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Analysis of fingerprint samples, testing various conditions, for forensic DNA
identification

Lana Ostojic, Elisa Wurmbach

PII: S1355-0306(16)30096-X
DOI: doi: 10.1016/j.scijus.2016.08.009
Reference: SCIJUS 620

To appear in: Science & Justice

Received date: 9 May 2016


Revised date: 25 August 2016
Accepted date: 30 August 2016

Please cite this article as: Lana Ostojic, Elisa Wurmbach, Analysis of fingerprint samples,
testing various conditions, for forensic DNA identification, Science & Justice (2016), doi:
10.1016/j.scijus.2016.08.009

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Analysis of fingerprint samples, testing various conditions, for forensic DNA

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identification

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Lana Ostojic and Elisa Wurmbach*
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Office of Chief Medical Examiner, Department of Forensic Biology, 421 East 26th Street,
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New York, N.Y. 10016, USA


EWurmbach@ocme.nyc.gov; Phone: 212-323-1498; Fax: 212-323-1570
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LOstojic@ocme.nyc.gov; Phone: 212-323-1468; Fax: 212-323-1570


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*Corresponding author
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Abstract
Fingerprints can be of tremendous value for forensic biology, since they can be collected
from a wide variety of evident types, such as handles of weapons, tools collected in
criminal cases, and objects with no apparent staining. DNA obtained from fingerprints
varies greatly in quality and quantity, which ultimately affects the quality of the resulting

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STR profiles. Additional difficulties can arise when fingerprint samples show mixed STR
profiles due to the handling of multiple persons. After applying a tested protocol for
sample collection (swabbing with 5% Triton X-100), DNA extraction (using an enzyme

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that works at elevated temperatures), and PCR amplification (AmpFlSTR® Identifiler®
using 31 cycles) extensive analysis was performed to better understand the challenges
inherent to fingerprint samples, with the ultimate goal of developing valuable profiles

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(≥50% complete). The impact of time on deposited fingerprints was investigated,
revealing that while the quality of profiles deteriorated, full STR profiles could still be
obtained from samples after 40 days of storage at room temperature. By comparing the

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STR profiles from fingerprints of the dominant versus the non-dominant hand, we found
a slightly better quality from the non-dominant hand, which was not always significant.
Substrates seem to have greater effects on fingerprints. Tests on glass, plastic, paper and
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metal (US Quarter dollar, made of Cu and Ni), common substrates in offices and homes,
showed best results for glass, followed by plastic and paper, while almost no profiles
were obtained from a Quarter dollar. Important for forensic casework, we also assessed
three-person mixtures of touched fingerprint samples. Unlike routinely used approaches
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for sampling evidence, the surface of an object (bottle) was sectioned into six equal parts
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and separate samples were taken from each section. The samples were processed
separately for DNA extraction and STR amplification. The results included a few single
source profiles and distinguishable two person mixtures. On average, this approach led to
two profiles ≥50% complete per touched object. Some STR profiles were obtained more
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than once thereby increasing the confidence.


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Keywords
forensic science; touched items; fingerprints; admixed samples; substrates; short tandem
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repeat

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1. Introduction
Numerous items recovered from crime scenes that are routinely examined by
forensic biology laboratories are most commonly associated with homicides, felony
assaults, sexual assaults, robberies, and burglaries. These items may include handled

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objects, such as weapons or tools that were touched by one or multiple persons. DNA
recovered from these items most likely originated from skin cells. Skin is remarkably

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dynamic; cells move from the basal to the upper epidermal layer of the skin as they
mature. During this process their cytoplasm condenses and becomes highly keratinized

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and their nuclear DNA becomes fragmented through apoptosis [1]. Daily, thousands of
skin cells are shed and transferred onto items the skin comes in contact with [2-5]. A

