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.

 

 

 

Control	Control

 

Control zinc	Control Phosphatesmo 15 s	Control Phosphatesmo 60 s	Control CheckMate® 15 s	Control CheckMate® 20 m

 

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

 

 

 

 

0-10 h

 

zinc pressed 0-10 h	Phosphatesmo 0-10 h	CheckMate® 0-10 h	PSA 0-10 h	zinc test of extract 0-10 h

 

zinc pressed 10-17 h	Phosphatesmo 10-17 h	CheckMate® 10-17 h	PSA 10-17 h

 

zinc pressed 17-34 h	Phosphatesmo 17-34 h	CheckMate® 17-34 h	PSA 17-34 h

 

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).