Vaginal swab specimen processing methods influence performance of rapid semen detection tests: a cautionary tale.
ABSTRACT Detection of semen biomarkers in vaginal fluid can be used to assess women's recent exposure to semen. Quantitative tests for detection of prostate-specific antigen (PSA) perform well, but are expensive and require specialized equipment. We assessed two rapid immunochromatographic strip tests for identification of semen in vaginal swabs.
We tested 581 vaginal swabs collected from 492 women. Vaginal secretions were eluted into saline, and PSA was measured using the quantitative IMx (Abbott Laboratories, Abbott Park, IL, USA) assay. Specimens were also tested using the ABAcard p30 test (Abacus Diagnostics, West Hills, CA, USA) for detection of PSA and RSID-Semen test (Independent Forensics, Hillside, IL, USA) for detection of semenogelin (Sg).
Vaginal swab extraction using saline was compatible with direct assessment of vaginal swab eluates using ABAcard for PSA detection, but not for Sg detection using RSID. The rapid PSA test detected 91% of specimens containing semen compared to 74% by the rapid Sg test.
Investigators are urged to optimize vaginal swab specimen preparation methods for performance of RSID or other tests to detect semen components other than PSA. Previously described methods for PSA testing are not uniformly applicable to other tests.
- [show abstract] [hide abstract]
ABSTRACT: Forty women participated in three clinic visits during which they were exposed to their partner's semen (10 microL, 100 microL, and 1 mL). At each visit they took vaginal fluid samples before exposure to their partner's semen, immediately after, and at 1, 24, and 48 h after exposure. PSA was measured with an enzyme-linked immunoassay. The mean PSA level for preexposure swabs ranged between 0.43 and 0.88 ng/mL. The mean PSA levels were 193 immediately after exposure to 10 microL, 472 after 100 microL, and 19,098 after 1 mL. The PSA levels declined within 1 h, and returned to background at 48 h. The findings confirm that our procedure is a sensitive and specific method for detecting recent semen exposure, and indicate that PSA levels depend on exposure intensity and time since exposure. Application of this method in condom efficacy studies provides objective evidence of condom failure that enhances the interpretation of self-report.Contraception 04/1999; 59(3):195-201. · 3.09 Impact Factor
Article: Objective markers of condom failure.[show abstract] [hide abstract]
ABSTRACT: Studies of condom efficacy rely on self-reported behavior. Objective markers of exposure to semen may provide a more valid assessment of condom failure and failure to use condoms. To compare three semen biomarkers: acid phosphatase (AP) activity, prostate specific antigen (PSA), and the human seminal plasma antigen (MHS-5). Twenty women were intravaginally inoculated with six measured, increasingly larger amounts of their partners' semen. Vaginal fluid was collected by the participant using swabs and tested. Background levels of PSA were low (0.00-1.25 ng/ml), background levels of AP were variable (0-350 U/l), and all preinoculation samples were negative for MHS-5. All postinoculation samples were positive for PSA, 64 of 117 (55%) for AP, and 14 of 120 (12%) for MHS-5. The PSA immunoassay was the best semen biomarker under these sampling and testing conditions.Sex Transm Dis 10/1998; 25(8):427-32. · 2.59 Impact Factor
- Journal of Forensic Sciences 02/1978; 23(1):106-15. · 1.24 Impact Factor
Original research article
Vaginal swab specimen processing methods influence performance of
rapid semen detection tests: a cautionary tale☆
Marcia M. Hobbs⁎,a, Markus J. Steinerb, Kimberly D. Richa, Maria F. Galloc,
Lee Warnerc, Maurizio Macalusoc
aUniversity of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
bFamily Health International, Research Triangle Park, NC 27713, USA
cCenters for Disease Control and Prevention, Atlanta, GA 30333, USA
Received 4 November 2009; revised 5 January 2010; accepted 24 February 2010
Background: Detection of semen biomarkers in vaginal fluid can be used to assess women's recent exposure to semen. Quantitative tests for
detection of prostate-specific antigen (PSA) perform well, but are expensive and require specialized equipment. We assessed two rapid
immunochromatographic strip tests for identification of semen in vaginal swabs.