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large portion of these skin cells/flakes do not contain nuclei. However, forensically
informative STR profiles can be obtained from touched samples [6, 7]. Many possible
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sources of DNA may contribute to these samples. In part, some of the condensed cells
still have nuclear DNA [6]. Skin flakes may serve as vectors for extracellular DNA
transferred from other body surfaces (eye, nose, and mouth) as well as from sebaceous
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and sweat glands [4, 8-11]. Therefore, cells from diverse areas may be present in so-
called ‘touch’ samples (reviewed in [10]). Consequently, it is likely to expect great
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variation in quality and quantity of DNA from such evidence [7, 12, 13]. In addition,
external conditions may affect the DNA recovery from touched evidence, such as (i) a
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time lag between deposition and evidence collection [14], (ii) surface of substrate, and
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(iii) the length of time of the contact to transfer cellular material (reviewed in [10]). It was
shown, despite poor DNA quantities, a routine contact for as little as 10 seconds can
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lead to 3 ng of DNA [15, 16]. Understanding the challenges inherent to touched DNA
samples, and designing a work flow to address them, will improve the quality of the STR
profiles obtained from such samples. This may be of crucial importance to some cases,
where touched evidence may be the only available source of information.
In casework, touched DNA samples are often difficult to interpret because of low
DNA amounts, DNA degradation and/or the contribution of DNA from multiple
individuals. DNA mixtures are likely because multiple individuals could have handled
items found at crime scenes prior to their collection as an item of evidence. Routinely
used methods of sample collection [16, 17] tend to generate admixed DNA samples,
which only can be used for comparisons and therefore do not provide as much
investigative information as single source or deconvoluted mixture DNA profiles.

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In this study, we used a tested protocol [12] for sample collection (swabbing
using 5% Triton X-100), DNA extraction (using an enzyme that works at elevated
temperatures), and PCR amplification (AmpFlSTR® Identifiler® using an increased cycle
number: 31) in an attempt to overcome some of the inherent challenges in developing

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STR profiles from touched items. We tested fingerprints stored for different time periods
and on commonly touched substrates. In addition, we assessed mixtures by sampling

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multiple sections of an item, generating several samples that are processed separately
[18]. To our knowledge, this is the first time that this approach was tested on touched

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three person mixtures.

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2. Material and Methods
2.1 Sample collection MA
This study was approved by the New York City Department of Health and Mental
Hygiene Institutional Review Board that oversees research involving human subjects for
the Office of Chief Medical Examiner (OCME) (IRB# 12-058). Volunteers who
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participated in this study read and signed the consent form before donating fingerprints.
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Each volunteer was assigned with a code to anonymize the sample. Six volunteers
contributed to this study.
Volunteers were asked to refrain from washing their hands for at least two hours
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prior to sampling for all experiments. Fingerprint samples were taken by pressing right
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and left thumbs for a few seconds (~ 3s) on various substrates that are commonly used
[glass (microscope slide, 25 mm x 75 mm, of which 19 mm were frosted for labeling),
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plastic (sheet protector made of polypropylene, Avery®), paper (office paper), and metal
(a US Quarter dollar, 24.26 mm in diameter, made of 91.67% Cu and 8.33% Ni)]. Prior to
collecting fingerprints, the substrates were decontaminated by immersing the entire
object in 10% bleach, followed by water, and 70% ethanol. The paper was UV irradiated
for 30 min in the NuAire biosafety cabinet (NuAire, Plymouth, MN). Fingerprints on
plastic, paper, and metal were stored in closed, decontaminated boxes for 3 days, to
allow for an effect on the deposited cellular material and to mimic casework samples.
Fingerprints on microscope slides were stored at room temperature in closed and open
decontaminated boxes in a laboratory, for the following time periods: 1, 3, 10, 20, and 40
days. For the mixture study, the body of an empty beer bottle (amber glass: height of
bottle: 23 cm, height of trunk: 12 cm, diameter of trunk: 6 cm) was held in the hand of
each volunteer for 60 seconds, allowing for the possibility of both the palm and fingers to

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touch the bottle. No further guidance was given. The trunk of the beer bottle, from which
the label was removed, was initially etched into six equivalent sections; each section
measured 6 cm x 6.3 cm. Three people held the bottle consecutively with their dominant
hand. The order of people touching the bottle was alternated. The beer bottle was kept

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unpackaged, on a laboratory bench at room temperature and was swabbed one day
following touching.

