Forensic Science International: Genetics Supplement Series 6 (2017) e9–e11

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Forensic Science International: Genetics Supplement Series

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

The separation of male and female: A comparison of seven protocols (P) T

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G. Schwerdtner , U. Germann, C. Cossu
Kantonsspital St. Gallen, Institute of Forensic Medicine, St. Gallen, Switzerland

A R T I C L E I N F O A B S T R A C T

Keywords: Differential lysis is an established method for vaginal swabs containing semen stains. However, it can be a
Differential lysis challenge to obtain a single-source male profile when vaginal swabs contain only low amounts of spermatozoa,
Spermatozoa as DNA from the perpetrator’s sperm cells tends to be obscured by the presence of an excess of DNA from
Epithelial cells epithelial cells. In our study we compared seven different protocols (Differex™, Sampletype i-sep®DL, Sampletype
Female:male ratio
i-sep®SQ, GEN-IAL® First-DNA all tissue kit, The Erase Sperm Isolation Kit, QIAcube Washing Station, in-House-
method) in order to determine the efficiency of sperm separation and DNA yield. In a first evaluation, a high
concentration of semen (about 1000 ng) was transferred onto vaginal swabs. The changes of the female:male
ratio in the sperm/non-sperm fractions comparing the standard extraction samples were used to determine
efficiency of separation. The recovery of spermatozoa was determined based on male DNA concentration in the
samples. Some methods showed significant improvement in the female:male ratio as well as the high male DNA
recovery and were therefore subsequentially investigated using decreasing amounts of semen.

1. Introduction 2.2. Sample preparation

Differential lysis is the established method for vaginal swabs con-
taining semen and was first described in 1985 by Gill et al. [1]. A
preliminary incubation that lyses epithelial cells is the first step, fol-
lowed by lysis of spermatozoa. Hereby the different lysis stability of
sperm cells compared to epithelial cells is used. The purpose of a dif-
ferential lysis is to obtain a mainly pure male DNA profile. Two es-
sential factors decides on success in separation: recovery of sperm DNA
and the change of female:male ratio starting from the mixed swab.
In this study we show the comparison of seven different protocols
(Differex™, Sampletype i-sep®DL, Sampletype i-sep®SQ, GEN-IAL® First-
DNA all tissue kit, The Erase Sperm Isolation Kit, QIAcube Washing
Station, in-House-method) in regards to the efficiency of sperm se-
paration and DNA yield.
2. Material and methods
2.1. Sample collection
Vaginal swabs were obtained from a single female volunteer col-
lected on different days. Semen was provided by a single volunteer
donor. The male DNA concentration was determined and defined
amounts were added.
Vaginal swabs were split into halves and 25 μl of semen (contain-
ing ≈ 1000 ng male DNA) were added to both parts. In further in-
vestigations a dilution series of semen (1:1, 1:4, 1:10, 1:20, 1:200) using
sodium chloride was used. 10 μl of each dilution was transferred on
each half of the swab. One half was used for differential lysis and the
other half swab was extracted with a standard extraction method
without differential lysis.
2.3. DNA-Extraction (standard extraction)
Since the amount of female DNA varies on the swab, the initial
amounts were determined by the PrepFiler Express™ DNA Extraction
Kit and AutoMate Express™ (Thermo Fisher). All extraction steps were
performed according the product manual.
2.4. Differential lysis
Differex™, Sampletype i-sep®DL, Sampletype i-sep®SQ, GEN-IAL®
First-DNA all tissue kit, The Erase Sperm Isolation Kit and QIAcube
Washing Station were processed following the manufacturer protocols.
In case a purification stepis recommended, the sperm and/or non-
spermfraction were purified with the AutoMate Express™ (Thermo
Fisher). For these experiments, we used a modified Gill-based protocol:
samples were treated with 0.1% SDS-solution and incubated. Sperm

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Corresponding author.
E-mail address: gesine.schwerdtner@kssg.ch (G. Schwerdtner).

http://dx.doi.org/10.1016/j.fsigss.2017.09.021
Received 24 August 2017; Accepted 10 September 2017
Available online 11 September 2017
1875-1768/ © 2017 Elsevier B.V. All rights reserved.
G. Schwerdtner et al. Forensic Science International: Genetics Supplement Series 6 (2017) e9–e11

Table 1
Female:male ratio in standard-extracted samples (SES) and corresponding sperm fraction samples (SF) and recovery of male DNA comparing standard-extracted samples (SES) and
corresponding sperm fraction samples (SF). (data are mean, n = 5).

