Confirmatory Tests for Semen
The presence of intact spermatozoa in a biological stain has historically been the conclusive test for semen; they are abundant in semen and should be plentiful. A lack of sperm doesn’t necessarily mean the stain isn’t semen, only that sperm are not present. The traditional method for sperm identification is to use the Christmas tree stain, which turns the tip of the sperm’s head pink, the bottom of the head dark red, the middle portion blue and the tail yellowish-green; skin cells stain green to blue green and are easily distinguished. See “In More Detail: Using the Christmas Tree Stain to Visualize Sperm” for a description of the method. An extract of the stain is dissolved in water, and then a portion is applied to a microscope slide, heat-fixed, and then colored with the Christmas tree stain. The slide is then scanned at magnifications of 400× to 1000×. Even if detached sperm heads are the only structure on the slide, and the Christmas tree stain assists in distinguishing them, this is still confirmative for the presence of semen. Phase contrast or dark field microscopy may also be used. The hematoxylin-eosin stain is also used but operates on the same premise (the stain colors are purple for heads and pink for the rest). In 1978, George Sensabaugh published a paper outlining the forensic use of a prostate specific antigen that he named p30 (p for prostate, 30 for its molecular weight of 30,000) (Sensabaugh, 1978). A method called enzyme-linked immunosorbent assay (ELISA) is typically used to detect p30 at levels as low as 0.005 ng/mL (the threshold for rectal samples is 2 ng/mL because of reactions with other sub stances found there). ELISA is based on the antigen–antibody reaction: The reagent is added to the filter paper or swab, and if an intense purple color is seen, a reaction has occurred and p30 is present. The labeled p30 is attached to the antibody. In ELISA, the label is an enzyme that catalyzes a reaction where the substrate changes color; the deeper or more intense the color, the more label, and therefore the more p30, is present. ELISA is a good method for mass-testing of samples; in forensic laboratories, ABA semen detection cards or similar types of tests are now used. Depending on the crime, it may be useful to determine the time since inter course (TSI) to assist in the sequence of events. Typically, this means the detection of spermatozoa, but because of natural variations, the timing is rarely exact. Motile (moving) sperm can survive in the vagina for about 3 h, ranging from 1 to 8 h; they survive longer in the cervix, up to several days in some cases. By the time a case gets to the forensic serologist, however, the sperm are rarely motile. Time lags inevitably occur between the occurrence of the crime, collection, submission, and analysis. Forensic serologists therefore look for intact sperm. These can persist in the vagina up to 26 h, and the heads alone can last up to 3 days; again, the persistence is greater in the cervix. Intact sperm can be found from 6 to 65 h post coitus in the rectum (or until the next bowel movement) but rarely more than 6 h in the mouth. Levels of p30 have been used to estimate TSI, and most p30 is eliminated within 24–27 h after intercourse.