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Fingerprints were swabbed under the Olympus SZX-16® stereomicroscope
(Olympus of the Americas, Central Valley, PA, USA) using a small portion of a sterile,

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UV irradiated cotton swab (Dynarex, Orangeburg, NY), held with reverse clamp
tweezers (Dumont N5 dissecting tweezers, Ted Pella, Stockholm, Sweden), and

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moistened with 4 µl of 5% Triton X-100 (Sigma-Aldrich, USA) [19]. A clean substrate was
swabbed alongside each batch of samples as negative control. If this substrate control
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tested positive for DNA the samples of this batch would be discarded.

2.2 DNA extraction and quantification


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DNA was isolated from swabbed samples using prepGEM® Tissue extraction kit
(Zygem, Corporation Ltd, New Zealand) following the manufacturer’s instructions.
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Briefly, swabs were incubated in 20 µl of prepGEM® tissue extraction mixture for 15 min
at 75oC, followed by 5 min at 95oC using a GenAmp 9700 thermal cycler (Life
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Technologies, Applied Biosystems, Foster City, CA) [12]. The extracted DNA was
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quantified using Alu-based real-time PCR for human DNA adapted from Nicklas and
Buel [20]. Two microliter of extracted DNA was used as template in a 25 µl reaction
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using SYBR-Green (Life Technologies Molecular Probes, Grand Island, NY) and Ampli-
Taq Gold (Life Technologies) on a Rotorgene™ Q (Qiagen, Valencia, CA). Samples
were quantified for the time course study as indicated and for the three-person mixtures.
For the comparison of left versus right fingerprints all samples were used regardless if
quantified or not, since right and left hand were always treated equally. The samples
were not quantified for the tests of various substrates.

2.3 Amplification of STR loci


All samples were amplified using AmpFlSTR® Identifiler® PCR Amplification Kit
for 31 cycles (Life Technologies Applied Biosystems, Foster City, CA), following the low-
template DNA testing protocol validated at the OCME [16]. The DNA of one sample was
extracted in 20 µl. If the sample was quantified (2µl), 5µl were used as template for the

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STR amplification, which was performed in triplicate (3x5µl = 15µl). If the sample was
directly used for STR amplification, 6µl were used as template (3x6µl = 18µl). Besides
the DNA template, each STR amplification contained 2.5 µl Primer Mix, 5 µl Reaction
Mix, and 0.5 µl AmpliTaq Gold DNA Polymerase (5 U/µl). A negative control was used

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for each STR amplification batch, and if positive, the entire batch was disregarded.

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2.4 Capillary electrophoresis and analysis
Amplified PCR products were separated on a 3130xl Genetic Analyzer (Life

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Technologies Applied Biosystems, Foster City, CA) at 3 kV for 20 seconds. Samples
with overblown signals were reinjected at 1 kV for 22 seconds, and samples with low

