InSite^® Semen Detection Kit

Abstract

This paper describes the development of the InSite^® Test Kit.  This kit is comprised of two main components:  acid phosphatase (AP) test strips and prostate specific antigen (PSA) test strips, which work together to provide evidence of semen on garments and other items.  The included AP strips were found to detect semen down to a 1/2000 dilution, whereas comparative testing with two other acid phosphatase (AP) tests and a zinc test showed that their limit of detection was 1/150-1/300. The PSA strips detected semen to a 1/500,000 dilution, which was approximately the same as semenogelin test strips used in comparative testing.  Semen which was discharged onto undergarments was detectable by the AP tests up to 17 h, by the zinc test up to 17 h, and by the PSA and semenogelin tests up to 36 h after intercourse.   The AP test gave a more dramatic color change than the zinc test with small amounts of semen and therefore was chosen for inclusion in the kit.  The zinc test, on the other hand, was more specific and would be superior when testing directly with a vaginal swab.  In contrast with spermatozoa, which can be found in the vagina more than seven days after intercourse,  the marker proteins PSA and semenogelin became undetectable after 36 hours.  We attribute this difference to the acidic pH of the vagina, among other factors.

Introduction

Conservative statistics indicate that about 14% of women and 22% of men have had affairs sometime in their marriage [Ref. 1].  According to a recent study by the Centers for Disease Control, about 4% of both married men and women had more than one sexual partner in the previous twelve months [Ref. 2].   This figure rises to 15% in the case of unmarried couples cohabiting.  These data indicate that infidelity is a significant problem in the United States, and there exists a need to objectively test spouses for sexual activity.  For women, one such test is for the presence of semen.

Results and Discussion

Acid phosphatase test strips were prepared according to a modification of the procedure of Babson [Ref. 6].  Zinc test strips were prepared according to the method of Hooft and van de Voorde, using various filter papers as substrate.  PSA, semenogelin and two other AP tests were obtained commercially as described below.  In order to measure the relative sensitivity of these different tests, and their ability to detect semen on undergarments, comparative studies were performed with semen dilutions and with analysis of garments after intercourse.

Semen dilutions

The sensitivity of the zinc strips was tested by analyzing a series of dilute semen samples, and acid phosphatase tests were carried out simultaneously for comparison.  Semen was diluted with deionized water to levels of 1/10, 1/50, 1/100, 1/150, 1/200, 1/300, 1/500, 1/1,000 and 1/2,000 and tested with zinc strips and three acid phosphatase tests (prototype InSite^®, CheckMate^® and Phosphatesmo KM brands).  The zinc strips proved to be sensitive to a 1/150 dilution, the prototype InSite test had a detection limit of 1/100, the CheckMate^® AP test was judged to have a detection limit of 1/150 and the Phosphatesmo KM test a detection limit of 1/300.  The AP tests were read after 15 seconds, but at high dilutions continued to slowly turn purple.  The results are shown in Fig. 1.  The zinc and InSite AP test strips in this experiment were prepared using Whatman Grade 1 filter paper.  The prototype InSite AP test (D) in this experiment turned out to be too weak, and the concentration of reagents in this strip later was increased.

The limit of detection of semen at a 1/150 dilution by the zinc spot test is generally consistent with the report of Hooft and van de Voorde, who reported a detection limit of 1/128 [Ref. 8].

The Phosphatesmo KM strips were clearly superior as a spot test for detecting semen in this experiment because of the dramatic color change, the small amount of enzyme needed for a reaction and the strip's overall design.  It was initially thought that, because of acidic conditions in the vagina, it might turn out that zinc would be the best test beyond a certain time frame, e.g. 12 hours.  This turned out not to be true, as shown below.  The Phosphatesmo KM strips were also somewhat expensive, at $5 each.

