With the increase in sensitivity of DNA profiling, determining the time-since-deposition (TSD) of human biological material on items of evidence has become highly pertinent in forensic cases to address activity level questions. We have developed a novel method to determine TSD of trace biological samples using morphological and autofluorescence profiles of individual epithelial cells measured non-destructively with flow cytometry prior to DNA profiling. Tested on trace epithelial samples from saliva and touch deposits over periods ranging from 1 day to over 1 year, we observed linear changes in autofluorescence intensity for most donors (R2>.80 for 12 out of 15 donors). Due to baseline autofluorescence variation among individuals, a binary prediction model was developed to classify TSD into specific intervals. Based on autofluorescent intensity samples with TSDs of less than 1 week can be determined without false positive classification of older samples, with a false negative rate of ~30% (i.e. TSD samples appearing older than they actually are).

We extended this technique to test whether cellular autofluorescence could be used to physically separate two-person saliva mixtures into their contributor cell populations. For this, 10 different saliva mixtures were created wherein each contributor’s saliva had different TSDs between ~ one week to ~ 10 months which was then subjected to autofluorescence analysis and cell separation with the Cytek Aurora™ Cell Sorter. In all mixtures tested, results showed that biological material from each contributor could be effectively separated from the mixture, and produce clearly interpretable DNA profiles corresponding to each donor. Specifically, mixture profiles with a contributor ratio ~ 1:1 yielded profiles in the sort fractions that were at least 2:1 for the correct donor, or in some cases became a single-source profile. In mixture samples with a clear major contributor, sorting produced a DNA profile that was clearly enriched for the minor contributor. 

Overall, autofluorescence and morphological signatures may be one of the first techniques for determining TSD for saliva and may be applicable to other bodily fluids. Additionally, these signatures enable the separation of older from newer depositions, allowing for the assignment of deposition timing to different contributors in a mixture and potentially improving mixture resolution prior to the application of mixture deconvolution techniques.

Authors

• James Morrow (Centre of Forensic Sciences, Canada)
• Gabrielle Wolfe (Virginia Commonwealth University, United States of America)
• Ariana DeCorte (Virginia Commonwealth University, United States of America)
• Monica Truong (Centre of Forensic Sciences, Canada)
• Cassandra Dodoo (Centre of Forensic Sciences, Canada)
• Jonathan Millman (Centre of Forensic Sciences, Canada)
• Christopher Ehrhardt (Virginia Commonwealth University, United States of America)