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signals were reinjected at 6 kV for 30 seconds. Data analysis was performed using
GeneMapper v. 4.0 software (Life Technologies Applied Biosystems). The peak
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amplitude threshold in GeneMapper was set to 75 RFU detection. Peak ratio cut off
value for tetra-nucleotide markers was set to 0.1.
The STR amplification was carried out in triplicates. Alleles that were present in
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at least two of three amplifications were considered part of the consensus profile. Only
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these alleles were assigned to the DNA profile of the sample [16]. Using in-house
developed interpretation guidelines for single source and mixed low template DNA
samples, the donor’s DNA profile was assigned [16]. For single-source samples,
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heterozygous alleles were determined based on the two tallest peaks observed in at
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least two of three amplifications, whereby the smaller peak must be ≥50% of the larger
peak. Homozygous alleles must appear in all three amplifications, in order to be
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assigned to a profile. Any additional peaks, repeating or not, must be <30% of the major
peak in order to call the locus a true homozygote [16]. For mixed samples, only clear
major components were used. Heterozygous alleles were assigned when they appeared
in all three amplifications and showed a peak balance greater than or equal to 50% in
two out of the three replicates. Homozygous alleles must also appear in all three
amplifications and must be clearly the major peak, while the minor peaks were <30%
[16].
Identifiler® kits amplify 15 autosomal loci plus Amelogenin, for a maximum of 30
autosomal alleles per donor. New York and the U.S. require a minimum of six core loci
for upload to SDIS (State DNA Index System) and ten to NDIS (National DNA Index
System). This study used percentages in order to compare the outcomes. The profile
percentage, or profile fraction, was obtained by dividing the number of alleles detected

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by the number of known alleles for that individual. A 70% threshold was determined,
which is rather a conservative approach, for database upload and a 50% threshold was
set for comparison of the STR profile to databases.

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2.5 Data analysis
Data analysis was performed using SPSS version 21 (IBM, Armonk, NY) and

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Microsoft Excel.

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3. Results
3.1 Time course

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A time course study was performed in order to assess the effects of time on
fingerprints, in terms of deterioration of the corresponding STR profiles. Fingerprints on
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glass slides were stored in either cleaned open or closed boxes for the following time
periods: 1, 3, 10, 20, and 40 days. Of 643 fingerprints, 25 mixtures were found and
excluded from data analysis.
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Figure 1 shows the profile fractions, obtained from 381 protected fingerprints that
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were stored in clean closed boxes for the time periods specified above. The profile
fractions varied greatly for all time points. Surprisingly, a great portion of fingerprints,
more than a quarter of those tested, showed profiles that were at least 70% complete
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after 40 days of storage. In addition, full profiles were also obtained for 5 (13.9%)
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samples. The shorter the storage time was, the higher the median percentage of the
obtained STR profiles; thus revealing a degree of degradation. ANOVA analysis showed
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significance between these time points and t-tests specified that the profile fractions of
day 1 and day 3 did not differ significantly (p=0.24), but differed significantly between
day 1 and day 10 (p=0.013), as well as for longer time periods (p<0.0001).
An additional 82 fingerprints, stored in open boxes, were also exposed to in-
house conditions of the laboratory area, e.g. dust, for specific time periods. Figure 2
shows the comparison of the corresponding profile fractions, which too varied greatly.
The direct comparison for 10 and 20 days is shown in Figure 2A. After 10 days, the
profile fractions of fingerprints stored in closed boxes seem to be higher than for
fingerprints stored in open boxes (Fig. 2A). A t-test revealed significance (p=0.033). This
difference was not seen after 20 days of storage (p=0.3).
For the tests above, the extracted DNA was quantified. Since we were working
with fingerprints, the problem is rather to have too little than too much DNA; therefore we

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decided to skip the DNA quantification and to consume the samples in amplification. For
another 155 fingerprints that were stored in open boxes, for the time periods 1, 3, and 20
days, the extracted DNA was not quantified. The comparison to storage in closed boxes
revealed a difference for one day, which was not seen after three and 20 days (Fig. 2B).

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A small difference was seen for 20 days storage in open boxes (compare green box-plot
of day 20 from Fig. 2A with 2B). From these results, we concluded that the difference

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between the protected storage in closed boxes and in open boxes in laboratories is
negligible. Nevertheless, it should be noted that after almost three weeks of storage

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more than 25 percent of the profiles were ≥70% complete.