Study Design: We tested 581 vaginal swabs collected from 492 women. Vaginal secretions were eluted into saline, and PSA was measured
using the quantitative IMx (Abbott Laboratories, Abbott Park, IL, USA) assay. Specimens were also tested using the ABAcard p30 test
(Abacus Diagnostics, West Hills, CA, USA) for detection of PSA and RSID-Semen test (Independent Forensics, Hillside, IL, USA) for
detection of semenogelin (Sg).
Results: Vaginal swab extraction using saline was compatible with direct assessment of vaginal swab eluates using ABAcard for PSA
detection, but not for Sg detection using RSID. The rapid PSA test detected 91% of specimens containing semen compared to 74% by the
rapid Sg test.
Conclusion: Investigators are urged to optimize vaginal swab specimen preparation methods for performance of RSID or other tests to detect
semen components other than PSA. Previously described methods for PSA testing are not uniformly applicable to other tests.
© 2010 Elsevier Inc. All rights reserved.
Keywords: Semen; Prostate-specific antigen; Human semenogelin protein; Vaginal swabs; Rapid test
Measurement of objective markers of semen exposure,
rather than reliance on self-reported behavior or mechanical
failure of barrier methods, can improve the accuracy of
studies designed to evaluate contraceptive efficacy. Detec-
tion of seminal biomarkers in vaginal secretions provides
objective evidence of a woman's recent exposure to semen.
Prostate-specific antigen (PSA, also known as p30) has been
validated as a reliable marker of semen exposure in studies of
vaginal specimens obtained after vaginal insemination with
different volumes of semen [1,2]. Seminal biomarkers have
long been used in forensic detection of semen in vaginal
specimens in sexual assault cases [3,4]. Both PSA, secreted
by the prostate , and semenogelin (Sg), the major seminal
vesicle secreted protein in human semen , are useful
markers for forensic identification of semen [7–11]. In
Contraception 82 (2010) 291–295
☆Funding: IMx test kits were generously donated by Abbott
Laboratories. This work was supported in part by the US National Institutes
of Health through the North Carolina Sexually Transmitted Infections and
Topical Microbicides Cooperative Research Center grant U19-AI031496
and a developmental award from the UNC Center for AIDS Research grant
P30-AI050410. Additional support was provided by the Centers for Disease
Control and Prevention (CDC) and the Contraceptive and Reproductive
Health Technologies Research and Utilization (CRTU) program of the
United States Agency for International Development (USAID) with funds
from cooperative agreement #GPO-A-OO-05-00022-00 through Family
Health International (FHI). Funding sources had no role in study design; in
the collection, analysis, and interpretation of data; or in the writing of the
report. The decision to submit the article for publication was approved by
the CDC and FHI. The findings and conclusions in this report are those of
the authors and do not necessarily represent the official positions of the
⁎Corresponding author. Tel.: +1 919 843 6893; fax: +1 919 843 1015.
E-mail address: firstname.lastname@example.org (M.M. Hobbs).
0010-7824/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
addition, Y-chromosome DNA contained within sperm cells
can be detected by polymerase chain reaction [12,13]. Rapid,
immunochromatographic strip tests for PSA and Sg are
We recently reported good performance of the rapid
ABAcard p30 test (Abacus Diagnostics, West Hills, CA,
USA) compared to a quantitative assay (IMx PSA, Abbott
Laboratories, Abbott Park, IL, USA) for detection of PSA in
vaginal swabs . In the present study, we compared and
contrasted ABAcard for detection of PSA and Rapid Stain
Identification test (RSID-Semen, Independent Forensics,
Hillside, IL, USA) for detection of Sg to identify semen in
vaginal swab specimens that had been specifically processed
using previously described methods for quantitative PSA
2. Materials and methods
2.1. Vaginal swab specimens
Vaginal swab specimens were obtained from women
participating in two different research studies that are
described in detail elsewhere . Briefly, 402 vaginal
swabs were from 313 women who participated in a study
comparing two methods of STI prevention and control
among sex workers in Dhaka, Bangladesh, conducted from
February 2005 through September 2006, and 179 vaginal
swabs were from women who participated in a study
comparing two interviewing techniques to obtain reports of
sexual behaviors among sexually active women in
Zimbabwe from November 2006 through January 2007.