Figure 1.  Serial Dilutions of Semen and Analysis with Zinc Strips and Acid Phosphatase Tests
A=Phosphatesmo KM;  B=Zinc strips;  C=CheckMate^® acid phosphatase test;  D=Early prototype InSite AP Test

Another series of zinc strips and InSite acid phosphatase strips were prepared.  The zinc strips were prepared according to the procedure of Hooft and van de Voorde using Whatman Grade 1, Whatman Grade 3 and VWR Grade 417 filter paper.  The AP strips were prepared according to a modification of Babson and contained approximately 5 mg citrate buffer, 1 mg 1-naphthyl phosphate and 1.6 mg Dye Fast Blue B salt per strip, using Whatman Grade 1 and Whatman Grade 3 filter paper.  A semen dilution series with these strips in shown in Fig 2.  The zinc strips proved to have a detection limit of 1/250, while the AP strips had a detection limit of 1/2000.  The InSite AP strips proved to be somewhat more sensitive then the Phosphatesmo strips used for comparison in this experiment, although they had a slight tan color.  (Perhaps this increased sensitivity can be attributed to a greater amount of reagent.)  Because of the dramatic purple color change upon exposure to acid phosphatase, however, this tan color (most likely due to degradation of the diazonium salt component) was judged not to be important.  Whatman Grade 3 paper seemed to give the best contrast with the zinc strips, whereas Whatman Grade 1 paper gave the best results among the AP strips.

Figure 2.  Serial Dilutions of Semen and Analysis with Zinc Strips and Acid Phosphatase Tests
Using different grades of filter paper (dilutions are listed by top, bottom)

The sensitivity of PSA strips from various manufacturers was measured using a similar dilution series, and a semenogelin immunochromatographic test was used simultaneously for comparison.  The results are shown in Fig 3.  Strips A, B and D are PSA, while strip B is semenogelin.  Both tests proved to be sensitive down to a dilution of 1/500,000 which is generally consistent with that previously reported [Ref. 9,10]. Since semenogelin strips were ten times more expensive than PSA strips, the latter were chosen for inclusion in the InSite kit.  The PSA strips were also judged to have better visibility than the semenogelin strips.

Figure 3.  Serial Dilutions of Semen and Analysis with PSA Strips and Semenogelin Strips from various Manufacturers
A= PSA;  B= Semenogelin;  C= PSA;  D= PSA.  Note:  PSA strips from Company "A" and Company "C" look identical and raw materials probably come from the same distributor.

Finally, a comparative study was conducted between seven different brands of PSA strips, as well as an RSID^TM semenogelin test, using the same semen dilution series as before. The results are shown in Fig 4.  Strips A-E, G and H are PSA, while F and I are semenogelin.  There were notable differences between brands.  The "E" brand was chosen for inclusion in the InSite kit because it seemed to have the best sensitivity and readability.  A few QC problems were noted with the "B" strips, which otherwise were identical with "E".  The RSID test ("I") did not work well under these conditions, probably because the required buffer solutions were not used to run the test, but plain water was used instead.  In contrast, the semenogelin strips from company "F" worked just fine.

Figure 4.  Serial dilutions of semen and comparative testing between brands of PSA and semenogelin strips.  A = PSA;  B = PSA;  C = PSA;  D = PSA;  E = PSA;  F = Semenogelin;  G = PSA;  H = PSA;  I = Independent Forensics RSID^TM (semenogelin).  Note:  PSA strips from Company "B" and Company "E" look identical.

Extraction of Garments

Control experiments were carried to demonstrate negative reactions.  Fig. 5 shows women's underwear with typical vaginal discharge, which was known to be negative for semen.  The zinc strip showed no color change when used to analyze these garments, nor did it show any time dependency.  The Phosphatesmo test, on the other hand, started to turn purple after 15 s, while the CheckMate^® test started to change after 10 minutes.  The InSite AP strips did not react at all (not pictured).  This demonstrates that vaginal acid phosphatase interferes with the detection of seminal AP and will give a false positive test in as little as 60 s.  The garments were analyzed by wetting the area of interest with 1-2 drops of water, and then pressing test strips against the areas (zinc and Phosphatesmo) or by pressing a filter paper circle against the area (followed by a CheckMate^® AP test).