Differex™ In-house method Sampletype i-sep®DL Sampletype i-sep®SQ GEN-IAL® First-DNA all tissue kit Erase Kit QIAcube Washing Station

♀:♂ ratio SES 3.7: 1 3.5: 1.2 6.9: 1 2.1: 1 6.3: 1 3.2: 1 5.5: 1
♀:♂ ratio SF 1: 3.5 1.1: 1.5 1: 10.3 1: 90.6 2.7: 1 1: 26.2 1: 40.4
Recovery in% 33 32 64 17 146 8 58

Table 2
Male DNA concentrations with associated female:male ratios in standard-extracted samples (SES) and corresponding sperm fraction samples (SF). (data are mean ± SD, n = 3).

Sampletype i-sep®DL Differex™ Erase Kit QIAcube Washing Station

A(dil.1:1) Male DNA in SES in ng 52.8 ± 13.4 58 ± 9.1 55.3 ± 20.4 27.7 ± 6.0
Female:male ratio SEF 79: 1 91: 1 98: 1 134: 1
Male DNA in SF in ng 24.2 ± 1.2 24.5 ± 7.5 2.7 ± 1.0 6.3 ± 3.3
Female:male ratio SF 1: 4.5 1.6: 1.5 1: 3.8 1: 6.3

B (dil. 1:4) Male DNA in SES in ng 27.2 ± 5.1 22.2 ± 4.5 18.7 ± 2.5 10.2 ± 6.4
Female:male ratio SEF 120: 1 195: 1 219: 1 552: 1
Male DNA in SF in ng 12.7 ± 0.6 5.5 ± 2.7 2.0 ± 0.0 2.2 ± 1.5
Female:male ratio SF 1: 3.5 1: 2.9 1: 3 1: 4

C (dil. 1:10) Male DNA in SES in ng 15.2 ± 1.8 8.2 ± 6.3 8.3 ± 1.8 5.7 ± 0.3
Female:male ratio SEF 286: 1 1551: 1 493: 1 789: 1
Male DNA in SF in ng 6.3 ± 0.8 2.7 ± 1.6 0.7 ± 0.3 2.0 ± 0.0
Female:male ratio SF 1: 2 3.8: 1 1: 1.3 1: 7

D (dil.1:20) Male DNA in SES in ng 1.3 ± 0.3 0.8 ± 0.3 0.5 ± 0.0 0.06 ± 0.02
Female:male ratio SEF 1648: 1 2765: 1 7217: 1 59618: 1
Male DNA in SF in ng 1.0 ± 0.0 1.2 ± 0.3 0.08 ± 0.02 0.03 ± 0.02
Female:male ratio SF 9.5: 1 7: 1 8.3: 1 22: 1

E (dil. 1:200) Male DNA in SES in ng 0.08 ± 0.02 0.05 ± 0.01 0.01 ± 0.01 0.001 ± 0.002
Female:male ratio SEF 4608: 1 79643: 1 284144: 1 359134: 1
Male DNA in SF in ng 0.1 ± 0.04 0.03 ± 0.03 ND ND
Female:male ratio SF 64: 1 184: 1 Indet. Indet.

