Journal of Forensic and Legal Medicine 61 (2019) 78–81

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Journal of Forensic and Legal Medicine

journal homepage: www.elsevier.com/locate/yjflm

Short communication

Successful analysis of a 100 years old semen stain generating a complete T

DNA STR profile
Alessandro Mameli, Carmine Maria Scudiero, Giovanni Delogu, Maria Elena Ghiani∗
Reparto Investigazioni Scientifiche Carabinieri di Cagliari, Piazza San Bartolomeo 29, 09126, Cagliari, Italy

A R T I C LE I N FO A B S T R A C T

Keywords: We performed forensic investigations on a handkerchief containing seminal fluid deposited 100 years earlier.
Forensic genetics The aim was to verify the possibility to achieve a complete genetic profile exceeding the limit of success reached
Aged semen stain until today with a partial semen stain profile stored up to 50 years. The current forensic methods were carried
Alternative Light Source out: Alternative Light Source (ALS), Prostate-Specific Antigen (PSA) Test, autosomal and Y-chromosome Short
Prostate-Specific Antigen Membrane Test
Tandem Repeats (STRs). ALS inspection was a crucial method in the success of the analysis. On a dried semen
Assays
stain, fluorescence remains constant for a very long time, allowing possibility to minimize the fabric sample to
Autosomal STRs
Y-chromosome STRs analyze, in order to preserve specimen for future defense guarantees or to protect historical finding. We achieved
positive results in 3 of 6 traces, PSA and DNA results were concordant, only those cuttings that tested positive for
PSA yielded DNA profiles. A complete male profile was generated by AmpFlSTR Identifiler Plus and 16 loci
profile by AmpFLSTR Yfiler. Considering some historical sources that attributed this handkerchief dated 1916, to
a famous Italian poet, the DNA profile found on the handkerchief was compared with a living male descendant,
and the comparison of the Y-STR between the two gave positive results, confirming the reliability of the out-
comes. The findings achieved by the current forensic method empower the application of this methodology to
other forensic cases, especially in past unresolved cases.

During an investigation following criminal actions, it has been
shown that body fluid traces can be essential as they are very in-
formative for the investigation itself. Moreover can often happen that
there is the need for biological fluid to be reinvestigated, in con-
sideration of current techniques in the forensic field to identify the
perpetrator several decades after the crime has occurred. Recent studies
of seminal fluid traces dated from 20 up to 50 years old, indicate that
with current forensic method it has been possible to succeed in the
analysis.1–6
The following study was performed to investigate a handkerchief
which contained biological traces of what was presumed to be seminal
fluid deposited 100 years earlier, using forensic examination methods
including: Alternative Light Source (ALS), Prostate-Specific Antigen
(PSA) Membrane Test Assays, autosomal and Y-chromosome Short
Tandem Repeats (STRs) analysis. Our aim is to verify whether it is
possible to exceed the limit of success achieved with semen stain stored
up to 50 years old1,6 where only a partial profile was obtained. It was
essential to preserve the handkerchief, being a historical find, it was
possible highlighting parts covering the highest content of biological
material in order to investigate the smallest fabric samples without
destroying the specimen. Molecular analysis generally involves the
destruction of material. It is important to minimize the conflict between
this fact and the need to preserve defense guarantees or to protect
historical specimen.
For the first time, from semen stain stored up to 100 years, we
achieved positive results carrying out a complete AmpFlSTR Identifiler
Plus profile and sixteen loci profile by AmpFLSTR Yfiler. In order to
guarantee the quality of acquired data which some historical sources
had attributed to this 1916 handkerchief to a famous Italian poet, the Y-
STR DNA profile found on the handkerchief was compared with a living
male descendant, and the comparison between the two gave positive
results, confirming the reliability of the outcomes.
1. Materials and methods
1.1. Samples and procedure
A white handkerchief dated to 1916, kept at room temperature in an
envelope within a crate, and letters which contained biological traces
presumed to be seminal fluid of the same age, has been attributed to a
famous Italian poet and also a swab of the poet's direct descendant (a
great grandson, informed consent was obtained) were subjected to

