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Analysis of DNA mixtures from sexual assault evidence is an ongoing challenge for DNA casework laboratories. To assist the forensic scientist address source and activity level propositions there is a significant need for new techniques that can provide information as to the source of DNA, particularly for sexual assault samples that do not involve semen. The goal of this study was to develop a new biological signature system that provides additional probative value to samples comprised of mixtures of epidermal and vaginal cells, as may be observed in cases involving digital penetration. Signatures were based on morphological and autofluorescence properties of individual cells collected through Imaging Flow Cytometry (IFC). Comparisons to reference cell populations from vaginal tissue and epidermal cells collected from hands showed strong multivariate differences across > 80 cellular measurements. These differences were used to build a predictive framework for classifying unknown cell populations as originating from epithelial cells associated with digital penetration or epidermal tissue. As part of the classification scheme, posterior probabilities of specific tissue group membership were calculated for each cell, along with multivariate similarity to that tissue type. We tested this approach on cell populations from reference tissue as well as mock casework samples involving hand swabbings following digital vaginal penetration. Many more cells classifying as non-epidermal tissue were detected in digital penetration hand swab samples than control hand swabbings. Minimum interpretation thresholds were developed to minimize false positives; these thresholds were also effective when screening licked hands, indicating the potential utility of this method for a variety of biological mixture types and depositional events relevant to forensic casework. Results showed that samples collected subsequent to digital penetration possessed markedly higher numbers of cells classifying as vaginal tissue as well as higher posterior probabilities for vaginal tissue (≥ 0.90) compared to cell populations collected from hands without prior contact with vaginal tissue. Additionally, digital penetration cell populations may be resolved from saliva cell populations and other non-target tissue types., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Crown Copyright © 2023. Published by Elsevier B.V. All rights reserved.)

This study investigated the transfer and persistence of salivary DNA under fingernails. This was performed to address a common alternate hypothesis presented to scientists in court, asserting that a relatively large quantity of DNA detected beneath the fingernails, typically from a victim of crime, originates from innocuous transfer of saliva in a casual setting. It was determined through these studies that contact with liquid saliva was an effective way to transfer foreign DNA beneath fingernails. However, when saliva was dried, DNA did not readily transfer through casual contact. When liquid saliva was placed directly beneath fingernails the amount of DNA detected from the saliva donor twenty-four hours later was several hundred-fold lower than the amount detected when sampling occurred immediately following deposition. Furthermore, when the recipients' hands were washed immediately following the deposition of liquid saliva beneath fingernails, the majority of foreign DNA was removed following one hand washing and all detectable foreign DNA was removed from most recipients' hands after three or six hand washings. This study demonstrates that casual contact with wet saliva can result in the transfer of substantial quantities of DNA beneath fingernails but that it does not typically persist for extended periods of time and is mostly removed if the hands are washed soon after deposition., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2023. Published by Elsevier B.V. All rights reserved.)

Empirical data obtained from controlled experiments is necessary to ensure that sound expert opinion evidence is provided regarding transfer and persistence of DNA in criminal proceedings. Knowledge in this area is also required at the outset of criminal investigations, to ensure that the proposed examinations can assist with answering questions that are relevant to forensic investigations. This study aimed to provide such data by examining the relative and absolute quantities of DNA deposited on items that are routinely submitted to the forensic laboratory by a habitual user, defined as someone who used it for ~1 week, and a subsequent one-time user. We found that the quantity of DNA deposited on routine household items spanned a broad range. The habitual user's DNA was detected on most items as the major donor, regardless of whether it was subsequently handled by another person for a short period of time. The one-time, short duration, user's DNA was detected on approximately two thirds of the items, albeit typically at quantities lower than the habitual user. Most of the household items we examined also had detectable DNA deposits from at least one other, unknown individual, typically in low quantities. Attempts to clean non-porous items with readily available household cleaners were partially effective but failed to completely eliminate detectable DNA from a habitual user in most cases., (Crown Copyright © 2022. Published by Elsevier B.V. All rights reserved.)

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., (Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.)