Estimation
of Prostate-Specific Antigen (PSA) Extraction Efficiency from Forensic
Samples Using the Seratecâ
PSA Semiquant Semiquantitative Membrane Test
Bill O. Gartside
Criminalist
Kevin J. Brewer
Criminalist Supervisor
Carmella L. Strong
Criminalist
Nebraska State Patrol Crime Laboratory
Lincoln, Nebraska
Introduction.......Methods
and Materials.......Results
Conclusions.......References
Introduction
Prostate-specific
antigen (PSA, also known as p30) is a glycoprotein produced by the
prostate gland that has been well characterized and validated in
the forensic science community as a marker for the presence of seminal
fluid (Hochmeister et al. 1999; Johnson and Kotowski 1993; Poyntz
and Martin 1984; Sensabaugh 1978). The Seratecâ
PSA Semiquant (Seratecâ
Diagnostica, Göttingen, Germany) membrane, and the OneStep
ABA cardâ p30 (Abacus
Diagnostics, West Hills, California) are immunochromatographic membrane
tests that allow for rapid, convenient, and highly sensitive analysis
for the presence of seminal fluid (Hochmeister et al. 1999). The
Seratecâ PSA Semiquant
test membrane also offers a semiquantitative test for prostate-specific
antigen by the use of an internal standard. A visible line, roughly
equivalent in intensity to a concentration of 4ng PSA/ml is available
on the test strip allowing a visual comparison with the test line
concentration (Seratecâ
Diagnostica 2000).
Methods
and Materials
In this study,
liquid semen sample dilutions were identified as being near the
4ng PSA/ml internal standard value. Forensic semen stains and swabs
were then prepared with known quantities of these semiquantified
semen samples. Semen from both a vasectomized individual and a nonvasectomized
individual were serially diluted to 1:1,000,000 with sterile water.
Swabs were prepared from the dilutions using approximately 150µl
of solution per swab by allowing the swab to saturate and then to
air-dry. Stains (approximately 44.5mm diameter, approximately 1555mm2
total area) were also prepared on clean cotton cloth using 500µl
of the semen dilutions and then allowed to air-dry.
Duplicate extractions
of approximately 1/3 of each swab (vasectomized semen only) were
performed in 250µl of sterile water for two hours at room
temperature. After centrifugation for one minute at 13,000g, 200µl
of the supernatant was removed and tested with the Seratecâ
PSA Semiquant membrane (x2) and the OneStep ABA cardâ
p30 (x1).
Duplicate extractions
of approximately 5mm square cuttings (25mm2)
from the center of the prepared semen stains (nonvasectomized semen
only) on cotton fabric were performed in 250µl of sterile
water for two hours at room temperature. After centrifugation for
one minute at 13,000g, 200µl of the supernatant was removed
and tested with the Seratecâ
PSA Semiquant membrane (x2) and the OneStep ABA cardâ
p30 (x1).
Duplicate extractions
of approximately 5mm square cuttings (25mm2)
from the center of the prepared semen stains (nonvasectomized semen
only) on cotton fabric were performed in 250µl of 1X HEPES
buffer (137.0mM NaCl, 5.0mM K Cl, 0.8mM Na2HPO4-2H2O, 5.5mM dextrose,
21.0mM HEPES) for two hours at room temperature. After centrifugation
for one minute at 13,000g, 200µl of the supernatant was removed
and tested with the Seratecâ
PSA Semiquant membrane (x2).
Two hundred
microliters of the prepared liquid semen dilutions were tested with
the Seratecâ PSA Semiquant
membrane (x1) and the OneStep ABA cardâ
p30 (x1).
Results
Estimation
of Semen PSA Concentration
The evaluation
of both the vasectomized and the nonvasectomized liquid semen dilutions
using the Seratecâ PSA
Semiquant membrane indicated that the concentration at 1:400,000
was greater than the 4ng PSA/ml internal standard marker and that
the concentration at 1:500,000 was less than the 4ng PSA/ml internal
standard marker. The 4ng PSA/ml internal standard equivalency point
was estimated at 1:450,000.
This estimation
was corroborated by the OneStep ABA cardâ
p30 results. The OneStep ABA cardâ
p30 has a published sensitivity of 4ng PSA/ml. The largest dilution
that gave a positive result with the nonvasectomized semen was 1:400,000.
The largest dilution that gave a positive result with the vasectomized
semen using the OneStep ABA cardâ
p30 membrane test was 1:500,000. The estimated dilution that is
roughly equivalent to 4ng PSA/ml (1:450,000) implies that the whole
semen samples contain approximately 1,800,000ng PSA/ml, well within
the published normal range (Abacus Diagnostics 2001; Seratecâ
Diagnostica 2000).
A PSA standard
(Sigma-Aldrich catalog number 3338) was diluted with 0.15M phosphate
buffered saline (Sigma-Aldrich catalog number P3813) to concentrations
from 8ng/ml to 2ng/ml and analyzed with the Seratecâ
PSA Semiquant membrane to assess the accuracy of the semiquant assay.
The ability of the assay to discriminate between these low concentrations
of prostate-specific antigen was assessed and found to be satisfactory
for the purposes of estimating extraction efficiencies. The accuracy
of the semiquant line was within a factor of two of the reported
approximation of 4ng PSA/ml (data not shown).
Estimation
of Extraction Efficiencies
Results using
the Seratecâ PSA Semiquant
membrane and the OneStep ABA cardâ
p30 were noted after ten minutes as either positive ([+] with control
and test lines present) or negative ([-] with control line present
and test line absent) using the individual manufacturer's instructions
(Abacus Diagnostics 2001; Seratecâ
Diagnostica 2000).