Women were not recruited based on the timing of recent
intercourse. However, most women (82%) reported vaginal
intercourse (with or without a condom) in the previous 48 h.
Women were recruited into the studies only after providing
written informed consent according to Good Clinical
Practice Guidelines. The Bangladesh study was approved
by the Biomedical Institutional Review Board (IRB) of the
University of North Carolina at Chapel Hill and by the IRB
of the International Center for Diarrhoeal Disease Research,
Bangladesh. The Zimbabwe study was approved by IRBs of
Family Health International, the University of North
Carolina at Chapel Hill, the University of California at San
Francisco and by the ethics review committees of the
Medical Research Council of Zimbabwe and the Medicines
Control Authority of Zimbabwe.
Vaginal specimens were collected on cotton-tipped swabs
(Falcon™ Screw Cap Single SWUBE™ applicator, Becton
Dickinson and Co., Sparks, MD, USA). Immediately after
collection, swabs were air dried, stored in screw-capped
tubes and shipped at ambient temperatures to the research
laboratory at the University of North Carolina at Chapel Hill.
For recovery of vaginal secretions, each swab was placed
into 3.0 mL phosphate-buffered saline, incubated at room
temperature for 15–30 min, agitated and pressed against the
side of the tube to elute the sample. Vaginal specimens were
centrifuged at 250×g for 10 min, and the supernatants were
removed from cell pellets and stored at −80°C until testing.
These conditions were chosen specifically for PSA detection
using a quantitative assay (IMx PSA; Abbott Laboratories)
to take advantage of previously established criteria for
detecting recent semen exposure based on PSA concentra-
tions, using these vaginal swab extraction methods [1,15–
17]. Vaginal swab eluates were stored frozen for variable
periods of time (up to 2 years) before testing; however, all
tests reported here were performed within a 6-week period.
Thus, comparisons between rates of semen detection using
different testing methods are valid; however, we cannot rule
out that semen components may have degraded during
2.2. Quantitative PSA testing
Supernatants (0.20 mL) from vaginal swab eluates were
tested using the IMx PSA assay (Abbott Laboratories). The
enzyme immunoassay measures PSA concentrations from
0.04 to 50 ng/mL; samples with initial test results N50 ng/mL
were diluted 1:100 with buffered saline and retested to obtain
PSA concentrations. Vaginal swab eluates containing ≤1.0
ng PSA/mL were considered negative; those containing N1.0
ng PSA/mL were considered positive for detection of semen,
as previously described .
2.3. Rapid PSA testing
The vaginal swab extraction procedure described above
using buffered saline was consistent with the manufacturer's
instructions for testing using ABAcard (Abacus Diagnos-
tics). We loaded 0.20 mL of vaginal swab eluate directly into
the sample well of the immunochromatographic strip test
cassette according to the instructions. After a 10-min
incubation at room temperature to allow sample migration
throughout the test strip, a positive result was indicated by
pink lines in both test and control areas. A negative result
was indicated by a line in the control area only. A control line
was visible in all tests with vaginal swab eluates,
documenting valid ABAcard results. Buffered saline alone
produced a visible line in the control area only. Buffered
saline containing 1 ng purified human PSA/mL produced a
faint, but consistently visible line in the test area, and lines
from solutions containing ≥5 ng PSA/mL were markedly
darker. For this analysis, specimens that produced any
visible line in the test area plus a line in the control area of the
strip were considered positive by the rapid PSA test. A
negative rapid PSA result was indicated by a line in the
control area only.