∗
Corresponding author.
E-mail addresses: alessandro.mameli@carabinieri.it (A. Mameli), ghiani.elena@gmail.com (M.E. Ghiani).

https://doi.org/10.1016/j.jflm.2018.11.008
Received 13 August 2018; Received in revised form 8 November 2018; Accepted 16 November 2018
Available online 19 November 2018
1752-928X/ © 2018 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.
A. Mameli et al.
forensic analyses. In order to preserve the find we identified the surface
of fabric that contained the highest amount of biological material. We
employed the most stringent analysis criteria handling the sample as it
was an ancient DNA.7 The laboratory is designed as described in
Goodwin et al.8 to reduce the possibility of introducing contamination,
the work flows goes through the different processes in one direction
starting with sample reception and finishing with the post-PCR analysis.
Our laboratory is accredited in compliance with European Standard
UNI CEI EN ISO/IEC 17025. DNA extractions were carried out in ded-
icates areas for the two classes of samples (specimen and a comparison
sample). Multiple extractions from the specimen have been performed
in order to guarantee the reproducibility of the result on a probably
degraded sample. All DNA extractions and PCR reactions included po-
sitive and negative controls, all steps of analysis were replicated at least
twice, quantification of target DNA were performed by real time (RT)
PCR.
The male descendant sample was analyzed only after the specimen
DNA analysis was completed.
1.2. Latent traces detection
In order to identify the biological traces in the handkerchief we
exploited the properties that body fluids, such as sperm, saliva and the
vaginal fluids exhibit under UV light, as they are naturally fluorescent.
To detect the areas with highest level of biological material, the
handkerchief underwent a Crimescope CS-16 inspection in a darkroom
with wavelength ranging from 415 to 490 nm.9 Six latent traces (la-
belled F1eF6, Fig. 1) were analyzed, and semen stains were success-
fully visualized at a wavelength of 445 nm. Approximately 1 cm2 of
each trace was cut and half piece of material was used for PSA test and
the rest for DNA analysis.
1.3. Confirmatory semen test
PSA-check-1 (VED-LAB, Parc du Londeau, 61 000 Alencon, France),
was performed on the fluorescence stain to detect prostate-specific
antigen using an immunochromatographic method.2 5-mm2 of each
fluorescence stain was cut from the handkerchief and placed in a micro
tube.
The fluid material from the handkerchief was extracted in 500 μl
isotonic sodium chloride solution (0,9%). 250 μl of the extracted solu-
tion was placed in a sample well, and the presence of prostate-specific
antigens determined after 10 min. Positive and negative controls were
used to validate the assay.
Fig. 1. Handkerchief containing biological traits, presumably seminal fluid;
withdrawals spots of F1, F2, F3, F4, F5 and F6 traces carried out in corre-
spondence of the luminescence highlighted by the CrimeScope CS-16.
79
Journal of Forensic and Legal Medicine 61 (2019) 78–81
1.4. DNA extraction
DNAs were isolated from the samples using a magnetic-particle
technology based on the EZ1 DNA Investigator Kit automated on the
instrument EZ1 Advanced XL (Qiagen).10
The six samples (F1eF6) from the handkerchief, about 5 mm2 of
fabric, were placed in a 2 ml sample tube, incubated in 190 μl of G2
digestion buffer (EZ1 DNA Investigator Kit, Qiagen) with 10 μl of pro-
teinase K at 56 °C for 18 h, rather than 15 min as prescribed in the in-
struction manual10 in a Thermomixer Confort (Eppendorf).
Each lysate was transferred into a new 2 ml sample tube without any
solid material. Samples were then loaded in the EZ1 Advanced XL in-
strument for automated DNA isolation, running the “DNA purification
Trace-protocol”.11 The DNA was eluted in a volume of 50 μl sterile
water.
The DNA sample was preserved from contamination with foreign
DNA, and a reagent blank was included in the extraction process and
was carried forward for all subsequent step analysis. DNA extractions
were carried out in dedicated areas (one laboratory for trace DNA and
another laboratory for comparison samples) meaning the specimen and
a comparison sample never came into contact.
1.5. DNA quantification
The quantity of the extracted DNA from both samples was de-
termined by real time PCR using the Quantifiler™ Human DNA
Quantification Kit (Applied Biosystems). The reactions were carried out
in an ABI PRISM 7500 Detection System (Applied Biosystems) ac-
cording to the manufacturer's instructions.12 The kit includes an in-
ternal PCR control formulated into each reaction to identify samples
that may contain PCR inhibitors.
1.6. STR and Y-STR profiling
3 DNA templates, 240 pg (F1 trace), 260 pg (F2 trace) and 290 pg
(F4 trace) were amplified using the AmpFlSTR Identifiler Plus kit, a STR
multiplex assay that amplifies 15 tetranucleotide repeat loci and the
Amelogenin gender-determining marker (Applied Biosystems).13 Y-STR
profiles were generated using the AmpFLSTR Yfiler PCR Amplification
kits, that amplifies 17 Y-STR loci (Applied Biosystems).14 All amplifi-
cations were carried out in a GeneAmp®PCRSystem 9700 (Applied
Biosystems) following the manufacturer's instructions.
Capillary electrophoresis of the amplicons was performed using the
3500 Genetic Analyzer System (Applied Biosystems), data collection
was conducted using the 3500 Series Data Collection Software 2
(Applied Biosystems), and data analysis was performed using the Gene
Mapper IDX v.1.3.1 software (Applied Biosystems) according to the
manufacturer's recommendations.15 PCRs were run for two aliquots of
each sample in different time. Size calling was carried out im-
plementing the Local Southern Method,15 and the peak amplification
threshold was set to 60 rfu.
1.7. Statistical analysis
The frequency of the genotype detected for the handkerchief was
calculated through the random match probability (RMP) using the
Italian allele frequencies available from the literature.16,17 The geno-
type frequency for each locus was calculated using p2 for the homo-
zygote and 2pq for the heterozygote loci (p and q are the allele fre-
quencies). The overall profile frequency was calculated by multiplying
the genotype frequency at each locus.18 To determine the frequency of
the Y haplotype a search of the YHRD database19 was performed.
2. Results
The white handkerchief revealed interesting luminescent areas
A. Mameli et al. Journal of Forensic and Legal Medicine 61 (2019) 78–81

