Science and Justice 51 (2011) 99–109

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Science and Justice

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / s c i j u s

Chemical enhancement of footwear impressions in blood on fabric – Part 1:

Protein stains
Kevin J. Farrugia a, Kathleen A. Savage a,⁎, Helen Bandey b, Niamh Nic Daéid a,⁎
a
Centre for Forensic Science, WestCHEM, Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow, G1 1XW, UK
b
Home Office Scientific Development Branch, Fingerprint & Footwear Forensic Group, Woodcock Hill, Sandridge, St. Albans, AL4 9HQ , UK

a r t i c l e i n f o a b s t r a c t

Article history: A range of protein stains were utilised for the enhancement of footwear impressions on a variety of fabric
Received 6 September 2010 types of different colours with blood as a contaminant. A semi-automated stamping device was used to deliver
Received in revised form 29 October 2010 test impressions at a set force to minimise the variability between impressions; multiple impressions were
Accepted 8 November 2010
produced and enhanced by each reagent to determine the repeatability of the enhancement. Results indicated
that while most protein stains used in this study successfully enhanced impressions in blood on light coloured
Keywords:
Footwear impressions
fabrics, background staining caused interference on natural fabrics. Enhancement on dark coloured fabrics
Blood was only achieved using fluorescent protein stains, as non-fluorescent protein stains provided poor contrast.
Chemical enhancement A further comparison was performed with commercially available protein staining solutions and solutions
Fabric prepared within the laboratory from the appropriate chemicals. Both solutions performed equally well,
Protein stain though it is recommended to use freshly prepared solutions whenever possible.
© 2010 Forensic Science Society. Published by Elsevier Ireland Ltd. All rights reserved.

1. Introduction latent fingerprints on paper and fabrics used radioactive sulfur

1.1. Enhancement of marks on fabrics
Commonly available textile materials have a wide compositional
range that includes naturally occurring materials, such as wool and
cotton, through to fully synthesised products such as nylon and
polyester. The properties, such as porosity and surface morphology, of
fabrics derived from these materials are highly variable and as a
consequence fabric is considered to be a difficult and challenging
surface for the chemical enhancement of marks such as fingerprints
and footwear.
Previous reported research relating to the recovery and enhance-
ment of impressions on fabric is limited to a few publications relating
to experiences in casework. The enhancement of latent impressions
has been reported with fabrics prepared from materials with a smooth
finish and fine weave but it is also possible to successfully enhance
marks on other types of fabric [1]. It has also been suggested that the
chemical enhancement of marks on fabric may cause background
staining [2], thus reducing the effectiveness of the methods, however,
Zauner [1] suggested that this may not always be the case. Initial
research in the early 1970s by the UK Home Office for the recovery of
⁎ Corresponding authors. Nic Daéid is to be contacted at Tel.: +44 1415484700; fax:
+44 1415482532. Savage, Tel.: + 44 1415482237; fax: + 44 1415482532.
E-mail addresses: kathleen.savage@strath.ac.uk (K.A. Savage),
n.nicdaeid@strath.ac.uk (N. Nic Daéid).
dioxide gas [3] with limited success, and the enhanced impressions
deteriorated over time. Spedding [4] however, suggested that the
radioactive sulfur reacted with lipids in the fingerprint, making it
potentially suitable for articles that had been immersed in water in a
manner similar to physical developer [5–11] and oil red O techniques
[12–18].
1.2. Enhancement of marks in blood using protein specific stains
The application of protein stains to impressions in blood is
generally performed in a three-step process involving fixing, staining
and de-staining.
1.2.1. Fixing
Fixing a blood impression before protein staining is necessary to
precipitate the basic proteins, so preventing leaching or diffusion of
the blood. There are a range of mechanisms by which proteins can be
fixed, such as cross-linking, dehydrating and precipitating [19].
Horobin [20] states that the ‘commonest mechanistic factor for all
fixatives is the disruption of the secondary and tertiary structure of
proteins via change in the balance of lipophilic/hydrophilic regions’.
Besides proteins, other molecules such as nucleic acids, lipids and
polysaccharides can be involved in the fixation process [21]. This
causes the solubility of the proteins in water to fall sharply, so
preventing diffusion. Hussain and Pounds [22] demonstrated that
fixing impressions in blood with 5-sulfosalicylic acid was safe,
effective and convenient. 5-sulfosalicylic acid precipitates proteins

