Inferring the presence of spermatozoa in forensic samples based on male DNA fractionation following differential extraction

To address sexual assault kit backlogs some laboratories in North America have implemented 'Direct to DNA' (DTD) approaches for the examination of relevant vaginal, oral, rectal and external genitalia swabs from sexual assault examination kits. Using this approach no preliminary serological screening for semen or spermatozoa is performed. Instead, swabs are directly subjected to differential extraction and quantitation using a dual quantification system. Decisions regarding the next steps in processing each sample are typically based on the quantity of male DNA detected, the fraction in which it is detected, and its ratio to the total human DNA in the sample. In the absence of serological results it remains of value in many cases to determine whether spermatozoa are present in the sample and whether the male DNA profile may be attributed to this body fluid. In this study we examine the distribution of male DNA from various body fluids between epithelial and spermatozoa fractions following differential extraction. Based on these results we identified criteria under which a DNA profile can be reliably attributed to spermatozoa. A total of 18 blood samples, 129 saliva samples, and 78 semen samples were processed. The maximum amount of male DNA observed in the sperm fraction of a spermatozoa-free sample was 10.4 ng and the maximum portion of total male DNA observed in the sperm fraction was 7.7%. In contrast, when a sample contained spermatozoa the minimum portion of total male DNA observed in the sperm fraction was 52.6%. This research supports a 50% threshold of male DNA in the sperm fraction following differential extraction as a conservative criterion under which the scientist at the Centre of Forensic Sciences (CFS) may infer that spermatozoa is present in a sample in the absence of serological results. This general threshold is applicable to all sample types (underwear, clothing, swabs, condoms) tested. We further demonstrate that, if the same male DNA profile is represented more prominently in the sperm fraction versus the epithelial fraction, the analyst should not fall into the intuitive trap of inferring the presence of spermatozoa in the sample. Instead, enrichment calculations must be based on the measured quantity of male and total DNA in each fraction and not the male:female DNA ratios observed in the DNA profiles.