another test from a similar population would not react, or if
it did it may do so at a different rate. The same distribution comes
from the number of substances other than blood that the given
reagent will react with.
Conclusion
It is almost never necessary to apply presumptive test reagents
directly to dried bloodstain evidence (33,34). However, with ex-
tremely small samples, or when testing large areas, it may be ne-
cessary to expose the potential bloodstains directly to presumptive
tests. Based on this, the best overall presumptive blood test in this
study was luminol. It had the greatest sensitivity and specificity. It
did not destroy the DNA, and it could be reapplied. Its only draw-
back is that it must be used in near or complete darkness. Leu-
chomalachite green was found to be as specific to blood as
luminol, but its sensitivity was 10 times less, and it destroyed
the DNA. Phenolphthalein had equal sensitivity to most of the
other tests, but was extremely unspecific, and the amount of re-
coverable DNA is reduced when this test is used. Hemastix
TM
were easy to transport and use, were sensitive, but not very spe-
cific although specificity could be increased if the strips were
looked at rather than the reaction on the stain. DNA was recovered
from stains exposed to Hemastix
TM
. Hemident
s
was specific and
sensitive, but destroyed DNA and so cannot be used where sub-
sequent DNA analysis is needed. Bluestar
r
had good sensitivity,
but very poor specificity. The need for complete darkness for use
further complicates this because even if a stain did not look like
blood, it would react in the same way and could be mistaken for
blood.
The dilutions of blood did not show any amplification of DNA,
but this could be because of the small quantity of template DNA
and the low number of cycles of PCR.
TABLE 5w
2
test for consistency results for sensitivity samples with
95% confidence.
Sensitivity v54
a50.05
w
4
2
(0.05) 59.49
Luminol LMG KM Hemastix
s
Hemident
TM
Bluestar
s
Luminol 18.12 12.04 0.00 12.76 8.89
LMG 18.12 2.73 18.12 2.00 4.41
KM 12.04 2.73 12.04 0.11 0.43
Hemastix
s
0.00 18.12 12.04 12.76 8.89
Hemident
TM
12.76 2.00 0.11 12.76 0.92
Bluestar
s
8.89 4.41 0.43 8.89 0.92
The null hypothesis was that the two samples originate from two popula-
tions with the same distributions. Numbers in italics reject the null hypothesis
at 95% confidence; KM, Kastle–Meyer; LMG, leuchomalachite green.
TABLE 6w
2
test for consistency results for specificity samples with 95% confidence.
Specificity v513
a50.05
w
2
13
(0.05) 522.36
Luminol LMG KM Hemastix
s
Hemident
TM
Bluestar
r
Luminol 13.76 83.08 58.12 14.59 184.17
LMG 13.76 65.01 35.07 18.44 155.28
KM 83.08 65.01 127.26 65.05 144.46
Hemastix
s
58.12 35.07 127.26 63.69 123.91
Hemident
TM
14.59 18.44 65.05 63.69 145.89
Bluestar
r
184.17 155.28 144.46 123.91 145.89
The null hypothesis was that the two samples originate from two populations with the same distributions. Numbers in italics reject the null hypothesisat
95% confidence; KM, Kastle–Meyer; LMG, leuchomalachite green.
108 JOURNAL OF FORENSIC SCIENCES
Acknowledgments
Thanks are due to WA Products for providing the Hemastix
s
,
Hemident
TM
, and Bluestar
r
reagents.
We thank the entire Centre for Forensic Science at Strathclyde
University for lab space and equipment.
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Additional information and reprint requests:
Niamh Nic Dae
´id, Ph.D.
Centre for Forensic Science
Department of Pure and Applied Chemistry
Strathclyde University
204 George Street
Glasgow, G1 1XW, U.K.
E-mail: n.nicdaeid@strath.ac.uk
TOBE ET AL. .AN EVALUATION OF SIX PRESUMPTIVE TESTS FOR BLOOD 109
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... Many analytical methods have been used to identify blood from different species. Traditional methods include immunochromatographic assays, redox reactions, and enzyme immunoassays, among others [1][2][3]. With progress in modern instrumentation and analytical methods, emerging technologies such as high-performance liquid chromatography [4], mass spectrometry [5], and DNA detection technology [6] can be used for blood identification. ...
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Laser tweezers Raman spectroscopy (LTRS) combines optical tweezers technology and Raman spectroscopy to obtain biomolecular compositional information from a single cell without invasion or destruction, so it can be used to "fingerprint" substances to characterize numerous types of biological cell samples. In the current study, LTRS was combined with two machine learning algorithms, principal component analysis (PCA)-linear discriminant analysis (LDA) and random forest, to achieve high-precision multi-species blood classification at the single-cell level. The accuracies of the two classification models were 96.60% and 96.84%, respectively. Meanwhile, compared with PCA-LDA and other classification algorithms, the random forest algorithm is proved to have significant advantages, which can directly explain the importance of spectral features at the molecular level.
