Chemical
Enhancement of Fingerprints in Blood: An Evaluation of Methods, EFFECTS
ON DNA, and Assessment of Chemical Hazards
Dr. J.D. DeHaan, J.D. Clark, T.F. Spear, R. Oswalt, S.S.
Barney, CA Department of Justice, Bureau of Forensic Services,
Sacramento, California
Introduction
Blood is commonly encountered as a transfer medium for
fingerprints at crime scenes. Sometimes, the residue retains
enough color to allow it to be photographed directly, but, more
often, the residue is so faint that its color (and thereby its
contrast) is so slight that ordinary light photography is
ineffective except on transparent or highly reflective sources.
The advent of tunable-wavelength light sources (Polilight et al)
allowed the latent print examiner to pick an examination
wavelength at which the residual hemoglobin most strongly
absorbs, thereby increasing its potential contrast, especially
on surfaces that reflect (or even fluoresce) at that wavelength.
Chemical methods for the enhancement of residual blood
fingerprints, have been successfully used for years.
Leucomalachite green, amido black, and ninhydrin chemically
react with components in blood to form a dark-colored dye
complex and have all been used successfully on light-colored or
transparent surfaces. Leucomalachite green and ninhydrin have
low background colors but are unsuitable for non-porous surfaces
as they run off, and either distort the print or fail to react
before detail can be photographed. Amido black is very sensitive
and works well on non-porous surfaces but its high background
color (light to medium blue) compromises contrast on porous
surfaces from which the stain cannot be removed by rinsing.
On dark surfaces, none of these color-producing reagent stains
could be guaranteed to produce detectable prints. Attempts to
use luminol had reportedly been of limited success since the
brief chemiluminescence created was weak, hard to photograph,
and failed to resolve fine ridge detail. Recent authors have
recommended new techniques for the development enhancement of
faint blood prints on various surfaces. In a 1995 review of
techniques, John Neuner, North Carolina SBI, suggested leuco
crystal violet [4,4',4"methylidynestris(N,N dimethyl
aniline)] (a reduced form of crystal violet) as a reagent for
developing dark-colored prints on light-colored backgrounds, and
merbromin for developing prints on dark backgrounds, exploiting
the merbromin-blood complex' fluorescing properties(1). He gave
no processing details or evaluation. Bodziak recommended leuco
crystal violet for the development of shoeprints in blood (2).
Maucieri and Monk recommended fluorescin (the reduced form of
fluorescein) as a potential candidate(3). Cheeseman and DiMeo
successfully applied fluorescin to bloody fingerprints in a
viscous medium to reduce running(4). Although it was not
mentioned in the literature, DFO (1,1 diazafluoren-1-one), with
its demonstrated sensitivity to amino acids in normal skin
secretions, was thought to have potential for blood prints.
Everse and Menzel suggested the use of merbromin (mercurochrome)
to develop fluorescing prints on non-porous surfaces(5). It must
be remembered that any blood-bearing surface - friction ridge
skin, tool, weapon, glove or shoe - can be of interest to the
crime scene examiner, so techniques developed for enhancement of
blood-residue fingerprints can be of use in many other
applications.
It was thought that a side-by-side comparison of the available
techniques on typical target surfaces would be of value. Their
sensitivity (using both serial dilution and sequential-touch
methods of producing concentration gradients) and applicability
to a variety of surfaces could be readily compared. In addition,
interference or visualization problems could be evaluated. Also,
since many blood bearing "fingerprint" surfaces may
also be subjected to DNA analysis, interferences with current
DNA methods could be evaluated using known human blood and
subjecting it to the harshest chemical exposure from each
method.