Table 1 In order to confirm results, two aliquots of each DNA extract were
Autosomal STR loci results on the handkerchief aged seminal traces and com- processed and all results were concordant. Amplification of the F1 and
parison data with the male live descendant. F2 traces resulted in a 9 loci for the F1 trace and 15 loci for the F2 trace
Loci STR F4 TRACE F1 TRACE F2 TRACE Male descendant (Table 2). The Y-STR profile from the F1 and F2 traces matched re-
spectively with the 9 and 15 loci of the profile from the F4 trace
D8S1179 10–14 10–14 10–14 13–14 (Table 2).
D21S11 29–29 29–29 29–29 29–30
The DNA from the reference sample of the poet's great grandson
D7S820 8–11 / / 8–10
CSF1PO 10–10 / / 10–12 gave a total yield of 9 ng. A full autosomal and Y profile was obtained.
D3S1358 15–15 15–15 15–15 15–15 The Y-STR profile of the F1, F2 and F4 traces matched respectively with
TH01 6–9 / 6–9 6–8 9, 15 and 16 Y-STR loci of the male-descendent (Table 2).
D13S317 9–13 / / 11–13
To determine the frequency of the poet Y haplotype a search of the
D16S539 12–13 / / 9–12
D2S1338 17–17 / / 17–20
YHRD database19 was performed. No match was found among 165 259
D19S433 13–14 13–14 13–14 13–14.2 haplotypes, which included 3366 Italian males.
D5S818 11–12 11–12 11–12 11–13 Moreover, there was no match of the STR and Y-STR profiles gen-
vWA 16–19 16–19 16–19 17–19 erated from the handkerchief latent traces with profiles in the labora-
TPOX 11–11 11–11 11–11 8–11
tory elimination database.
D18S51 13–16 / / 15–17
FGA 21–28 21–28 21–28 22–23
Amelogenin XY XY XY XY
3. Discussion