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3.2 Analysis of fingerprints from right versus left hands
Three right handed volunteers contributed to this study. The 618 samples (643
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minus 25 mixtures) were analyzed separately, in order to find out whether the dominant
hand tends to deposit more or less cellular material.
The fractions of DNA profiles obtained from the left hands were more complete
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than from the right hands for all participants (see Suppl. Figure 1). T-tests revealed that
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the difference was not significant (p=0.206) for volunteer 1, but was significant for the
other two (p=0.001 for volunteer 2 and p=0.020 for volunteer 3). The differences of the
obtained profile fractions between left and right thumb were moderate. The profile
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fractions of volunteer 1 and 3 were similar, but lower than from volunteer 2.
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3.3 Fingerprints on various substrates


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To test the effect of various substrates on cellular material, fingerprints were


placed on glass (microscope slide), metal (a US Quarter dollar, made of 91.67% Cu and
8.33% Ni), paper, and plastic (sheet protector made of polypropylene). In order to allow
for an impact of these substrates and to mimic casework samples, fingerprints were
swabbed after three days of storage at room temperature. We just showed that three
days storage can result in slightly less complete STR profiles; however the difference
was not significant (Fig. 1). Each substrate was tested with 32 fingerprints. From 128
samples, five mixtures were detected and excluded from analysis. Seven STR profiles,
six from glass and one from plastic, were overblown and reinjected (see Material and
Methods: 2.4 Capillary electrophoresis and analysis). The completeness of DNA profiles
ranged widely. From each of these substrates, except metal, full profiles were obtained.
Over half of the fingerprints on glass slides led to ≥70% complete STR profiles. By

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comparing Figures 1 and 3 (3 days on glass), no difference can be seen in the outcome.
Over a quarter of fingerprints on plastic led to ≥70% profiles, but noticeably fewer were
generated from paper (Figure 3). Almost no profiles were obtained from metal (Figure 3).

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3.4 Analysis of three-person mixtures
To assess mixtures of touched items, an empty beer bottle, a typical casework

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evidence item, was used. The trunk was etched into six equally sized sections, which
each measured 6.0 cm x 6.3 cm. This sizing, bigger than the glass slides used before,

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was chosen in order to increase the probability of obtaining enough cellular material for
database eligible STR profiles.

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The bottle was cleaned thoroughly. Three volunteers held the bottle for 60 s
consecutively. Throughout the study the order of volunteers was changed, creating six
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series. Each section was swabbed entirely, generating six samples per bottle. The
samples were processed separately for DNA extraction and PCR amplification, which
was done in triplicate. The study included thirty-six bottles, generating 216 samples.
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Data analysis revealed DNA mixtures for all bottles. The six samples generated
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per bottle led to a good estimate of the total number of contributors that touched the
bottle. Twenty-six of 36 bottles revealed three distinct different people (Table 1). For
eight additional bottles, two people were detected plus an indication of a third person.
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As noted before, the percentage of profiles detected varied a great deal.


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However, for this study only profiles that were ≥ 50% complete were considered: ≥50-
69% for potential database comparison, and ≥70% for uploading. As shown in Table 1,
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over 90% of the tested 36 bottles resulted in informative STR profiles.


Data analysis utilized the results of the three PCR amplifications per sample and
followed the high sensitivity interpretation protocol of single source and mixed samples,
developed at the OCME [16]. Possible outcomes included: inconclusive, 2 or 3 person
mixture and single source. Table 2 shows the outcome of the six samples per bottle.
From all samples generated in this study (n=216), most were two-person mixtures
(n=159), of which some could be used for deconvolution, only if the major contributor
could clearly be determined. All single-source profiles (n=24) and a few of the three-
person mixtures (n=20), led to informative profiles (Table 2).
From the majority of bottles (n=21) multiple STR profiles were obtained and often
profiles of the same person were obtained (n=18). This is specifically valuable if profiles
were below 70% complete (see bottles 2 and 16 in Table 2). Altogether 74 profiles

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(≥50% complete) were obtained from 36 bottles, which equals 2 profiles on average per
bottle.
Considering the order of touching, it was found that the DNA of the last person
who touched the bottle was mostly detected in three of the six series: A-B-C (bottles 1-