1355-0306/$ – see front matter © 2010 Forensic Science Society. Published by Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.scijus.2010.11.001
100 K.J. Farrugia et al. / Science and Justice 51 (2011) 99–109

Fig. 1. Chemical structure of acid black 1 (acidic dye).

present in blood by the formation of insoluble salts or complexes and sprayed with or immersed in a de-staining solution and allowed to dry
by disruption of the protein structure [19,23]. before examination visually and under a variety of light sources.

1.3. The use of protein stains for impressions in blood

1.2.2. Protein specific staining
Biological stains and dyes have been widely used to impart colour
Protein stains will not detect constituents normally present in
to plant or animal tissues. Dyes can be described as coloured organic
latent fingerprints but will react with amines or other groups within
aromatic molecules with conjugated bonds and large systems of
all proteins present in blood and other body fluids to yield a coloured
delocalised electrons providing visible colour and permit molecular
complex [30,31]. In general, these chemicals are cheap, easy to apply
binding to a material [19,20,24–26]. In order to avoid ambiguity, dyes
and can be used for porous and non porous items. There are numerous
are described by a Colour Index (C.I.) number which is a five-digit
protein stains available [32], however, the HOSDB [30] recommends
number associated with each dye available in the market and has been
using acid black 1 (AB1), acid violet 17 (AV17) and fluorescent acid
developed by the Society of Dyers and Colourists (Bradford, UK) and
yellow 7 (AY7). AB1 was also recommended on paper for the
the American Association of Textile Chemists and Colourists (Lowell,
enhancement of footwear impressions in blood [33]. In 2000,
Massachusetts) [27].
HOSDB [34] suggested the use of a water/ethanol/acetic acid
Dyes can be separated into natural, acidic and basic groups. Natural
(WEAA) based AB1 rather than the previous methanol (highly toxic
dyes, such as haematoxylin, are directly extracted from animal or
and flammable) or water based formulations. Further research by
plant material. Acidic and basic dyes are in fact not acidic or basic in
Sears et al. [35] has demonstrated that most protein stains behaved in
nature and their terminology relates to their usage for dyeing textiles
a similar manner to AB1 and thus could be prepared using the WEAA
under acidic or basic conditions [28,29]. Acidic dyes (example in
solvent system.
Fig. 1) possess coloured anions in association with colourless cations
Impressions in blood enhanced with protein stains can be lifted
whereas basic dyes possess coloured cations in association with
with a white gelatin lifter [36]. A lifted impression in blood that has
colourless anions. Under acidic conditions, the protein is dyed by an
been treated with acid violet 19 (AV19) can fluoresce under green
acidic dye whereas under basic conditions the protein is dyed by a
light (band pass filter 473–548 nm at 1% cut-on and cut-off points
basic dye (Fig. 2). Stains prepared for application to proteins are often
respectively) when viewed with a long pass 549 nm (1% cut-on point)
modified in order to facilitate the formation of appropriate charges of
viewing filter and can thus enhance weak traces of protein containing
the anion and cation. For example, the UK Home Office Scientific
material, even when present on dark surfaces [36,37]. Using a weak
Development Branch (HOSDB) [30] formulation of protein stains
solution of acid violet 19 (diluted 100 times) allows for fluorescence
includes the addition of acetic acid to provide optimal conditions for
observation of impressions in blood directly on the substrate [33].
acidic dyes.
Non-fluorescent stains have a limited enhancement effect on dark
surfaces as the contrast is very poor. The performance of AY7 has been
1.2.3. De-staining shown to improve with lighter deposits of blood and enhancement is
De-staining is a simple procedure used to remove the excess dye viewed as fluorescent green-yellow by excitation with a blue-green
containing solution from the surface of interest. The material is either excitation source (band pass filter 385–509 nm 1% cut-on and cut-off

Fig. 2. Dye reactions under different pH conditions.