Experimental
A folded paper towel "touch pad" was dampened with
fresh whole human blood (anti-coagulant added, but no
preservative) until touching it just produced an even film of
blood on the tips of the fingers. Three fingers (index, middle,
ring), were touched simultaneously on the touch pad and then
immediately touched repeatedly to the target surface. The use of
three fingers gives some control over finger-to-finger touch
variations without having to create another test target. Test
targets were a variety of porous, non-porous and semi-porous
surfaces typical of the surfaces on which bloody prints are
found in casework. The targets consisted of: porous: white
typing paper, black construction paper, brown bag paper, bare
wood (balsa); semi-porous: white contact (shelf) paper, textured
gypsum board painted with a semi-gloss latex wall paint,
weathered fiberglass panel; and non-porous: smooth-surface glass
bottles, plastic soft drink bottles, and aluminum soft drink
cans. The used or weathered targets were cleaned in hot water and
allowed to dry before prints were placed on them. All blood
prints were placed on all targets by the same person on the same
day.
It had been previously found by the authors during the
preparation of test targets for latent print training exercises
that sequential touches of a surface provides a convenient,
reproducible gradient of concentration of the transfer medium.
No matter what the medium is: blood, grease, or natural skin
oils, each touch removes a percentage of the medium, leaving
less on the skin for the next touch (until it is replenished).
Multiple sequential touches, thus, produce a series of target
transfers, each having less medium than the one just before it.
A single target then offers a range of concentrations for a
single chemical treatment to react with. Multiple duplicate
targets were prepared for each reagent to be tested as
appropriate. For instance, ninhydrin and DFO were to be used
only on porous or semi-porous targets. Tests would be conducted
1 day, 10 days and 30 days after prints were deposited. The
blood-printed targets were allowed to dry for approximately 18
hours at room temperature (68°F, 22°C) and humidity (~40%)
until first processing.
In addition, whole human blood was serially diluted in DI water
from 1:10 to 1:1,000,000. Single drops of these dilutions were
then placed on white typing paper and allowed to dry at ~30°C
for 4 hours before test reagent solutions were applied.
The following reagent solutions were made for these tests:
Ninhydrin:
0.6% solution in acetone
Amido black (AB):
0.2% solution in methanol/glacial acetic acid (9:1)
Methanol/glacial acetic acid (9:1) rinse
DFO:
Stock Solution:
0.5% diaza-fluoren-1-one in methanol/acetic acid (9:1)
Working Solution: 60ml stock solution + 10ml 2-propanol + 50ml
acetone + 50ml xylene + 830ml petroleum ether
Fluorescin:
Stock Solution: Dissolve 1g fluorescein in 10%
NaOH with 10g Zinc metal. Boil until clear and
pale in color
Working Solution: 5% stock solution in water
Leuco Crystal Violet (LCV):
Dissolve 10gm 5-sulfosalicylic acid (Aldrich 24700-6)
3.7g sodium acetate
1g Leuco crystal violet (Aldrich 21921-5) in 500 ml 3%
hydrogen peroxide
Merbromin:
Stock Solution: 0.45g merbromin in (Aldrich 19959-1) 100ml
ethanol, 15ml formic acid, 10g mossy zinc. Reflux until clear
and pale in color.
Working Solution A: 10ml stock/30ml acetone
Working Solution B: 10% hydrogen peroxide/acetone (1:9)
Each target was treated according to the best established
method for each reagent (or according to recommendations of
authors discussing the newer techniques), as follows:
Ninhydrin: Applied by immersion (dipping) the target into the
reagent solution for 5s, then allowing excess to drain away. Air
dried and then developed at room temperature and humidity for 7
days prior to reading, or developed in 70°C/70% humidity for 2
hrs.
DFO: Applied by immersion, two applications, allowed to air dry
between. Heated at 85°C in a dry oven and evaluated using 532nm
laser.
Amido Black: Applied using a squeeze bottle, rinse applied with
squeeze bottle, then cold tap water rinse.
Leuco crystal violet: Applied using a fine-mist aerosol
applicator in a high-draft fume hood. Allowed to develop for 30
seconds, then rinsed with cold tap water.
Fluorescin: Applied using a fine-mist aerosol applicator in a
high-draft fume hood. Allowed to develop at room temperature and
then examined using UV and laser light sources.
Merbromin: Applied using a fine-mist aerosol applicator, (both
solutions) allowed to dry at room temperature, and then examined
using a laser light source.