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6), B-C-A (bottles 19-24), and B-A-C (bottles 31-36), see Table 2. However, donor C
contributed to most profiles in five of the six series, even when being the first person to

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have touched the bottle: A-B-C, B-A-C, C-B-A, C-A-B, and A-C-B. For the remaining
series it was person A, who contributed to most profiles, being also the last person to

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have touched the bottle: B-C-A. Further, by counting the profiles per person (A: n=22, B:
n=9, and C: n=43), it seems that person C shed most cellular material leading to the

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most STR profiles, followed by persons A and B. Therefore, we assume that the order of
touching is not as relevant as the amount of cellular material that is deposited by
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touching, contributing to informative STR profiles.

4. Discussion
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In this study, we thoroughly examined touched items (e.g. fingerprints) for their
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ability to produce informative DNA profiles within forensic casework. Over 700
fingerprints and over 200 mixed samples from 36 bottles were processed. A previously
tested protocol was used to swab fingerprints of substrates, extract DNA and amplify
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STR profiles [12] with the goal of obtaining ≥70% profiles that were considered usable
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for databases in the U.S. such as CODIS [25]. It was shown that approximately 70% of
the fingerprints processed with this protocol were at least to 70% complete [12]. This
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outcome is comparable to other recent protocols, where 71% of the processed


fingerprints led to database eligible profiles [7]. Extracted DNA from touched items,
processed through routine workflow, showed a great variation in quality and quantity [12,
13], which was also confirmed in this study. For that reason fingerprints are considered
challenging and potentially unreliable for forensic DNA testing, especially if samples
contain DNA of more than one person [26, 27].
For each extraction and amplification batch a negative control was added and if
tested positive the samples would have been discarded. Approximately 4% of mixtures
were detected from both batches (25/643 and 5/128) and disregarded. We assume,
since the negative controls were negative, one of the reasons for occurrence of these
mixtures could be secondary DNA transfer. This rate is low compared to other studies,
where the secondary transfer ranged from 10% to 85% [2, 11, 28-31], but could be a

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consequence of the protocol we utilized. The amplification was performed in triplicate


and a consensus profile (alleles must be present in at least two of three amplifications)
was used along with the High Sensitivity interpretation guidelines to assign alleles [16].
This interpretation method eliminates spurious alleles and considers the effects of

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increased allele drop out [32]. The 4% rate is in concordance with an earlier study we
performed [12].

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The effect of time on fingerprints was investigated by evaluating the resulting
STR profile quality, since there is limited research on this topic. However, successful

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DNA typing from touched objects was reported 24 h after deposition [29], which was
confirmed by this study. Full profiles were even received after 40 days of deposition

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(protected in-house storage), which is in concordance with an earlier study [14].
However, since we tested several time points we could show that the portion of
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database eligible profiles, decreased over time. A slight decrease was noticed after three
days, but was not significant, while time periods exceeding 10 days were. We further
tested the influence of the laboratory environment, by storing the fingerprints in open
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boxes, but concluded that there is no difference, possibly because the air was filtered,
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reducing dust and pollution.


Since we collected thumb prints from left and right hands, we were able to
compare their DNA profile quality. Three right-handed individuals were tested. The left,
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the non-dominant hands, resulted in slightly more complete profiles, which was not
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always significant. Possible abrasion may happen on the more frequently used,
dominant hand, while the non-dominant hand may still carry material suitable for DNA
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testing. However, conversely, the dominant hand is also often used for touching the
face, or other areas that contain sebaceous glands, thereby transferring DNA [4, 10, 11].
Therefore, no conclusion can be drawn.
More important for DNA recovery and the quality of the subsequent STR profiles
is the impact of substrates. Four substrates, glass, paper, plastic (sheet protector made
of polypropylene) and metal (US Quarter dollar) - materials that are commonly used in
households and offices - were tested. Glass led to the best STR profile quality, followed
by plastic and paper. Almost no profiles were obtained from metal, which could be due to
the possibility that Cu- and Ni-ions provoke DNA degradation [21] and the metals bind
and damage DNA [22, 23]. The approximate size of a thumb-print was 3 cm x 2 cm (6
cm2), revealing that the surface area of the US Quarter dollar was slightly smaller (4.62
cm2). Previous studies also documented best DNA recovery from glass, when comparing