 K.J. Farrugia et al. / Science and Justice 51 (2011) 99–109
Fig. 3. Semi-automated stamping device.
points respectively) and viewed with a long pass 510 nm filter (1%
cut-on point) [38,39]. Nonetheless, it has been reported that AY7 is
not suitable for porous surfaces such as paper, since the dye cannot be
washed off during the de-staining procedure [39].
The use of protein stains can potentially interfere with the
examination of ‘handwriting, ink, paper and indented impressions,
body fluids including DNA profiling, fibres, hairs, paint and other types
of evidence’ [30]. The correct sequence of forensic tests needs to be
established to maximise the recovery of evidence. Nonetheless,
research by the Royal Canadian Mounted Police (RCMP) [2] showed
that seven different protein stains and heme-reacting chemicals used
for enhancing blood impressions did not have any detrimental effects
on the potential for successful DNA analysis. Even aged blood
impressions (up to 14 days old) submitted to long term exposure to
enhancing chemicals (up to 54 days) yielded excellent STR results
Fig. 4. A Colour Checker Chart before (left) and after (right) colour calibration.
101
using the Profiler PlusTM multiplex system. Other chemical techniques
were successful in enhancing blood spatter and impressions after
having been exposed to temperatures exceeding 800 °C [40].
To date no studies have been reported which comprehensively
compare the range of available protein stains on the enhancement of
repetitive marks in blood prepared under the exact same conditions
and across a variety of fabric types. This study examines the
effectiveness of 11 protein staining techniques to enhance repetitive
marks made in blood on nine different fabric types. The fabrics
investigated included natural and synthetic materials of a range of
colour and porosity. The study also evaluates the effects that the
preparation of the staining reagents and ageing of the marks has on
the resultant enhancement.
2. Materials and methods
2.1. Deposition of the footwear impressions and preparation of the test
marks
It can be argued that robust comparisons of footwear enhance-
ment techniques can only be made if the test footwear impressions
have been prepared in the same manner where these factors have
been controlled in each case. The objective of this work was the
comparison of the ability of various protein stains to enhance the
footwear mark, rather than directly mimic operational conditions
normally encountered. Only when repeatability of the quality of the
footwear impression produced is controlled (such that there was no
variation from mark to mark) could a direct comparison of the various
stains be robustly and reliably achieved.
Variables introduced during the preparation of test footwear
impressions include the force exerted by the footwear sole on the
receiving surface as the footwear impression is made [41]. In this
work the force applied to the receiving surface by the blood
contaminated footwear was precisely controlled using a rig developed
and calibrated for that purpose and presented in Fig. 3 [42]. The device
was calibrated to deliver a force comparable with the average force
used in a stamping action (3500 Newton) as determined through
trials conducted with live volunteers. It should also be noted that the
impression produced represents a stamping action rather than a
walking action.
Other influencing factors on the quality of the mark (excluding
variations associated with the receiving surface) include the amount
and composition of blood on the footwear sole prior to being transferred
to the receiving surface and the actual amount of contaminant
transferred to the substrate. The application of blood to the footwear
sole and the subsequent transfer of blood to a substrate are challenging
to control during experimental trials. Stepping into a pool of blood
followed by stepping onto the fabric resulted in a heavy blood-stained
102 K.J. Farrugia et al. / Science and Justice 51 (2011) 99–109

and overloaded footwear impression. The following method however, Table 1

yielded reasonably weak but importantly, repeatable blood impressions List of protein stains.

