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Conclusion
InSite® Semen Detection Kit

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 (RSIDTM 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 a pending U.S. patent.

 
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