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to metal and wood [33]. However, the metal was not specified [33]. It would be
interesting, for future studies, to compare various metals, such as gold, silver and
messing. These metals mimic crime scene items but also bare different chemical
reactivity. It was also shown that DNA could be recovered from paper [34], whereby a

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targeted sampling may improve the outcome for casework [35]. The substrates we
tested had rather smooth surfaces. Recently, more jagged and textured items were

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tested by holding the object firmly in a fist, leading to higher DNA yields from wood
(piece of timber) and fabric than from glass [2], demonstrating that more research is

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needed to reach to more consistent results.
Obtaining forensically useful STR profiles from admixed fingerprint samples is

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still a challenge, in particular when dealing with initially small or trace amounts of cellular
material [26, 27]. If similar amounts of donors’ DNA are present, the resulting STR
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profiles cannot be deconvoluted to their individual contributors [36].
In order to increase the value of admixed DNA evidence, sampling of multiple
sections was performed. Some studies emphasized the importance of sectioning
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evidence and processing these samples separately [10, 18, 37]. We used a beer bottle,
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of which the trunk was sectioned into 6 equally sized segments, which was touched
consecutively by 3 persons for 1 min. These sections were larger than a single
fingerprint, because some parts of these sections remained untouched. It was shown
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that DNA shed from the palm was significantly less than from two fingers [24]. The
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bigger the section is, the higher the chance of having a mixture that cannot be resolved.
On the other hand, with smaller sections, the chances of encountering too little material
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for a usable result are higher.


We tested 36 bottles, generating over 200 samples. All bottles revealed mixtures.
Most samples identified two contributors, but when considering all six samples per
bottle, the presence of three persons was indicated. Thus, sampling multiple sections led
to an accurate estimation of the numbers of contributors.
This approach simplified the complex mixtures by reducing the number of
contributors for most samples. Over 90% of the bottles resulted in informative STR
profiles, and 75% produced profiles that were ≥70% complete. Many bottles revealed
several profiles that belonged to the same person, increasing the confidence of the
outcome. We proved, to our knowledge for the first time, that this approach could resolve
admixed fingerprint samples by providing individual profiles. It would be interesting to

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test this approach on mixtures consisting of more than three people and on other items
found at crime scenes.
Taken together, this study provided valuable facts, when taking and processing
touched items or fingerprints as evidence.

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5. Conclusions

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Over 900 fingerprint samples were examined to gain more insight in these
challenging samples with the goal of obtaining ≥50% STR profiles.

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The effect time has on fingerprints is significant, however, even after 40 days full
profiles can be obtained. The effect substrates have on fingerprints seems to be

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considerable: certain metals lead to DNA degradation therefore no STR profiles will be
obtained, while the effect of other tested substrates was low: glass was better than
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plastic and plastic was better than paper. Sampling multiple sections can help to resolve
mixed touched evidence, by reducing the complexity of the sample.
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7. Conflict of interest
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The authors have declared no conflict of interest.


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Tables

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Table 1: Number of contributors and different STR profiles per bottle
# of contributors identified # of # of
STR profiles

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per bottle bottles bottles

3 26 3 different profiles ≥50% 0

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≥70% 5
2 with a hint of a third 8 2 different profiles
≥50-69% 3

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≥70% 19
1 profile
2
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0 profiles - 3
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Table 2: Detailed outcome of three-person mixture study


Outcome
Series of Number of different persons:
2 3
persons Bottle Single database eligible profiles
Inconclusive persons persons
touching source (%)*
mixture mixture

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1 2 4 1: C (67)
2 4 2 2: A (61,50), B (93)
3 6 2: A (82), C (80,80,70,70)
A-B-C

