Comparison of Semi-Automated and Manual Differential Separation Methods for Mock Sexual Assault Samples

In the event of a sexual assault, separation of suspect from victim DNA is possible through differential extraction if the evidentiary mixture consists of female epithelial cells and male spermatozoa. The basis of differential extraction is the differing properties of epithelial and sperm cell membranes, specifically the comparative sturdiness of the proteins making up the sperm head containing the male DNA. Effective retrieval and separation of male and female DNA fractions is important in generating high-quality STR profiles that can be utilized in identifying suspects in a sexual assault case. Differential extraction has been conventionally performed using Proteinase K (PK) to lyse epithelial cells, then dithiothreitol (DTT) to lyse sperm cells after the fractions were separated through centrifugation. The protocol for differential separation currently used by the FBI Laboratory is a semi-automated variation of this method using the QIAcube and EZ1 Advanced XL. SpermX is a manual differential separation method from Innogenomics that uses a novel nanofiber matrix to trap and separate sperm from nonsperm cells. Three comparisons were performed to observe the ability of the FBI and SpermX differential extraction methods. The comparisons examined samples with different semen donors, volumes of mixture (2 uL, 10 uL, 20 uL, 50 uL), and stain materials (condoms, 100% cotton, 95% cotton/5% spandex, 100% polyester, and 100% denim). The samples’ DNA quantity, degradation index, male to female ratio, proportion of mixture DNA profiles, and allele percentages were then used to observe differences between the FBI and SpermX differential extraction methods. Differences were observed in the DNA quantity and proportion of mixture profiles. A higher female fraction DNA quantity was generally observed when the FBI method was used across all comparisons, and a lower proportion of mixtures was observed for the donor comparison male fractions and the sensitivity comparison female fractions (