In this study, researchers use novel cellular signatures for front-end differentiation of contributor cell populations and estimation of DNA content.
This project develops a new method for screening trace biological samples for the number of contributors and DNA content based on the presence and relative abundance of key protein and hormone targets within cell populations. This research could increase the probative value of many types of mixture samples and reduce case-working bottlenecks associated with complicated interpretation protocols for DNA mixtures. The authors present a novel workflow for analyzing biological evidence samples that (1) estimates the number of contributors in a mixture based upon flow cytometry histogram profiles, (2) estimates the human-specific DNA content in the sample based upon fluorescent signal intensities, and (3) differentiates cell populations in the mixture based on contributor-specific attributes. The results include a novel set of contributor-specific signatures with the potential to utilize novel intracellular targets to selectively tag cell populations. All aspects of this workflow are inherently non-destructive; thus, these results can be instrumental in bridging the gap between the demonstrated utility of fluorescence-based cell labelling and its relevance for forensic casework. A series of ‘touch’ cell samples were generated by taking direct swabs of an individual’s palmar surface or by having participants handle various substrates. Replicate samples from the same contributor were also collected to assess intra-donor variability. Overall, results showed that some contributor cell populations showed higher binding affinities for testosterone-probes as assessed by comparing the median fluorescence intensity for the entire cell population as well as the mode fluorescence intensity, which corresponded to the peak in each fluorescence histogram. The authors also tested the binding efficiency and differentiation potential of antibody probes targeting structural alleles of cytokeratin molecules. The authors found that extending the incubation time, using higher concentration of blocking buffer to reduce non-specific binding, and standardizing the ratio of antibody probe to cell concentration effectively decreased non-specific binding.