from mark to mark and was used within this work. Protein specific stains
A tray measuring 0.33 × 0.23 × 0.06 m was lined with two
Name Alternative name Colour Supplier Fluorescent
Kimberley® blue double ply tissues covering the whole base. 50 mL index
of swine blood was poured over the tissues. The tray was then pushed
Acid Black 1 Amido Black 10B 20470 BVDA No
against the sole of the footwear attached to the rig in a walking (AB1)
motion. The same motion was repeated twice on clean tissues to Acid Violet 17 Coomassie Violet 42650 BVDA No
remove excess blood before releasing the foot onto the fabric. (AV17) R-200
Six individual repeat footwear marks were prepared as described Acid Yellow 7 Brilliant Sulfoflavine 56205 BVDA Yes
(AY7)
for all tests undertaken in this work. This provided a means by which Acid Violet 19 Hungarian Red 42685 Acros Yes
the repeatability of the deposited impressions within each test set (AV19)
could be assured thus building a robustness into the study that is Acid Yellow 23 Tartrazine 19140 Sigma- No
lacking in much of the reported research within the area. (AY23) Aldrich
Acid Blue 1 Patent Blue VF 42045 Acros No
All impressions were allowed to age for seven days before
(ABlu1)
enhancement with the various protein stains. Photography of all Acid Blue 83 Coomassie Brilliant 42660 BVDA No
impressions was performed, using a Canon EOS 300D [sensor size (AB83) Blue R
22.7 × 15.1 mm (3.42 cm²)], immediately after the impression was Acid Red 71 Crocein Scarlet 7B 27165 BVDA No
prepared, after seven days, after chemical treatment and during (AR71)
Acid Green 50 Lissamine Green B 44090 Acros No
fluorescence examination if required. Before the application of blood (AG50)
to the footwear sole, dry and wet blanks were carried out for each Acid Red 52 Sulforhodamine B 45100 TCI Europe Yes
protein stain sample set for each receiving surface. A dry blank (AR52)
included stamping on the fabric without any blood on the footwear Solvent Green 7 Pyranine 59040 TCI Europe Yes
(SG7)
sole, ensuring the footwear sole was dry. A wet blank included
stepping on a distilled water-soaked tissue before stamping on the
fabric. The blanks ensured that the staining observed was not due to
additives, such as plasticisers, in the footwear sole or impurities in the
water. (right) calibration. Note that there are only minor changes, however,
after calibration a true representation of what the camera sensor sees
2.2. Fluorescence photography is obtained. These calibration settings will only be valid for the camera
utilised to photograph the Colour Checker. Once the calibration
Fluorescent enhancement of footwear impressions was photo- settings are saved, they can be applied to multiple images through
graphed with the camera setting on Program mode (P), where the Photoshop software [43,44].
shutter speed and aperture (depth of field) were determined
automatically by the camera. The exposure time varied due to the 2.4. Protein stain formulation
nature of the substrate. For example, a very short exposure time (fast
shutter speed and small aperture) was needed for white fabrics due to A full list of protein stains and fabrics utilised in the study are
the bright background fluorescence from the fabric. A fast shutter presented in Tables 1 and 2. Protein stains were prepared using the
speed is open for a short period of time and allows less light to reach water/ethanol/acetic acid (WEAA) formulation [30,35].
the camera sensor. In contrast, dark fabrics required longer exposure
times thus having a slow shutter speed to allow more light to reach 2.4.1. Fixing solution
the camera sensor. A general camera setting could not be adopted for 23 g of 5-sulfosalicylic acid dihydrate (Acros) was dissolved and
fluorescent photography as the background fluorescence varied from stirred in 1 L of distilled water. This was used to fix the impressions in
fabric to fabric. Photographing a dark image in Program mode will blood by immersion for a minimum period of 5 min.
increase the shutter speed and the aperture (i.e. depth of field
decreases) which may increase the occurrence of blurring in the
picture. The use of P mode in this study provided unblurred
photographs that were similar or more sensitive, to what was
observed with the human eye using the appropriate viewing filters. Table 2
List of fabrics.
2.3. Computer monitor and colour calibration Fabric Supplier

White cotton [CD13] WBL Whaleys Bradford Ltd.