Like all immunoassays that depend on antigen–antibody
interactions, the ABAcard test and the RSID test (described
below) are subject to potential interference in the presence of
excess antigen, which impairs immune complex formation.
As a result of this so-called high-dose hook effect, high
concentrations of the analyte can give false-negative results.
The threshold concentrations at which rapid semen test
292M.M. Hobbs et al. / Contraception 82 (2010) 291–295
results may be subject to the high-dose hook effect have not
been established by the manufacturers. However, our
unpublished observations suggest that this threshold may
be between 2000 and 5000 ng PSA/mL for the ABAcard.
2.4. Rapid semenogelin testing
It is important to note that the vaginal swab extraction
procedure described above using buffered saline does not
conform with the manufacturer's instructions for testing with
the Rapid Stain Identification test (RSID-Semen, Indepen-
dent Forensics), which specifies the use of RSID-Semen
Extraction Buffer. Nevertheless, we wished to determine
whether the test could be used for detection of Sg in these
specimens, as extraction with buffered saline is commonly
used for PSA testing from vaginal swabs. In initial
experiments, buffered saline alone or buffered saline diluted
1:5 in RSID Semen Running Buffer according to the
manufacturer's instructions was applied to the sample well
of the rapid test strip as directed. Samples that produced a
visible line in test and control areas of the strip were
considered positive by the rapid Sg test; those with a line in
the control area only were considered negative. Buffered
saline alone produced uniform false-positive results with
visible lines in control and test areas of the strip, whereas
buffered saline diluted 1:5 in running buffer was negative.
Accordingly, the vaginal specimens, which had already been
extracted in buffered saline, were diluted 1:5 in running
buffer for RSID testing. A control line was visible in all tests
with diluted vaginal swab eluates.
2.5. Calculations and statistical analyses
As a result of the requirement for specimen dilution and
the loading characteristics of the RSID test device, the
amount of vaginal swab eluate tested by RSID was 10-fold
lower (fivefold dilution and half the volume loaded into the
sample well) than the corresponding specimen tested using
the ABAcard. To account for the additional dilution of
specimens required for RSID testing, PSA concentrations in
specimens were adjusted to 1/10 the values determined by
the quantitative assay in the undiluted vaginal swab eluates
for analysis of RSID results.
The kappa statistic for multiple raters was calculated
using the MAGREE macro from SAS/STAT Software
(release 6.11 TS020). Among four independent evaluators,
interreader reliability was substantial for both rapid tests with
a kappa score of 0.97 (SE, 0.02) for ABAcard with a subset
of 402 vaginal swab specimens and 0.78 (SE, 0.03) for RSID
with a subset of 207 specimens.
Ninety-five percent confidence intervals (95% CI)
for proportions were calculated according to Wilson
. Differences between proportions were assessed by
the z-test using Sigma Stat for Windows version 3.5
(Systat Software, Inc.); p values b.05 were considered
With the use of the quantitative IMx assay for PSA
detection, 194 (33.4%) of 581 vaginal swab specimens
extracted in buffered saline contained N1 ng PSA/mL
(Table 1). Both rapid tests identified positives among
specimens containing ≤1 ng PSA/mL; Sg detection by
RSID was higher among specimens with ≤1 ng PSA/mL
than PSA detection by ABAcard. Among specimens with
N1.0 ng PSA/mL, PSA detection by ABAcard was
significantly higher than Sg detection by RSID. For vaginal
swab specimens with N1.0 ng PSA/mL, we also compared
the proportion of rapid test positives among those with low,
moderate and high semen exposure categories as previously
defined [15,16] (Table 2). Both ABAcard and RSID detected
semen in all specimens containing high levels of PSA. All
low and moderate PSA positives were also positive by
ABAcard, whereas RSID detected Sg in significantly fewer
specimens with low PSA concentrations.