Upon completion of the chemical treatment of the test targets,
the resulting prints were evaluated visually by two experienced
latent print examiners for intensity (detectable color against
the background) and clarity (readability of developed ridge
detail). For color-producing reagents: (ninhydrin, LCV, AB), the
evaluations were carried out in normal room light. For
fluorescing reagents (merbromin, DFO, fluorescin) the
evaluations were carried out using illumination of appropriate
wavelength and using suitable barrier filters when needed. The
prints were rated according to the number of the sequential
touch that demonstrated detectable color on fluorescence and
identifiable ridge detail. Each target, then would have a two
digit rating, such as 4/3 or 6/6. This rating takes into account
background coloration or reactivity as well as loss of clarity
by solvent action.
Evaluations of the sequential touch targets were repeated at 10
day intervals, up to 40 days after treatment. Results are
presented in Tables 1 and 2, in the next section.
The serial dilution targets were rated in a similar fashion with
each target being rated on a scale of 0 to 5+ for intensity
(color) against a white background. See Tables 1 and 2.
Since the possible source of the blood associated with a bloody
fingerprint or shoe impression could be an important issue, the
impact of these six reagents on the ability to successfully type
DNA from bloodstains using 14 PCR-based markers was examined.
Given that the blood associated with an evidence impression can
be quite limited, PCR-based markers were chosen since they are
one of the most sensitive typing techniques available. In
addition, successful amplification of DNA with several of these
primer sets and characteristic human typing profiles are
considered to be sufficient analytical information to establish
that the analyzed DNA is of human /primate origin.
Bloodstains were prepared by adding 30ml of whole, human blood
(anti-coagulant added, no preservative) to clean cotton swabs.
These stains were allowed to dry for 24 hours at room
temperature and two bloodstained swabs were each dipped into one
of the following reagents: merbromin, amido black, fluorescin,
LCV, DFO, or ninhydrin reagents. Three similarly prepared
bloodstains were used as control samples and were not treated
with the fingerprint reagents. Following this treatment, the
bloodstains were maintained for 11 days at room temperature and
then stored frozen (-15°C) for 20 days.
The DNA was extracted from all bloodstains by digestion with
Proteinase K, purification with phenol/chloroform and
concentration through microfiltration. The quality of the
resulting DNA was evaluated by running the extracted DNA in a 1%
agarose gel. With the exception of the samples treated with
merbromin or ninhydrin, no significant degradation was noted in
the DNA obtained from the treated bloodstains or the untreated
control stains. The DNA from the bloodstains treated with
merbromin or ninhydrin displayed high molecular weight DNA with
an associated faint smear of degraded DNA. Following the agarose
gel evaluation, the DNA was prepared for amplification in the
following PCR Amplification Kits from PE Applied Biosystems
(Foster City, CA.): DQA1/PolymarkerÔ, AmpliFLP D1S80Ô,
AmpFLSTRÔ GreenI and AmpFLSTRÔ Blue. Using the protocol
provided by the manufacturer, 2ng to 5ng of DNA (in a 20ul fixed
volume solution containing 160ng of BSA) was added to the
amplification cocktails for the DQA1/Polymarker and D1S80 loci.
The amplified DNA from these 7 loci (D1S80, DQA1, LDLR, GYPA,
HBGG, D7S8 and GC) was then typed by either immobilized probe
typing strips or polyacrylamide gel electrophoresis. Following
the manufacturer's protocol, approximately 1.0 ng of DNA was
added to the AmpFLSTRÔ GreenI and Blue amplification cocktails.
The amplified DNA from these 7 loci (Green1: Amelogenin, THO1,
TPOX, CSF1PO and Blue: D3S1358, VWA, FGA) was typed using
capillary electrophoresis on an ABI Prism 310 Genetic Analyzer.
The resulting data was analyzed using GeneScanÔ /GenotyperÔ
software.
Results The results of color-developing reagents on
sequential touch targets are presented in Table 1. The results
of fluorescing reagents on sequential touch targets are
presented in Table 2.
Table
3: Results: Color Reagents
Serial
Dilution: Blood on White Paper