Computer monitor and colour calibration were carried out as Plain weave; 19 warp threads/cm;
explained in the literature using Adobe® Photoshop CS3/CS4 software 10 weft threads/cm
[43–46]. The calibration was important enabling the images to be Black cotton [CD13D] WBL Whaleys Bradford Ltd.
viewed correctly on a computer monitor. The monitor calibration was Plain weave; 19 warp threads/cm;
10 weft threads/cm
performed using Adobe® Gamma within Photoshop and following the
Patterned cotton [SF2360/B] WBL Whaleys Bradford Ltd.
software instructions. Colour calibration was performed by photo- Twill weave; 19 warp threads/cm;
graphing a Gretag Macbeth Colour Checker and then running a 19 weft threads/cm
calibrator script (obtained from http://fors.net/chromoholics) White polyester taffeta [SF25] WBL Whaleys Bradford Ltd.
through Photoshop software as explained by Evening [43,44]. The Black polyester taffeta [SF25A] WBL Whaleys Bradford Ltd.
White nylon (82%)/lycra (18%) [SF28] WBL Whaleys Bradford Ltd.
Colour Checker is a checkerboard array (approximately A4 size) of 24 Black nylon (82%)/lycra (18%) [SF27] WBL Whaleys Bradford Ltd.
scientifically prepared coloured squares in a wide range of colours. Blue denim [Rialto Indigo] Mandors, Glasgow, UK
Once the calibration script has finished running through Photoshop, Twill weave; 25 warp threads/cm;
the calibration settings are saved and named with a descriptive name. 19 weft threads/cm
Brown bovine leather The Clyde Leather Co., Glasgow, UK
Fig. 4 shows a picture of the Colour Checker before (left) and after
 K.J. Farrugia et al. / Science and Justice 51 (2011) 99–109 103

Table 3
Excitation wavelength and viewing filters for a Mason Vactron Quaser 40 for
fluorescent protein stains.

Chemical Excitation Excitation Viewing filter/ Viewing

name wavelength/nm filters nm filter

AY7 385–509 Blue 510 Yellow/

orange
AV19 473–548 Green 549 Orange
SG7 350–469 Violet/Blue 476 Yellow
AR52 503–591 Green/ 593 Red
Yellow

2.4.2. Staining solution

1 g of the appropriate protein stain was stirred for at least 30 min
in a solution of 50 mL of acetic acid (Sigma), 250 mL of absolute
ethanol (Sigma) and 700 mL of distilled water. This was used to stain
the impressions in blood under test by immersion for a minimum
period of 5 min. All of the formulations have a shelf-life of at least
12 months if refrigerated. AB83 and AR71 were removed from the list
of techniques in the early stages of the experimental work as their
enhancement potential and colour was deemed to be similar or of
lesser quality to AV17 and AV19 respectively.
2.4.3. De-staining solution
The final de-staining methodology used in this study was to first
rinse under running tap water for several minutes to remove the
excess dye (as suggested by Bodziak [47]) followed by immersion in a
de-staining solution of 50 mL of acetic acid, 250 mL of ethanol and
700 mL of distilled water. HOSDB, however, advises that the use of
water rinsing prior to immersing in a de-staining solution may re-
solubilise the proteins in the impression in blood leading to diffusion.
The use of tap water could be a practical solution provided diffusion is
minimal or non-existent.
2.4.4. Fluorescence observations
The appropriate excitation wavelengths and viewing filters which
were utilised for observation of the fluorescent protein stains are
presented in Table 3. Fluorescence examination was performed using
a Mason Vactron Quaser 40 and a Foster and Freeman Crime-Lite® 2.
The wavelength ranges represented in Table 3 show the 1% cut-on and
cut-off points respectively [48]. Other light sources may use
wavelengths representing the 50% point or the peak wavelength.
Fig. 6. Visualisation of a footwear impression in blood on black polyester during
fixation.
2.5. Comparison of freshly prepared and commercially available
pre-mixed protein stains
Most protein stains are commercially available as pre-mixed or
‘ready-made’ solutions. A comparison was performed between
commercially available solutions and solutions prepared from raw
chemicals on marks prepared on white cotton only. The protein stains
included in this study were acid black 1 (AB1), acid violet 17 (AV17),
acid violet 19 (AV19), crocein scarlet (AR71) and coomassie blue
(AB83). Pre-mixed solutions were purchased from BVDA
(Netherlands) and fresh solutions were prepared according to the
HOSDB formulations [35,38]. BVDA solutions of fix and protein stains
were sprayed on the blood impression (as recommended by BVDA)
whereas freshly prepared solutions were applied by immersion (as
recommended by HOSDB). For this part of the study, the impressions
in blood were prepared by using half a footwear sole followed by
pressing on a blood-soaked tissue and then twice on a clean tissue
before stamping on the fabric using hand force rather than the
footwear stamping device.
2.5.1. Effect of ageing
Footwear impressions in blood can deteriorate over time, even in
indoor or sheltered environments [49]. A mini-study was devised to
compare the enhancement of footwear impressions in blood with