In order to determine how long semen can be detected on underwear, the undergarments of a volunteer were analyzed at periods of 0-10, 10-17 and 17-34 hours after intercourse.  Fresh cotton underwear was worn during each time period.  The garments were analyzed using zinc strips, three acid phosphatase tests (InSite^® strips, CheckMate^® and Phosphatesmo KM brands), PSA strips and semenogelin strips.  The results from the zinc and AP tests are shown in Fig. 6-7.  The two acid phosphatase strips again proved to be sensitive and convenient, yielding a dramatic purple reaction after a few seconds.  The CheckMate^® test took somewhat longer to turn blue, the reaction rate being dependent on the freshness of reagents.  The zinc test turned red when pressed against the suspect area, but the result was not as dramatic as the acid phosphatase tests.  A PSA test was performed for confirmation, showing a strongly positive PSA test after 10 h (after 17 h the AP tests were done first and soaked up inordinate amounts of material, which explains the weak PSA).  The zinc test was very slightly positive after 34 h, while Phosphatesmo and CheckMate^® were judged to be negative.  The PSA test was also very slightly positive.  (UV light was found not to be useful in visualizing semen stains.  These stains appeared not to be fluorescent.)

The results from the PSA and semenogelin tests are shown in Fig. 8.  PSA was detectable up to 32 h after intercourse, whereas semenogelin was detectable only up to 20 h.  In addition, the PSA strip had slightly better visibility.  Since PSA strips are 1/10th the price of semenogelin strips, the former were chosen for inclusion in the InSite kit.

The original paper by Hooft et al [Ref. 3] stated that the zinc test was more sensitive and specific than the classical AP test.  This test was based on analyzing vaginal swabs from a gynecologist's office, and also evidentiary material from alleged sexual assaults (probably also vaginal swabs).  This method is much different from analyzing women's underwear.  Vaginal swabs analyze source material deep in the vagina, whereas underwear has absorbed whatever leaks out.  Zinc is probably much more concentrated in the vagina.  In addition, there was an unknown time period between sample collection and laboratory analysis of the swabs, during which time seminal proteins could have become denatured or cleaved, releasing the zinc bound to them. Thus, these two methods really aren't comparable.

Although laboratory testing with zinc chloride has shown that the limit of detection of the strips is approximately 0.01 mg/mL of zinc, the limit of detection of semen is 1/200, corresponding to a free zinc concentration of about 1 mg/mL (assuming 200mg/mL in semen).  This means that zinc is probably 99% bound to proteins and other compounds in semen. This is consistent with literature reports that zinc tends to form complexes with other components of semen and that after ejaculation, 50% is bound to seminal vesicle proteins [Ref. 7].

These results indicate that semen could be detected by the AP and zinc tests up to 17 h, and by the PSA and semenogelin tests up to 36 h after intercourse.  These data are consistent with those reported in the literature, where it is reported that AP is generally not detectable beyond 12-18 h after intercourse [Ref. 11], that PSA levels return to baseline by 48 h [Ref. 12], and that semenogelin has been detected up to 47 h [Ref. 13].  This experiment was repeated twice with identical results.

The volunteer still had semen visible in her vagina after 36 h as a stringy white substance; however, no marker proteins could be detected.  We attribute this observation to the denaturization of seminal proteins by the acidic pH of the vagina (in a fashion similar to egg whites when they are cooked), among other factors.

Fig. 5.  Control specimens of women's underwear showing typical vaginal discharge with negative test results.

Fig. 6.  Tests of women's underwear with zinc and AP tests, plus PSA for confirmation (note:  there is no visual difference between control and test undergarments).