possibly containing biological traces. The most intense luminescent Increasingly in forensic science it is necessary to work with trace
areas (F1; F2; and F4) 3 of 6 (Fig. 1) showed the presence of prostate- amounts of DNA as there are unresolved cases that are reopened
specific antigens, confirming the hypothesis that the tissue contained comprising traces which previously had given unreliable or no results.
seminal fluid. Today, the current technologies can be of great assistance in solving
DNA was extracted from all the latent traces (F1eF6) but only three such cases. The aim of this study was to evaluate the profiling of a 100
(F1; F2; F4), gave positive results in the real-time PCR quantification. years old seminal stain using the following forensic methods: ASL
The eluted DNA results were 24 pg/μl (F1 trace), 26 pg/μl (F2 trace) (Alternative Light Source), PSA antibody–antigen reactions, autosomal
and 29 pg/μl (F4 trace). Although there is no rigid definition of low and Y-chromosome STRs, in the context that until today successful STR
template level DNA (LTDNA), some authors define it as < 100 pg of results had only been obtained with seminal stain samples dated at most
input DNA,20 while others use a threshold of 200 pg (21,22), we treated to 50 years.1,6
the extracts as low template DNA and amplified two aliquots of each Due to their optical properties, biological stains often fluoresce
extract, as suggested by Gill et al. and Caragine et al.20,23 when they highlighted with light of certain wavelengths (with or
A complete male profile was generated from the F4 trace (Table 1). without the additional use of filters).24 One method by which trace
The profile is typical of a degraded sample, with a gradual reduction in biological evidence may be identified is via the use of an Alternative
the RFUs as the amplicons increase in size. At some loci (D7S820 and Light Source (ALS). Seminal fluid stains fluorescent on a broad excita-
D13S317) there is evidence of heterozygote allelic peak imbalance. tion spectrum25–29. Using alternative light source (ALS) inspection was
The frequency of the 15-loci STR profile was 9.09 × 10−21 (i.e. 1 in a crucial method for the detection of biological stains on the hand-
1119). kerchief. It is known that dry biological stains can fluoresce for a long
Amplification of the F1 and F2 extracts resulted in a partial profile time after deposition. Our work has confirmed that biological traces can
confirmed by a consensus approach of the two replicates, with 9 loci for be detected even after a century.
the F1 trace and 10 loci for the F2 trace (Table 1). As described by Hochmeister et al.,2 PSA membrane test assays offer
A 16 loci Y-STR profile (Table 2) was produced from the F4 trace. high sensitivity and provide a rapid approach for identification of
The profile showed evidence of a degraded sample with over-amplifi- seminal fluid. Moreover, the authors obtained positive results on semen
cation of the smaller loci. The successful amplification declines with the stains on cotton cloth stored at room temperature for up to 30 years. For
size of the allele, with the result of a locus drop out event for DYS392. the above reasons we investigated the most intense luminescent areas
by an antibody–antigen reaction PSA test. Our positive results for 3 of 6
Table 2
traces showed it is possible to highlight semen stain stored for 100
Y-STR profiles from the white handkerchief aged seminal traces and the com- years. PSA and DNA results were concordant, only those cuttings that
parison data of the male live descendant. tested positive for PSA yielded DNA profiles.
Kido et al.3 reported that all loci (9 loci) were typed from seminal
Marker Allele F4 Allele F1 Allele F2 Allele male
TRACE TRACE TRACE descendant
stains stored for up to 25 years, using a commercial kit (AmpFLSTR
Profiler Kit). Nakanishi et al.5 confirmed that STR analysis using
DYS456 15 15 15 15 AmpFlSTR Identifiler™ could be applied to old seminal stains, and all
DYS389I 13 13 13 13 sixteen loci were detected in a 33-years-old sample, and five of sixteen
DYS390 23 23 23 23
loci were detected in a 56-years old sample. We obtained a complete
DYS389II 31 / / 31
DYS458 16 16 16 16 AmpFlSTR Identifiler Plus profile and a 16 loci profile by AmpFLSTR
DYS19 14 / 14 14 Yfiler, demonstrating that DNA can persist in dried biological materials
DYS385a/b 13–18 13–18 13–18 13–18 for many years, decades and even centuries. This result was possible
DYS393 12 12 12 12
since we increased number of samples taken from the handkerchief for
DYS391 10 10 10 10
DYS439 11 / 11 11 testing in order to have a better chance of success and to test the single
DYS635 22 / 22 22 origin of the samples. In order to guarantee the quality of acquired data,
DYS392 / / / 11 the DNA profile found on the handkerchief was compared with a re-
Y GATA H4 11 11 11 11 ference sample. The comparison between the YSTR profiles of the two
DYS437 15 / 15 15
gave positive results, confirming the reliability of the results outcomes.
DYS438 9 / 9 9
DYS448 20 / 20 20