Fig. 5. Enhancement of a footwear impression in blood on black polyester with AV17:

(a) blood impression before enhancement; (b) AV17 enhancement under white light; Fig. 7. Enhancement of a footwear impression in blood on leather with AB1: (a) before
(c) visualisation of (b) using oblique lighting. enhancement; (b) after enhancement.
104 K.J. Farrugia et al. / Science and Justice 51 (2011) 99–109
Fig. 8. Enhancement of a footwear impression in blood with ABlu1: (a) white cotton;
(b) white nylon/lycra.
AB1, AV17 and AV19 on white cotton that had been aged for 1, 7, 10
and 28 days. For this part of the study the impressions in blood were
prepared as described above using hand force.
2.5.2. Diminishing series
A diminishing series was prepared by stepping on a blood soaked
tissue (50 mL of blood) and then using the test rig to produce ten
impressions in blood for each fabric with the first one being the most
blood-stained (in this case no excess blood was removed in the
manner previously described). The impressions were left to air dry for
one week before treatment. The impressions were cut into four
through the centre of the mark and enhanced with four different
reagents: acid yellow 7 (AY7), leuco crystal violet (LCV), leuco
Fig. 9. Enhancement of a footwear impression in blood on black nylon/lycra: (a) blood impression before enhancement; (b) enhancement with AY7 under white light; (c) AY7
fluorescence using Blue Crime-Lite™; (d) AY7 fluorescence using a Quaser 40 385–509 nm excitation filter and viewed with a 510 nm viewing filter.
malachite green (LMG) and luminol. AY7 enhancement results are
presented in this paper whereas the other enhancement results are
presented in part 2 of this study.
3. Results and discussion
The experimental approach taken for this work was done so as to
produce repeatable footwear impressions rather than directly mimic
operational conditions. This methodology was chosen so that the
ability of the enhancement techniques could be directly compared
across the same and different fabric types.
3.1. Comparison of different protein stains
All protein stains provided visual colour enhancement on light
coloured fabrics. Colour enhancement was minimal on black and dark
coloured fabrics due to poor contrast, however, fluorescent stains
provided the added advantage of fluorescence. No enhancement was
obtained on the dry or wet blanks indicating that the staining was not
due to additives in the footwear sole or components in water.
3.1.1. Fabric type
The footwear impression in blood on black polyester exhibited
some additional advantages when enhanced with protein stains.
Under normal white lighting, the protein stain enhancement was very
weak. However, with oblique lighting (Crime-Lite® 80 L) the
footwear impression could be visualised and is illustrated in Fig. 5.
The use of oblique lighting prior to treatment with protein stains did
not provide visualisation as the blood appeared to absorb the lighting.
The marks on black polyester were also visualised during fixation
(Fig. 6) when the black polyester was immersed in the fixing solution
of 2% 5-sulfosalicylic acid. This was not observed for the other black
fabrics (nylon/lycra or cotton).
Footwear marks produced on leather left a faint indentation of the
footwear sole in the fabric material and the footwear impression in
blood was clearly visible when compared with similar marks on the
other fabrics as illustrated in Fig. 7. Enhancement of the impressions
in blood on leather with each of the protein stains appeared to
 K.J. Farrugia et al. / Science and Justice 51 (2011) 99–109 105

Fig. 10. Enhancement of a footwear impression in blood on black cotton with: (a) AY7; (b) AV19; (c) AR52 and (d) SG7.