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4 6 1: B (80)
5 4 2 1: C (100,90)
6 4 2 1: C (100,93,87)

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7 5 1 1: C (93)
8 4 1 1 1: A (100,71,68)
9 2 2 2 1: C (97,93,63)

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C-B-A
10 5 1 2: A (100,100,50), B (89)
11 3 3 1: C (100,100,100,97)
12 3 3 2: A (86), B (60)
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13 1 4 1 2: A (100), C (57)
14 5 1 1: B (100,53)
15 1 4 1
C-A-B
16 2 4 1: C (57,50)
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17 3 1 2 1: A (100,79,79,64)
18 5 1 1: C (100,90,77,77)
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19 5 1
20 6 2: A (100,68), B (100)
21 4 2 2: A (71), B (100)
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B-C-A
22 6 1: A (86,50)
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23 4 1 1 1: C (77)
24 5 1 1: C (87)
25 1 5 1: C (87,83,73)
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26 6 1: C (60)
27 1 4 1 1: C (63)
A-C-B
28 1 5 1: C (83)
29 5 1 1: A (57)
30 6 1: C (80,73,60)
31 2 2 2 1: C (100,100,100)
32 1 5
33 5 1 1: A (100)
B-A-C
34 1 3 2 1: C (79)
35 2 3 1 1: C (63)
36 5 1 2: B (100), C (83,57)
Total 13 159 20 24
* Only STR profiles ≥50% are listed; letters in bold indicate that at least one profile ≥70%

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Figure legends:

Figure 1. Time course study of protected fingerprints, stored in closed boxes at room
temperature: X-axis: storage time in days: 1 (n=66), 3 (n=65), 10 (n=67), 20 (n=147),

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and 40 (n=36); Y-axis: fraction of STR profiles. Following DNA extraction, the samples
were quantified and 5 µl was used for each PCR amplification, which was done in

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triplicate.

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Figure 2. Comparison of storage conditions: A: blue bars (dark gray): storage in closed
boxes; green bars (light gray): storage in open boxes (5 µl DNA template for STR

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amplification); X-axis: storage time in days: 10 (closed: n=67, open: n=40), 20 (closed:
n=147, open: n=42); B: blue bars (dark gray): storage in closed boxes; green bars (light
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gray): storage in open boxes, sample consumed (6 µl DNA template for STR
amplification); X-axis: storage time in days: 1 (closed: n=66, open: n=42), 3 (closed:
n=65, open: n=52), and 20 (closed: n=147, open: n=61); Y-axis (both panels): fraction of
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STR profiles.
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Figure 3. Fingerprints on various substrates: X-axis: substrates: glass (microscope slide,


n=32), metal (US Quarter dollar made of Cu and Ni, n=32), paper (n=32) and plastic
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(sheet protector made of polypropylene, n=27); Y-axis: fraction of STR profiles.


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Acknowledgment
Parts of this project were supported by award No. 2012-DN-BX-K043 funded by
the National Institute of Justice, Office of Justice Programs, U.S. Department of Justice.

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The opinions, findings, and conclusions or recommendations expressed in this
publication/program/exhibition are those of the authors and do not necessarily reflect

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those of the Department of Justice.
We are grateful to Stacey A. Klempner, Rosni A. Patel, Boramchan Lim, Michael

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B. Goldstein, Nicholas F. Kaaya, Feleicia Lotmore and Elizabeth C. Valle for their
assistance in sample collection, DNA extraction, amplification, capillary electrophoresis

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and data interpretation of the results. We are very thankful to Kyra McKay for careful
reading of this manuscript. MA
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Highlights:

 After 40 days, full profiles were obtained from fingerprints, despite degradation.
 Best results were obtained from glass, followed by plastic, and paper.

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 Sampling multiple sections reduced complexity of touched mixed objects.
 Assessment of three-person mixtures led to database eligible STR profiles.

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