Because detection of either Sg or PSA is consistent
with the presence of semen, we conducted a secondary
analysis in which any vaginal swab specimen containing
Sg detected by a positive RSID test or N1.0 ng PSA/mL
detected by the quantitative assay was considered to be
positive for semen. ABAcard detected 92% of all positives
(194/212) compared to 74% detected by RSID (113/152,
pb.001, z-ratio for proportions).
We compared the qualitative detection of two different
semen biomarkers, PSA and Sg, in vaginal swab specimens
Rapid test results for qualitative detection of semen biomarkers in vaginal swab specimens eluted into phosphate-buffered saline
PSA detected with ABAcardSg detected with RSID p valued
Total Number positive (%, 95% CI)Totalc
Number positive (%, 95% CI)
16 (4%, 3–7%)
194 (100%, 98–100%)
53 (11%, 9–15%)
59 (60%, 50–69%)
aCutoff for dichotomous PSA results to define semen exposure, as previously described .
bDetermined using the quantitative IMx PSA assay.
cSpecimen totals in qualitative categories based on PSA concentration in vaginal swab eluates diluted 1:10 for RSID testing.
dProbability of z-ratio for proportion of specimens positive with ABAcard vs. RSID.
293M.M. Hobbs et al. / Contraception 82 (2010) 291–295
extracted in buffered saline using commercially available
rapid immunochromatographic strip tests. In addition, PSA
concentrations in these specimens were determined using a
quantitative enzyme immunoassay. Using these very same
vaginal swab specimens, we recently reported that the
ABAcard rapid test is 100% sensitive (95% CI, 98–100%)
and 96% specific (95% CI, 93–97%) compared to the
quantitative test in detecting N1.0 ng PSA/mL vaginal swab
eluate . Although the quantitative IMx PSA assay may
be considered the ‘gold standard’ for detection of PSA, it is
not a valid comparator for the RSID test, which detects Sg, a
biochemically distinct analyte. Without a reference test for
detection of Sg, and with acknowledged deviations from the
manufacturer's directions for specimen processing, we did
not attempt to define the performance characteristics of the
RSID test for Sg. Rather, we elected to describe the
percentage of specimens with positive ABAcard or RSID
test results and stratify those observations based on PSA
concentrations in the specimens.
In this comparison of two rapid immunochromatographic
strip tests for detection of semen in vaginal swab specimens,
PSA was detected by ABAcard significantly more frequently
than Sg was detected by RSID. However, the study has
important limitations that must be considered in interpreting
these results. Specimen preparation was compatible with
direct assessment of vaginal swab eluates using ABAcard for
PSA detection, but for Sg detection using RSID, acceptable
test performance required fivefold dilution of samples with
running buffer supplied with the test. The imperfect
specimen preparation for use with RSID likely reduced the
sensitivity of the test. To compensate for specimen dilution
and lower test volume (necessitated by a smaller sample well
in the RSID test device compared to the ABAcard), we
adjusted PSA concentrations to 1/10 the actual values
determined by the quantitative assay for analysis of RSID
test results. However, this adjustment could not compensate
for impaired RSID sensitivity that may have resulted from
the use of a different extraction buffer. Thus, the apparent
greater sensitivity of the rapid PSA test compared to the Sg
test for detection of semen in vaginal swabs must be
considered to be preliminary and specific for the specimen
preparation methods used in our study. However, these
methods are used commonly, and our experience may
benefit others considering the use of RSID for detection of
semen in vaginal fluid.