obliterate the impression and the stain did not wash off during the
de-staining procedure.
3.1.2. De-staining
The protein stains excess washed off very well during the de-
staining procedure from synthetic fabrics when compared to the
natural fabrics (Fig. 8). Background staining was more prominent on
cotton and this can be explained by the fact that synthetic fibres, such
as polyester, are hydrophobic and thus will repel acidic dyes [50]. The
protein stain was almost completely washed off from white polyester
after de-staining. This was also observed on blank tests.
3.1.3. Fluorescence
In general fluorescence did not further enhance impressions on the
light coloured fabrics and in some circumstances the bright
fluorescence of white fabrics obscured the impression, potentially
due to optical brighteners in white fabrics [51,52]. Fluorescent AY7
provided excellent results on dark coloured fabrics when illuminated
with blue light. Background staining occurred on light, coloured and
natural fabrics such as white and patterned cotton after AY7
treatment, however, no background staining was observed on dark-
coloured fabrics. This study showed vivid visual yellow enhancement
on impressions in blood on light coloured fabrics in contrast to
previous research by the HOSDB [39] that had concluded that AY7 did
not impart any visual colour unless dyeing times were greatly
increased. The HOSDB [39] study had also highlighted the fact that
AY7 was unsuitable for porous surfaces such as paper (fabric surfaces
were not included in the HOSDB study) as it was impossible to wash
off the dye from the background. However, the opposite was found to
be true in this study on fabrics where most of the dye washed off
easily. During the de-staining procedure, AY7 washed off easily from
synthetic fabrics (less porous than natural fabrics) such as polyester
when compared to the more porous fabrics such as cotton, however,
the footwear impression was still clearly visible on porous fabrics.

Fig. 11. AY7 fluorescence enhancement of footwear impression in blood on coloured denim: (a) blue; (b) bright blue; (c) grey; (d) black; (e) red.
106 K.J. Farrugia et al. / Science and Justice 51 (2011) 99–109
Fig. 12. Enhancement of a footwear impression in blood on black cotton with AV19:
(a) under white light; (b) under Quaser green light (473–548 nm).
HOSDB confirmed that their current research on blood impressions on
fabrics and AY7 agree with these results, however, the use of AY7 on
paper exhibited extensive background staining. AY7 fluorescence
enhancement of impressions in blood on black fabrics provided
excellent results (Fig. 9) although the fluorescence on denim and
leather was very weak. Fluorescence using a Foster and Freeman
Crime-Lite® 2 with a blue excitation source and a Mason Vactron
Quaser 40 with a blue excitation filter (385–509 nm) and viewed with
a yellow/orange filter (510 nm) showed similar results. The Crime-
Lite® has the added advantage of being small, portable and battery-
powered compared to the bulky, heavy Quaser which requires a
connection to an electrical output. The use of a laser source with a
specific excitation wavelength such as a blue laser at 460 nm may
create a stronger response for the fluorescence of AY7 [53]. The
fluorescence observed from enhancement with protein stains AV19,
AR52 and SG7 behaved similar to but not as vivid as AY7, and visual/
background staining with SG7 was minimal or non-existent. The use
of SG7 and AR52 has not previously been reported in the literature for
the enhancement of impressions in blood. Fig. 10 shows the
Fig. 13. Enhancement of a footwear impression in blood on white cotton: (a) blood impression; (b) enhancement of (a) with BVDA AV17; (c) blood impression; (d) enhancement of
(c) with fresh AV17 solution.
fluorescence enhancement of footwear impressions in blood on
black cotton with AY7, AV19, AR52 and SG7.
It was not initially clear why the fluorescence on denim and
leather was weak compared to the black fabrics. The enhancement
with AY7 on denim was repeated using other coloured denim fabrics
(bright blue, dark blue, faint blue, red, black and grey). AY7
fluorescence enhancement for all these fabrics was still weak
suggesting the use of indigo and vat dyes, commonly used in dyeing
denim [54–58], might interfere with the AY7 fluorescence. Fig. 11
shows the AY7 fluorescence enhancement of blood impressions on
differently coloured denim.
Recent research has suggested that a lifted blood impression that
had been treated with AV19 and lifted with a white gelatin lifter
fluoresced under green light [33,37]. Furthermore, a 1:100 dilution of
the AV19 solution could provide direct fluorescence without lifting.
The gelatin lifter was left on the blood impression for at least 15–
30 min before removing [36]. Longer periods of up to 2 h were tested
but no further improvement on fluorescence was observed. In this
study, preliminary studies showed that no enhancement was
achieved by lifting with a white gelatin lifter after staining with
AV19 or other protein stains on any type of fabric. The use of a BVDA
high-resolution gel lifter scanner did not provide any improvement.
Diluting the AV19 stain solution by a factor of 100, however, produced
a weak fluorescence by exciting with a green light (band pass filter
473–548 nm at 1% cut-on and cut-off points respectively) and viewed
with a long pass 549 nm filter (1% cut-on point) (Fig. 12). Photog-
raphy of the original impression is highly recommended before any
chemical enhancement or lifting takes place [47].
3.2. Comparison of freshly prepared and commercially available
pre-mixed protein stains
Both the commercially prepared and laboratory prepared solutions
successfully enhanced all of the blood impressions prepared on white
cotton with no discernable differences. The pre-mixed BVDA
solutions, specifically designed to be applied by spraying, however
had no indication of their shelf-life or an expiry date. Sensitivity of
these methods was not tested via a depletion series. A footwear
impression in blood before and after enhancement with AV17 from
BVDA and laboratory prepared AV17 is presented in Fig. 13. A number
of studies [33,59] related to the chemical enhancement of footwear
impressions utilised half of a footwear sole which was applied by hand
pressure only. Fig. 13a and c show the difference in the repeatability of
the footwear impression in blood when prepared by using half of a
footwear sole applied using hand pressure only. This highlights the
 K.J. Farrugia et al. / Science and Justice 51 (2011) 99–109 107