Bold values represent the best values of each dilution.

cells were pelleted by centrifugation. After removing the supernatant a
second lysis with Proteinase K, Chelex and DTT was performed.
2.5. DNA quantification/amplification
DNA quantification of all samples was determined by Quantifiler™
Trio DNA Quantification Kit using a 7500 Real-Time PCR System (both
Thermo Fisher). All samples were amplified using the AmpFLSTR™
NGM SElect™ PCR Amplification Kit (Thermo Fisher).
3. Results
All described protocols were compared in regards to DNA yield and
change of female-to-male ratio. The female:male ratio of standard-ex-
tracted samples (SES) was used as a reference for the enrichment of
male DNA in sperm fraction samples (SF).
In all seven tested protocols we observed improvements in fema-
le:male ratio. Samples extracted with Sampletype i-sep®SQ showed the
highest improvement, the ratio enhanced on average 264 times. Second
highest improvement ratio was determined using the QIAcube Washing
Station (188 times) followed by the Erase protocol (88 times). The
Sampletype i-sep®DL samples and in-House-method reached an im-
provement of female-to-male ratio of 73 times resp. 5 times. With the
GEN-IAL® First-DNA all tissue kit an improvement of 3.6 times was
gained but a change in female-to-male ratio could not be reached
(Table 1).
The recovery of male DNA was determined by dividing average of
total male DNA in the standard-extracted samples and sperm fraction
samples for each differential extraction (Table 1 upper part). In samples
extracted using Sampletype i-sep®DL and QIAcube Washing Station
protocols, we observed the highest recovery of male DNA yield (64%
and 58% respectively). Samples extracted with Differex™, In-House-
method, Sampletype i-sep®SQ and Erase Kit showed recovery rates of
33%, 32%, 17% and 8%; corresponding to male DNA yields of 219 ng,
247 ng, 124 ng and 45 ng. The sperm fraction of GEN-IAL® First-DNA all
tissue kit achieved more male DNA (+ 46%) than standard-extracted
samples, therefore the recovery achieved values over 100%.
For further experiments with decreasing amounts of semen, four
methods were selected. The choice for Differex™, Sampletype i-sep®DL,
Erase Kit and QIAcube Washing Station was based on the combination
of female-to-male ratio improvement and DNA yield. We decided to
investigate the Sampletype i-sep®DL method instead of the Sampletype
i-sep®SQ, because of two reasons: (A) sperm DNA recovery was sig-
nificant superior with the i-sep®DL method (B) although the improve-
ment in female:male ratio was better in i-sep®SQ method, but com-
paring the electropherograms no significant difference was observed.
For samples prepared with serial semen dilution (1:1, 1:4, 1:10,
1:20, 1:200), we observed large fluctuations within the methods. For
1:1 dilution (dil. A) the median values of male DNA recovery in sperm
fraction varied between 24.5 (Differex™) and 2.7 ng (Erase). Except
Differex™ extracted samples, in each method the female:male ratio
changed in favor to the male component. Up to 1:200 dilution (dil. E)
all methods showed an improvement of female-to-male ratio (Table 2).
Up to 1:10 dilution (dil. C) Sampletype i-sep®DL, Erase and QIAcube
Washing Station processed samples changed the female:male ratio in
favor to the male component. For samples prepared with 1:20 diluted
semen (dil. D), the recovery of male DNA varied between 1.2 (Dif-
ferex™) and 0.03 ng (QIAcube Washing Station), that corresponds to a
7:1 and 22:1 ratio. The sperm fraction of 1:200 diluted semen samples
(dilution E) contained 0.1 (Sampletype i-sep®DL) and 0.03 ng (Dif-
ferex™) of male DNA. In Sperm fraction samples extracted with Erase
and QIAcube Washing Station, no male DNA could be detected.

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G. Schwerdtner et al. Forensic Science International: Genetics Supplement Series 6 (2017) e9–e11

4. Conclusion Conflict of interest

The differential lysis has been, and remains, a challenge for forensic None
laboratories. Our study shows that the efficiency varies greatly within
the individual methods as well as between methods. However, the References
amount of sperm cells on the swab as well as the starting female:male
ratio was shown to be decisive for the success of the work-up. [1] P. Gill, A.J. Jeffreys, D.J. Werrett, Forensic application of DNA ‘fingerprints’, Nature
318 (1984) 577–579.

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