Migration patterns of seminaI fluid components and spermatozoa in semen stains exposed to water and blood
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Typically, semen testing involves presumptive and confirmatory tests to determine the region in which a semen stain has been deposited prior to initiating DNA analysis. However, previous research showed that the soluble components of seminal fluid, but not spermatozoa, migrated from their original location on cotton cloth upon exposure to porcine decomposition fluids and rainfall/dew6. This indicates that preliminary testing and detection techniques may result in areas being sampled that will not yield a successful DNA profile. The present study assesses how various amounts of water or blood affect migration patterns of seminal fluid components using traditional serological screening methods as well as DNA analysis. The effects of exposing a semen stain to water over the course of several days are also investigated. The final component of the study evaluates whether the presence of acid phosphatase (AP) Spot reagent had any detrimental effects on subsequent antigen P30 (P30) testing, Kernechtrot Picroindigocarmine (KPIC) sperm staining or DNA analysis. Neat semen was deposited onto swatches from cotton sheets and allowed to dry before being sprayed with 2 mL, 5 mL, or 10 mL of water or blood. The swatches were allowed to dry while lying flat, at 45°, or at 90°. Three of the swatches were sprayed directly with AP Spot reagent to determine any potential interference with subsequent P30 and DNA testing. After the water or blood was dry, the swatches were viewed with an alternate light source (ALS) at 450 nm using orange barrier filter goggles. Three-millimeter fabric punches were collected from each swatch in at least thirteen locations (one from the center of the stain and four at 1 cm, 4 cm, and 7 cm from the perimeter of the stain in multiple directions), and were extracted for two hours prior to testing for the presence of P30. Additional fabric punches were collected from each P30 positive location to be used for DNA analysis. AP testing showed positive results beyond the original semen stain with an average distance of 1-3 cm from the perimeter of the original region of deposition (ORD) for all swatches except those moistened with blood. AP mapping was performed on the swatches moistened with blood and negative results were obtained. Positive P30 results were obtained for all swatches with an average distance of 1-3 cm from the ORD. The angle at which the swatch was positioned influenced the direction(s) that the soluble components migrated; however the amount of water (or blood) the swatch was exposed to had a much greater effect on the distance of migration. Microscopic examination of slides made from the extracts of each fabric punch revealed minimal spermatozoa migration for all swatches; the majority of the samples outside of the ORD showed no spermatozoa, although a few showed a single sperm cell. These findings demonstrate that the soluble components of semen stains that often aid in detection migrated when exposed to moisture, while sperm cells containing genetic material largely remained in their original location. The DNA analysis results confirmed the lack of spermatozoa migration. Full DNA profiles were obtained from within the ORD of the flat and 90° swatches. The samples from outside of the ORD produced either partial profiles (maximum dropout rate of 97%) or no profile. If case circumstances suggest that evidence has been exposed to water, multiple regions should be tested in order to maximize the possibility of identifying semen and obtaining a DNA profile. AP Spot reagent was not found to have detrimental effects on P30 testing, sperm staining or DNA analysis. Therefore, direct application of AP Spot reagent could be used for larger pieces of evidence where the location of a stain is unknown. This would eliminate the careful documentation needed for chemical mapping and the reliance on the transfer of acid phosphatase from one substrate to another.