importance of the footwear stamping device for preparing repeatable
test footwear impressions.
3.3. Effect of ageing
In general, the enhanced blood impressions appeared visually
similar after the different ageing periods had elapsed and no obvious
changes were observed. Ageing the impressions in blood for seven
days was practical. This ageing period also reflects, to some degree,
the number of days which commonly elapse between the collection of
the item and subsequent chemical processing (as opposed to
biological or DNA analysis).
3.4. Diminishing series
The diminishing series work was carried out using AY7 only as the
best responding reagent for the blood marks in this study. AY7
enhancement for the diminishing series of impressions made in blood
was not possible beyond the third impression. Fluorescence improved
the contrast on black coloured fabrics and slightly weakened the
contrast on light coloured fabrics. An interesting observation was that
the first impression on denim and leather fluoresced strongly,
compared to the second impression and the rest of the series. This
suggests that fluorescence was weak on denim and leather in previous
studies because of the absence of blood in the impression rather than
an effect of the surface and that previous results were obtained as a
result of the poor ability of these fabrics to retain blood.
This weakly enhanced diminishing series can be explained by the
ability of fabrics to retain the blood. There is in fact a relationship
between the type of fabric and the retention of bloodstains; Cox [60]
observed that blood was absent from synthetic fabrics (acetate,
polyester and nylon) but present in all cotton fabrics after washing in
a washing machine. The results of the diminishing series are
illustrated in Fig. 14. The AY7 fluorescence on white cotton causes

Fig. 14. AY7 fluorescence enhancement for a diminishing series in blood for: black cotton, white cotton and denim.
108 K.J. Farrugia et al. / Science and Justice 51 (2011) 99–109
the blood to appear as black and the background as yellow when the
usual expectation for AY7 is for the blood to appear as bright yellow
and the background dark. This is most probably due to optical
brighteners used to impart a bright white colour and introduced
during the manufacture of white fabrics. As discussed previously,
bright AY7 fluorescence on white fabrics appeared to obscure the
impression.
4. Conclusion
The results clearly showed that AY7 is the most suitable protein
stain for footwear impressions made in blood and deposited onto dark
fabrics. Limited fluorescence was observed for similar marks on denim
and leather. Comparable but weaker results were obtained with other
fluorescent protein stains. Other protein stains performed in a similar
manner on light coloured fabrics, however, the sensitivity was not
tested via a depletion series. The authors believe that the HOSDB
recommendation of protein stains AB1, AB17 and AY7 is appropriate
for such substrates. Their use is not strictly limited to non-porous
substrates, however, it is highly recommended to test an area of the
substrate beforehand away from the blood impression. Furthermore,
observations with photography under different lighting conditions
and fluorescence, before and after chemical enhancement and lifting,
should be included in routine analysis as a great improvement in
contrast can be obtained.
Acknowledgements
The authors would like to thank HOSDB, EPSRC and the University
of Strathclyde for their continued financial support. This work is also
partially funded by the Malta Government Scholarship Scheme. The
authors would also like to express their thanks to the anonymous
reviewers for their helpful comments.
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