80
A. Mameli et al.
4. Conclusion
After 100 years, latent traces of seminal stain, can still be identified.
DNA was extracted of such quantity and quality as to assure a complete
male profile generated by AmpFlSTR Identifiler Plus and 16 loci profile
by AmpFLSTR Yfiler, this one was compared to a live direct descendant,
ensuring reliability of results.
Ethical disclosures
All procedures performed in studies involving human participants
were in accordance with the ethical standards of the institutional and/
or national research committee and with the 1964 Helsinki declaration
and its later amendments or comparable ethical standards.
The authors have no financial conflict or disclosures to make in
regards to this research.
Funding
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-profit sectors.
Acknowledgments
We are grateful to Professor Giordano Bruno Guerri, for entrusting
us the DNA identification of the handkerchief semen stain.
The authors gratefully acknowledge receiving advice from Professor
R. John Mitchell (Office of Molecular Sci, La Trobe University,
Melbourne Australia) for his assistance in the manuscript preparation
and English revision.
References
1. Bini C, Ceccardi S, Trento C, et al. Analysis of aged seminal stains by current forensic
DNA approach. Forensic Sci Int Genet Supp Series. 2015;5:e248–e249.
2. Hochmeister MN, Budowle B, Rudin O, et al. Evaluation of prostate-specific antigen
(PSA) membrane test assays for the forensic identification of seminal fluid. J Forensic
Sci. 1999 Sep;44(5):1057–1060.
3. Kido A, Hara M, Yamamoto Y, et al. Nine short tandem repeat loci analysis in aged
semen stains using the AmpFLSTR Profiler Kit and description of a new vWA variant
allele. Leg Med. 2003 Jun;5(2):93–96.
4. Lalueza-Fox C, Giglie E, Bini C, et al. Genetic analysis of the presumptive blood from
Louis XVI, king of France. Forensic Sci Int Genet. 2011;5:459–463.
5. Nakanishi H, Hara M, Takahashi S, Takada A, Saito K. Evaluation of forensic ex-
amination of extremely aged seminal stains. Leg Med. 2014 Sep;16(5):303–307
81
Journal of Forensic and Legal Medicine 61 (2019) 78–81
0.1016/j.legalmed.2014.04.002. Epub 2014 May 6.
6. Tsukada K, Shimizu M, Kurasawa Y, Kasahara K. DNA typing using AmpFlSTR Y-filer
for very long-term stored specimens. Leg Med. 2009;11:S466–S467. https://doi.org/
10.1016/j.legalmed.2009.01.057.
7. Cooper A, Poinar HN. Ancient DNA: do it right or not at all. Science.
2000;289(5482):1139.
8. Goodwin W, Linacre A, Hadi S. The PCR laboratory. An Introduction to Forensic
Genetics. Chichester, UK: Wiley-Blackwell; 2011:60–62 978-0470710197.
9. Crimescope CS-16, Application Manual.
10. QIAmp,DNA EZ1 Investigator Kit Handbook, Qiagen.
11. EZ1 Advanced XL User Manual, Qiagen.
12. Applied Biosystems. QuantifilerTM Human DNA 292 Quantification Kit User Guide.