This study was further complicated by comparing
different individual semen components as markers of this
complex biological specimen. Although both Sg and PSA
are invariably present in human semen, individual semen
specimens with a detectable level of one component may not
necessarily contain a similarly detectable concentration of
the other. Sg is a natural substrate for cleavage by the serine
protease PSA , and the concentration of Sg is inversely
correlated with PSA concentration in seminal plasma,
independent of the level of PSA protease activity . Sg
cleavage products are detectable by monoclonal antibodies
against Sg [10,19], and the capture antibody in the RSID test
does detect cleaved Sg (K. Reich, Independent Forensics,
personal communication). Thus, Sg that had been cleaved by
PSA in a specimen would still theoretically be detectable by
RSID, and specimens in this study with high PSA
concentrations were positive with RSID. Sg concentrations
(4–68 mg Sg/mL)  are generally higher than PSA
concentrations (0.2–5.5 mg PSA/mL) in seminal fluid
[3,21]. Therefore, specimens containing semen in which
Sg but not PSA can be detected are to be expected. Indeed,
such specimens were identified in our study and accounted
for approximately 10% of all positive vaginal swab samples
in which either Sg or N1.0 ng PSA/mL was detected.
However, PSA was detected in the absence of Sg in 25% of
positive vaginal swab specimens. Suboptimal specimen
preparation for the RSID test, as discussed above, may
account for the failure to detect Sg in these samples. We did
not dilute vaginal swab eluates beyond 1:5 with the running
buffer supplied with the test kits for fear of compromising
RSID sensitivity even further; however, it is possible that
specimens with high concentrations of Sg could have
produced false-negative results. According to the RSID
product insert, 20-fold dilution of samples containing large
amounts (3–50 μL) of pure semen eliminates false negatives
resulting from the high-dose hook effect. We estimate that
the dilution of semen in secretions in the vagina and further
into the buffered saline used for extraction of material from
vaginal swabs resulted in a 500- to 5000-fold dilution of
semen in samples tested by RSID. Thus, it is unlikely that the
high-dose hook effect affected the RSID results. However,
Rapid test results for detection of semen biomarkers in vaginal swab specimens eluted into phosphate-buffered saline, stratified by PSA concentration
TotalNumber positive (%, 95% CI)Totalc
Number positive (%, 95% CI)
129 (100%, 97–100%)
28 (100%, 88–100%)
37 (100%, 91–100%)
42 (54%, 43–64%)
13 (81%, 57–93%)
4 (100%, 51–100%)
NS, Not significant.
aSemen exposure categories among specimens containing N1 ng PSA/mL vaginal swab eluate, as previously described [16,17].
bDetermined using the quantitative IMx PSA assay.
cSpecimen totals in qualitative categories based on PSA concentration in vaginal swab eluates diluted 1:10 for RSID testing.
dProbability of z-ratio for proportion of specimens positive with ABAcard vs. RSID.
294M.M. Hobbs et al. / Contraception 82 (2010) 291–295
we cannot rule out that some false-negative results may have
occurred if specimens contained unusually high concentra-
tions of Sg.
There are several advantages to using PSA as a marker of
semen exposure. The use of PSA allows investigators to
capitalize on previous work characterizing the kinetics of
PSA clearance from vaginal swab specimens prepared in the
same way that was used in our study. We do not know the
kinetics of Sg clearance or degradation in the vagina or
whether the presence of PSA and Sg in vaginal fluid from
women with recent semen exposure is correlated. Studies are
needed to provide the necessary context for interpreting Sg
detection in vaginal swab specimens: How long after semen
exposure can Sg be detected in vaginal fluid? How soon does
Sg disappear compared to PSA? What concentrations of Sg
correspond to semen exposure resulting from problems with
condom use? We urge investigators to optimize vaginal swab
specimen preparation methods for the performance of RSID
or other semen detection tests and not rely on previously
described methods for PSA testing. Research needs include
comparison of specimens collected in buffered saline and
For rapid PSA detection, ABAcard is an appropriate, simple
and relatively inexpensive test to identify a biological marker
of recent semen exposure in vaginal swabs .
The authors thank Robert Krysiak and Dana Lapple who
assisted with laboratory testing.
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