Fig. 7.   Additional tests of women's underwear (5 separate pairs) at intervals after intercourse with zinc and AP tests

Figure 8.  Extraction of cotton underwear at intervals after sexual intercourse and testing for PSA and semenogelin.  Three separate trials are shown.

Extraction of Absorptive Pads

In order to determine whether a woman's menstrual period affects the ability to detect PSA, the absorptive pads of a volunteer on her menstrual period were analyzed at various intervals after intercourse.  The pads were analyzed using PSA strips.  The results are shown in Fig. 9-10.  PSA was detectable up to 36 h after intercourse, which was approximately the same result obtained by analyzing undergarments while not on a menstrual period.  These results indicate that a woman's menstrual period does not interfere with the immunochromatographic detection of PSA.

Figure 9.  Extraction of menstrual absorptive pads at intervals after sexual intercourse.  Two separate trials are shown.

Conclusion

AP test strips have been prepared based on the classic reaction first reported by Babson.  These strips proved to be sensitive and convenient to use, and were able to detect semen which was discharged onto a woman's undergarment up to 17 h after intercourse.  The AP strips gave a more dramatic color change than zinc strips with actual specimens of used underwear, and therefore were chosen for inclusion in the InSite kit.  PSA was found to be the best marker protein for immunochromatographic detection of semen, based mainly on cost.  Product evaluation of PSA strips from several manufacturers allowed identification of the best brand of strip.  These strips can detect semen which has been discharged up to 36 h after intercourse.  It is recommended that the AP strips be used first to test an item, providing presumptive evidence of semen, followed by the more sensitive and specific PSA test for confirmatory evidence.

Summary

Zinc test strips have been prepared according to the method of Hooft and van de Voorde.  These strips were found to detect semen to a dilution of 1/250.  Acid phosphatase strips were prepared according to a modification of the procedure of Babson, and had a detection limit of 1/2000.  The CheckMate^® and Phosphatesmo AP tests had detection limits of 1/150 and 1/300, respectively.  There was no difference in sensitivity between the PSA and semenogelin strips, which both detected semen to a dilution of 1/500,000.  Testing of cotton undergarments showed that semen discharged onto them could be detected by the AP and zinc tests up to 17 h, and by the PSA and semenogelin tests up to 36 h after intercourse.  Semen was still visible in the volunteer's vagina after this time.  These results suggest that marker proteins such as PSA and semenogelin are denatured to undetectable levels by 48 h after intercourse, possibly due to the acidic pH of the vagina, and other factors such as oxidation by hydrogen peroxide and enzymatic cleavage by other seminal proteases.

Experimental

Materials.  Zinc test strips were prepared according to the procedure of Hooft and van de Voorde, using Whatman No.1, No. 3 and VWR No. 417  filter papers as substrates.  Acid phosphatase test strips were prepared according to a modification of the method of Babson [Ref. 6] using the same filter papers as above and mounted to a plastic backing as an assembly.  PSA test strips were obtained from several proprietary suppliers.  AP test kits were obtained from Evergreen Industries (CheckMate^® brand) or CTL Scientific Supply (Phosphatesmo KM), respectively.  Semenogelin strips were obtained from Independent Forensics of Illinois (RSID^TM cassettes) or a proprietary supplier (strips), respectively.

Zinc test:  A 2-5 drop aliquot of deionized water was placed on a suspect area of a garment, and a zinc strip was pressed against it.  A color change from yellow to pink was a POSITIVE test.  Alternatively, a cotton-tipped swab was placed against the wetted area of the garment, and then the garment was pressed gently around the swab in order to saturate the swab with solution.  This was done in several places on the garment.  Then, the swab was pressed against a zinc test strip.  This procedure yielded a more easily visualized, higher-contrast spot.  It also avoided leaving any stain on the garment.

