Differentiating Touch Epidermal Cells from Blood Cells in Single Source and Mixture Samples using Flow Cytometry

Bloodstains are a common piece of forensic evidence that can be found at crime scenes such as murders and sexual assaults. One factor that can complicate the analysis of bloodstains is the presence of epithelial cells within the same sample. Epithelial cells, particularly those transferred through skin contact, cannot be presumptively tested for and can create mixture DNA profiles that are difficult to interpret. Flow cytometry has the potential to provide probative information for biological samples that may contain ‘touch’ epithelial cells and/or blood because it is capable of determining morphological information such as the cell’s size and the components found within the cell. In order to determine if flow cytometry can differentiate touch cells from blood, single source touch cell and blood cell samples as well as mixtures were analyzed using flow cytometry to characterize autofluorescence profiles of individual cells. Single source samples were analyzed under with red, yellow, and green fluorescence. Profiles were then used to generate criteria for detecting each cell type in an unknown sample. The reliability of these signatures was assessed using a series of single source tissue samples and mixture samples. DNA analysis was also performed on mixtures to determine if the number of male blood cells in the overall sample corresponded with the A:Y ratio. Results showed that yellow and red fluorescent channels combined with a large cell gate (>8,000) were best for differentiating single source blood and touch epithelial samples. However, it was still difficult to determine the presence of blood in the mixtures created. Results from DNA analysis showed that the A:Y ratio did not correspond to the expected mixture ratio particular at lower blood concentrations. Overall, this indicates that flow cytometry-based characterizations may be capable of differentiating touch cells from blood for some single source samples, but further research is needed to apply these signatures for mixture samples.