We compared qualitative detection of two different semen biomarkers, PSA and Sg, in vaginal swab specimens extracted in buffered saline using commercially available rapid immuno-chromatographic 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 >1.0 ng PSA/mL vaginal swab eluate[14
]. 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 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 5-fold 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 one-tenth 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 [6
], and the concentration of Sg is inversely correlated with PSA concentration in seminal plasma, independent of the level of PSA protease activity [19
]. Sg cleavage products are detectable by monoclonal antibodies against Sg [10
], 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) [20
] are generally higher than PSA concentrations (0.2 – 5.5 mg PSA/mL) in seminal fluid [3
]. 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 >1.0 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, we cannot rule out that some false-negative results may have occurred if specimens contained unusually high concentrations 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 are 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 RSID or other buffers for detection of the two semen markers. 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 [14