AP test:  A 2-5 drop aliquot of deionized water was placed on a suspect area of a garment, and an AP test strip was pressed against it.  A color change to bright purple within 15 s was a POSITIVE test.  Alternatively, a cotton-tipped swab was placed against the wetted area of the garment, and then the garment was pressed gently around the swab in order to saturate the swab with solution.  This was done in several places on the garment.  Then, the swab was pressed against an AP test strip.  This procedure yielded an easily visualized, high-contrast spot.  It also avoided leaving any stain on the garment.

PSA test.  A 15-mL aliquot of water was placed in a coffee cup.  The suspect area (i.e. crotch) of a pair of cotton underwear was extracted in the cup by repeatedly allowing water to soak in, then pressing it out.  Finally, the garment was wrung out into the cup.  A PSA test strip then was placed into the cup.  It was necessary to tilt the cup on edge to immerse the strip.  Care was taken not to immerse the strip above the marker line.  After 10 minutes, the strip was removed and laid on a clean, dry surface.  The strip was read after an additional 10 minutes.  Resolution continued to improve for 30 minutes after the strip was removed from the coffee cup, but tended to decrease after that.  A POSITIVE test was indicated by two lines as shown in Fig. 10.  A strongly positive test was clearly visible within two minutes, while a weakly positive test took 20 minutes (after immersion) to become evident.

Absorptive pads were tested by placing 25 mL of water into the coffee cup (for a full pad) or 10 mL for a mini-pad, and repeatedly extracting the pad manually.  Then, the pad was wrung out into the cup and discarded.  The PSA test was carried out as usual.

NOTE:  latex gloves were used for these procedures.

  Figure 10.  Simplified diagram for performing a PSA test.

References

1. Laumann, E. O. et al.  "The Social Organization of Sexuality:  Sexual Practices in the United States";  University of Chicago Press:  Chicago, 1994, p. 216.

2. Mosher, W. D. et al. "Sexual Behavior and Selected Health Measures: Men and Women 15-44 Years of Age, United States, 2002,"  Advance Data 2005, 362, p. 10.  (http://www.cdc.gov/nchs/data/ad/ad362.pdf.)

3. Hooft, P. J. and van de Voorde, H. P. "Bayesian Evaluation of the Modified Zinc Test and the Acid Phosphatase Spot Test for Forensic Semen Investigation," American Journal of Forensic Medicine & Pathology 1997, 18, 45-49.

4. Pilch, B. and Mann, M. Genome Biology 2006, 7:R40.

5. Tanaka, M. et al  FEBS Letters 2004, 571, 197-204.

6. Babson, A. L. et al, Am. J. Clin. Path. 1959, 32, pp. 88-91.

7. Owen, D. H. and Katz, D. F. Journal of Andrology 2005, 26, 459-469.

8. Hooft, P.; van de Voorde, H. and van Dijck, P. Forensic Science International 1992, 53, 131-133.

9. Pang, B. C. M. and Cheung, B. K. K.  Forensic Science International 2007, 169, 27-31.

10. Gartside, B. O.; Brewer, K. J. and Strong, C. L.  Forensic Science Communications 2003, 5 (2).  (http://www.fbi.gov/hq/lab/fsc/backissu/april2003/gartside.htm)

11. Khaldi, N. et al.  J. Forensic Sci. 2004, 49 (4), p. 1-5.  http://www.hawaii.edu/hivandaids/Evaluation_of_Three_Rapid_Detection_Forensic_Id_of_Seminal_Fluid_in_Rape_Cases.pdf

12. SERATEC GmbH.  "PSA in Body Fluids--an overview for users of the SERATEC PSA SEMIQUANT Tests."

13. Keil, W.; Bachus, J. and Tröger, H. D. Int. J. Legal Medicine 1996, 108 (4), p. 186-190.

This paper was published on March 19, 2008.  The InSite^® Semen Detection Kit and certain technology described in this paper is covered under U.S. Pat. 8,137,956 and other patents pending.