Abstract:
The main objectives of this research were to investigate improvements to sperm recovery from rayon swabs and archived glass slides and to evaluate the effectiveness of Sperm Hy-Liter™ PI for sperm searching and profile quality. Forensic casework evidence often comprises mixtures of sperm and epithelial cells, with sperm often being the cells of interest in a sexual assault investigation. Therefore, maximising sperm recovery from samples, expediting their identification, and optimising their separation from epithelial cells prior to downstream DNA analysis are important steps that contribute to the success of DNA profiling of semen stained samples in casework. Rayon swabs are currently used for the collection of forensic evidence during the medical examination of individuals and cellular material from items and crime scenes. ESR currently uses the Differex™ method for the recovery and separation of sperm from semen stained samples. However, as little was known about the effectiveness of this method in recovering sperm from rayon swabs, investigations were undertaken to determine its efficacy. It was determined that approximately 62% of the sperm DNA present remains on the rayon swabs following Differex™ treatment. Technical modifications were made to the Differex™ method and alternative methods were tested in an attempt to improve sperm recovery from rayon swabs. Alternative swab types were also tested, comparing sperm recovery to that obtained from rayon swabs. This testing determined that rayon swabs and Differex™ were currently the most suitable sampling substrate and sperm recovery method, respectively, for the analysis of semen stained swabs. Investigations were also undertaken into the effects of storage temperature and time on sperm recovery, with the results indicating that the current storage practises at ESR were appropriate. The current method for cell recovery from histologically stained glass slides for DNA analysis involves the use of harmful chemicals and vigorous cell scraping. With the possibility of cell loss, a safer alternative cell recovery method was investigated. Mount Quick, a mounting medium, was tested for its ability to recover and transfer cells from glass slides to a slide compatible with laser microdissection. Such a method would be beneficial to the DNA analysis of slides containing mixed cell samples with low numbers of sperm. Alternatively, if sperm numbers were high, direct analysis of Mount Quick, or a PEN membrane, following the recovery of cells from a glass slide could be undertaken. Near complete male DNA profiling results were obtained from the sperm fraction after the Differex™ DNA IQ™ analysis of the Mount Quick and PEN membrane, and also from the Mount Quick and PEN membrane after direct DNA IQ™ analysis. The poor DNA profiling results obtained after standard DNA testing of laser microdissected post-coital sperm indicate that a more sensitive DNA test, such as Low Copy Number (LCN) DNA analysis, is required for the analysis of sperm dissected from these samples. The findings from the testing of Mount Quick indicate that it should be considered as a suitable replacement to the current glass slide scraping method. ESR’s current method for identifying sperm, particularly when numbers are low, can be challenging and time-consuming. An evaluation of a fluorescence-based test, Sperm Hy-Liter™ PI, was performed to assess its suitability for sperm searching and the profile quality of Sperm Hy-Liter™ PI stained sperm using LMD DNA analysis. Preliminary results indicate that Sperm Hy-Liter™ PI may have a place in ESR’s Forensic Biology Group, however more work would be required before it could be used routinely.