Optical differentiation of cell populations in contact cell mixtures using flow cytometry
Creators: name: Stanciu, Cristina E.
name: Kwon, Ye Jin
name: Bustamante, Eduardo E.
name: M. Katherine Philpott
name: Ehrhardt, Christopher J.
Publisher: name: F1000Research
Year: 2016

description: Analysis of contact or ‘touch’ mixtures is a significant problem for DNA caseworking laboratories. The presence of cells from multiple contributors in an evidence sample can produce STR profiles that are difficult to interpret and may ultimately lead to its total loss. Although many protocols for cell separation exist, most cannot be applied to touch mixture samples that contain only one cell type. New methods are therefore needed that can differentiate epidermal cell populations from different contributors and allow them to be physically isolated from the mixture prior to DNA profiling. One promising strategy is to target the allelic diversity in protein structures within cells from different individuals but this approach has not been explicitly tested on forensically relevant touch samples. Therefore, the goal of this project was to survey biochemical variation in epidermal cell populations and identify specific features that may be used to differentially label and then sort individual cell populations from a touch mixture.<br><br>Cell characterizations initially focused on two different protein systems, Human Leukocyte Antigen (HLA) Complex and Cytokeratin (CK) Filaments. Hybridization experiments using allele-specific HLA probes showed that surface antigens on contact epidermal cells are largely unreactive, suggesting that they cannot be used to differentiate cell populations in a mixture. Epidermal cells were also hybridized with Cytokeratin probes AE1 which targets intermediate filaments within the cell. Fluorescence levels of AE1 hybridized cells were observed to vary across donors with individual differences ranging between 10 to 40% in median fluorescence. Lastly, variations in red autofluorescence profiles (650nm) were examined as a potential signature for distinguishing contributor cell populations. Results showed that many pairs of contributors exhibited distinct differences in red autofluorescence profiles. However, profile variation was also observed from cell populations from the same individual sampled on different days, suggesting that autofluorescence may not be a contributor-specific phenomenon.<br><br>To test whether these observed optical differences could be used as the basis for a cell separation workflow, cell populations from a two person touch mixture were analyzed for their red autofluorescence profiles, separated into two fractions corresponding using Fluorescence Activated Cell Sorting (FACS), a high throughput technique for isolating cells based on their optical properties, and then subjected to STR profiling. Results showed that touch epidermal cells could be sorted, intact, with high efficiency (~95%). STR typing of the DNA fractions provided partial to full profiles that often confirmed separation of individual contributors from the mixture. Overall this research introduces a new strategy for resolving touch mixtures that involves physical separation of cell populations before DNA analysis. The technique can assist forensic caseworking units by providing an alternative to complex profile interpretation procedures, thereby reducing analytical bottlenecks and loss of evidence.