Article
For the past 7 years, Matrix Assisted Laser Desorption Ionisation Mass Spectrometry (MALDI MS) based methods have been developed and published for the forensic detection of blood in stains and fingermarks. However, in the view of adoption in an operational context, further investigation into the capabilities and limitations of this approach must be conducted. The refinement and testing of this approach must also be tailored to the requirements of the end users, enabling them to address the specific circumstances most encountered in a forensic scenario. The present study delves deeper into the assessment of the applicability of MALDI MS based strategy for the reliable and robust detection of human blood through: (i) a semi-qualitative assessment of the sensitivity of the method, (ii) a wider investigation of the compatibility of the method with the prior application of commonly used presumptive tests and (iii) assessment of the specificity of the method (when blood is present in mixture with other biofluids) and of its robustness, by assessing blood detection from a range of porous materials. The findings strengthen the evidence supporting the adoption of MALDI MS based approaches as a confirmatory test for the forensic detection of human blood in an operational context.
Article
Tetramethylbenzidine based chemical reagent test strips are often used in forensic science as a presumptive test for blood. These tests are designed as urinalysis test strips and include brands such as Combur®, HENSOTest®, Hemastix ®, MultiStix® and Chemstrip®. They are used because they are simple to apply, stable, temperature tolerant and cost effective. The addition of a chelating agent, ethylenediaminetetraacetic acid increases the selectivity of this presumptive test for blood. This is a method validation for the hemoglobin chemical reagent test strip with EDTA. A range of substances, metal compounds, chemical solutions, blood and mixtures were tested in this method validation. The chelation with EDTA successfully prevented non-blood (false) positive results from all the substances tested and consistently produced a positive result for blood on a variety of surfaces. This study has shown that this method is capable of discriminating a blood stain on copper metal surfaces and eliminate the positive results generated by clean-up solutions such as hydrogen peroxide, which usually produce a positive result for most other presumptive tests for blood. This modified method is a simple, effective and reliable test for blood stains. A variety of variations were evaluated in this study. The simplest method of application was spraying the surface of the stain with a 0.5 M EDTA solution and testing the surface of the stain and only requires a spray bottle of 0.5 M EDTA and the chemical reagent test strip. This spray approach is rugged and can be applied to horizontal, vertical and underside surfaces and requires little additional training. Overall, this study provides the forensic science community with an improved method more easily used, stored, transported and selective for blood, than luminol and safer than TMB.
Article
Biological materials found at a crime scene are crucially important evidence for forensic investigation because they provide contextual information about a crime and can be linked to the donor-individuals through combination with DNA analysis. Applications of vibrational spectroscopy to forensic biological analysis have been emerging because of its advantageous characteristics such as the non-destructivity, rapid measurement, and quantitative evaluation, compared to most current methods based on histological observation or biochemical techniques. This review presents an overview of recent developments in vibrational spectroscopy for forensic biological analysis. We also emphasize chemometric techniques, which can elicit reliable and advanced analytical outputs from highly complex spectral data from forensic biological materials. The analytical subjects addressed herein include body fluids, hair, soft tissue, bones, and bioagents. Promising applications for various analytical purposes in forensic biology are presented. Simultaneously, future avenues of study requiring further investigation are discussed.
Article
From a forensic perspective, a presumptive test, one which indicates the presence or absence of a certain target material such as blood, is an invaluable tool. Among these tests, there are different specificities, sensitivities, and shelf lives. The accuracy of a test is an algebraic combination of the specificity and sensitivity of the test. Each test has limitations as given by its false positive and false negative rates. The aim of this study was to illustrate how the false positive and false negative rates are to be properly determined using a simulation study for the phenolphthalein test. New presumptive tests must be properly evaluated/validated through testing of commonly encountered household items and other potentially probative items usually found at crime scenes, however, the makeup of test sets must appropriately capture all error rates. In order to correctly use these results when the test is applied to an unknown sample recovered at a crime scene, the error rates cannot be applied directly to estimate whether or not the sample is actually the analyte of interest. In a validation study, the forensic scientist calculates the false positive rate as the p(Positive Reaction|Blood), whereas at the scene, the crime scene investigator wishes to determine the p(Blood|Positive Reaction). All crime scene investigators need to ensure that the conditional is not transposed when interpreting such results. Furthermore, this work provides a model for the assessment of a multiple test diagnostic system intended for investigators.
Article
The presence of blood and its origin was studied on various types of cloth, immersed in water, for up to 20 days. Blood could be detected by the benzidine and phenolphthalein reactions until at least 20 days, whereas the haemochromogen test produced no crystals after any period of water immersion, with any type of cloth. The species origin could be demonstrated with woollen cloth after up to five days of water immersion whereas with other types of cloth, it could be detected until after from six hours to two days of water immersion.
Article
It has been known for many years that blood shows considerable peroxidase like enzymic activity and therefore in the presence of hydrogen peroxide will give highly coloured products with certain substrates and, in particular, with amines such as benzidine. The carcinogenic nature of benzidine, however, dictates a search for a satisfactory alternative substrate such as phenolphthalein which is believed to be non carcinogenic. This study, designed to ascertain the relative merits of using benzidine and phenolphthalein as reagents in indicator tests for blood, centres primarily on the sensitivity, stability and specificity of the two substrates. Despite the fact that the three stage phenolphthalein presumptive test for blood appears to be somewhat less sensitive than the conventional benzidine test, results indicate that it has several advantages in terms of specificity and stability. Results also indicate that the enzyme peroxidase, which is widely distributed in plants, does not contribute to false positive results in the three stage phenolphthalein indicator test for bloodstains.