Utilizing differential extraction thresholds to deduce the existence of spermatozoa in forensic casework samples

Testing of evidence in an alleged sexual assault case not only seeks to address the question of who was involved, but also looks to answer the question of what biological source provided the DNA. It can be difficult to obtain positive serological data on challenging samples such as laundered items, low-level DNA samples, or sexual assault kit swabs obtained after a prolonged interval from the time of assault. In the absence of confirmatory serological results, an expert witness often cannot speak to the biological source of the DNA. In order to determine quantitation thresholds which could be used to deduce the presence of spermatozoa (sperm) within a sample, we evaluated the fractionation of male DNA utilizing our laboratory’s differential extraction method. Study samples included serial dilutions of semen and semen/saliva mixtures, post-coital and laundered samples as well as casework data from 1,729 samples that were processed using a differential extraction. Based on this data, it was determined that a sample which had at least 200 picograms of male DNA and at least 10% of the total male DNA in fraction 2 (F2, also known as the sperm-enriched or sperm fraction) could be reported as positive for the presence of sperm. No false positive results were obtained from the study-generated samples when using these thresholds to infer the presence of sperm. Additionally, samples that contained sperm, but were negative using traditional serological methods, could be detected. However, not all sperm-containing samples fractionated above both thresholds; therefore, serological testing may still be necessary to minimize false negative results. The thresholds developed here, proved reliable to deduce the presence of sperm in real casework samples.