Foster City (CA): Applied Biosystems; 2003.
13. Applied Biosystems. AmpFlSTR® Identifiler® Plus PCR Amplification Kit. User’s
Guide.
14. Applied Biosystems. AmpFlSTR® Yfiler® PCR Amplification Kit. User Guide.
15. Applied Biosystems. GeneMapper® ID-X Software Version 1.3.1. Reference Guide.
16. Berti A, Brisignelli F, Bosetti A, et al. Allele frequencies of the new European
Standard Set (ESS) loci in the Italian population. Forensic Sci Int Genet.
2011;5(5):548–549.
17. Garofano L, Pizzamiglio M, Vecchio C, et al. Italian population on thirteen short
tandem repeat loci: HUMTH01, D21S11, D18S51, HUMVWFA31, HUMFIBRA,
D8S1179, HUMTPOX, HUMCSF1PO, D16S539, D7S820, D13S317, D5S818,
D3S1358. Forensic Sci Int. 1998;97:53–60.
18. Butler JM. Fundamentals of Forensic DNA Typing. San Diego, CA: Academic Press;
2010.
19. https://yhrd.org/search.
20. Gill P, Whitaker JP, Flaxman C, Brown N, Buckleton JS. An investigation of the rigor
of interpretation rules for STR's derived from less that 100 pg of DNA. Forensic Sci Int.
2000;112.
21. Caddy B, Taylor DR, Lincare AM. A review of the science of low template DNA
analysis. . Available from: http://police.homeoffice.gov.uk/publications/
operational-policing/Review_of_Low_Template_DNA_1.pdf.
22. Budowle B, Van Daal A. Validity of low copy number typing and applications to
forensic science. Croat Med J. 2009;50:207–217. https://doi.org/10.3325/cmj.2009.
50.207 Medline:19480017.
23. Caragine T, Mikulasovich R, Tamariz J, et al. Validation of testing and interpretation
protocols for Low Template DNA analysis using AmpFlSTR Identifiler. Croat Med J.
2009;50:250–267. https://doi.org/10.3325/cmj.2009.50.250.
24. Sterzik V, Hinderberger P, Panzer S, Bohnert M. Visualizing old biological traces on
different materials without using chemicals. Int J Leg Med. 2018 Jan;132(1):35–41.
https://doi.org/10.1007/s00414-017-1678-3.
25. Vandenberg N, van Oorschot RAH. The use of Polilight in the detection of seminal
fluid, saliva, and bloodstains and comparison with conventional chemical-based
screening tests. J Forensic Sci. 2006;51:361–370.
26. Kobus HJ, Silenieks E. Scharnberg J Improving the effectiveness of fluorescence for
the detection of semen stains on fabrics. J Forensic Sci. 2002;47:1–5.
27. Stoilovic M. Detection of semen and blood stains using Polilight as a light source.
Forensic Sci Int. 1991;51:289–296.
28. Lincoln C, McBride P, Turbett G, Garbin C, MacDonald E. The use of an alternative
light source to detect semen in clinical forensic medicine. J Clin Forensic Med.
2006;13:215–218.
29. Virkler K. Lednev I Analysis of body fluids for forensic purposes: from laboratory
testing to non-destructive rapid confirmatory identification at a crime scene. Forensic
Sci Int. 2009;188:1–17.