Positive test
lines that were identified with the Seratecâ
PSA Semiquant membranes were further evaluated for their intensity
in comparison to the 4ng/ml semiquant line. The test line was noted
as being more intense (T > q), equally as intense (T = Q), or
less intense (t < Q) than the 4ng PSA/ml semiquant line.
Results of the
liquid semen dilutions are shown in Table
1.
Results of the
semen-stained swabs are shown in Table
2.
The PSA concentration
of the undiluted semen was estimated at approximately 1,800,000ng
PSA/ml; therefore, the total available PSA per prepared swab can
be estimated at 0.15ml x 1,800,000ng/ml = 270,000ng/swab. Using
1/3 of a swab and a 0.250ml extraction volume, the total available
PSA concentration becomes approximately 360,000ng/ml. At 100 percent
extraction efficiency, a 1:90,000 dilution could be expected to
have a PSA concentration of approximately 4ng/ml.
The evaluation
of the extractions from the swabs indicated that the 4ng PSA/ml
internal standard was roughly equivalent to the results obtained
with the swab prepared with a 1:100 semen dilution. This indicates
that approximately 4ng/ml of the available 360,000ng/ml PSA was
extracted, implying a 1/900, or approximately 0.11 percent, PSA
extraction efficiency from these swabs.
Results of the
semen stains on cotton cloth are shown in Table
3.
At the approximately
1,800,000ngPSA/ml undiluted semen concentration estimated above,
the total available PSA per prepared ~ 44.5mm diameter stain can
be estimated at 0.5ml x 1,800,000ng/ml = 900,000ng/stain. Using
approximately 1/62 of the stains (25 mm2
/ ~1555 mm2), and a 0.250ml extraction
volume, the total available PSA concentration becomes approximately
58,000ng/ml. At a 100 percent extraction efficiency, a 1:14,500
dilution could be expected to have a PSA concentration of approximately
4ng/ml.
The evaluation
of the extractions from the stains extracted in sterile water indicated
that the 4ng PSA/ml internal standard was roughly equivalent to
the results obtained with the stain prepared with a 1:50 semen dilution.
This implies an approximately 1/290, or 0.34 percent, PSA extraction
efficiency from these prepared stains.
The evaluation
of the extractions from the stains extracted in 1X HEPES buffer
indicated that the dilution that was closest to the 4ng PSA/ml internal
standard was the stain prepared with a 1:100 semen dilution. At
this dilution the 4ng/ml standard line was slightly less intense
that the test line, hence an estimate of a 1:150 semen dilution
for equivalence to the semiquant line was used. This estimation
would imply an approximately 1/97, or 1.03 percent, PSA extraction
efficiency from these prepared stains.
It was noted
that positive results were not obtained at lower concentrations
when using the HEPES buffer despite the three-fold higher estimate
of PSA extraction efficiency over extractions using sterile water
at the 4ng PSA/ml equivalence level.
Conclusions
Comparison of
the stain and swab extracts with the liquid semen sensitivity results
shows that it can be estimated that one percent or less of the PSA
applied to the swabs and stains was able to be extracted. This estimation
of PSA extraction efficiency indicates that the yield from common
forensic substrates is one percent or less of the estimated total
available PSA. Whereas the sensitivity of immunochromatographic
cassettes using diluted liquid semen samples is quite high, the
PSA extraction efficiency when analyzing liquid extracts from dried
semen dilutions is low enough to limit their usefulness. This study
shows that a more efficient extraction process would be beneficial
when applying these types of membrane tests to forensic science
applications.
If these extraction
efficiency estimations are also valid for prostate-specific antigen
found in adult male postejaculate urine and blood serum, then concerns
regarding positive test results from these sources may be undeserved.
Liquid postejaculate urine samples are reported to have a mean value
of 260ng PSA/ml, and this must be considered when analyzing liquid
samples (Abacus Diagnostics 2001; Seratecâ
Diagnostica 2000). However, if dried samples are submitted and extraction
efficiencies are one percent or less, these same samples would yield
less than 2.6ng PSA/ml. This PSA concentration is much less of a
concern as a possible confounding factor during evidence examination
because it is less than the stated sensitivity of the OneStep
ABA cardâ p30 and
less than the semiquant line of the Seratecâ
PSA Semiquant membrane.
References
Abacus Diagnostics.
OneStep ABA Cardâ.
Abacus Diagnostics, West Hills, California, 2001.
Hochmeister,
M. N., Borer, U., Budowle, B., Dirnhofer, R., Gehrig, C., Rudin,
O., and Thali, M. Evaluation of prostate-specific antigen (PSA)
membrane test assays for the forensic identification of seminal
fluid, Journal of Forensic Sciences (1999) 44:1057-1060.
Johnson E. D.
and Kotowski T. M. Detection of prostate-specific antigen by ELISA,
Journal of Forensic Sciences (1993) 38:250-258.
Poyntz, F. M.
and Martin, P. D. Comparison of PSA and acid phosphatase levels
in postcoital vaginal swabs from donor and casework studies, Forensic
Science International (1984) 24:17-25.
Sensabaugh G.
Isolation and characterization of a semen-specific protein from
human seminal plasma: A potential new marker for semen identification,
Journal of Forensic Sciences (1978) 23:106-15.
Seratecâ
Diagnostica. Seratecâ
PSA Semiquant Membrane Test for Detection of Seminal Fluid.
Seratecâ Diagnostica,
Göttingen, Germany, 2000.
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