Separation of epithelial cell mixtures using fluorescently labeled antibodies and flow cytometry
Creators: name: Stanciu, Cristina E.
name: Kwon, Ye Jin
name: Ingram, Sarah R.
name: Bustamante, Eduardo E.
name: Sturgill, Jamie
name: Seashols-Williams, Sarah
name: Cruz, Tracey Dawson
name: Ehrhardt, Christopher J.
Publisher: name: F1000Research
Year: 2016

description: DNA mixtures are consistently problematic for forensic caseworking units as they yield profiles that require extensive interpretation. Many laboratory methods have been developed to facilitate the separation of certain components of a biological mixture prior to PCR amplification and STR profiling. However, most of these have been optimized for mixtures of two cell types with different physical and chemical properties (i.e. sperm and epithelial cells). Few separation techniques have been demonstrated on 'touch' or trace biological mixtures that contain epithelial cells from multiple individuals. Our goal with this research was to examine Flourescence Activated Cell Sorting (FACS) as a strategy for separating cell populations within epithelial mixtures including touch samples. Due to its efficacy in earlier studies, we chose an antibody probe targeting the A*02 allele within the Human Leukocyte Antigen (HLA) Complex for all experiments. Antibody hybridizations were conducted on both buccal cell mixtures and touch epithelial cell mixtures. Results from buccal cell mixture hybridization showed that this cell type has intrinsically high levels of fluorescence which can interfere with the signal originating from specific probe-antigen interactions on the cell surface. However, detailed statistical analysis of fluorescence histograms did indicate greater shifts in cell fluorescence when donor cell populations were either heterozygous or homozygous for the A*02 allele. This suggests that separation of labeled cell populations is still possible even if antibody reactivity is not strictly a function of HLA genotype. In the second experiment, hybridization of the A*02 probe was tested against contact epithelial cells. Cells were sampled from the stratum corneum of the epidermal layer from four different individuals representing a range of HLA types. Hybridization results showed little to no interaction with the cell surface across all donors tested. This suggests that HLA antibody probes have the potential to resolve complex mixtures containing different sources of epithelial cells (buccal cells from skin) which may aid in the analysis of certain types of forensic cases. Other signatures of epithelial cells have been analyzed by flow cytometry, leading to an enrichment of DNA from one contributor in a mixture. Intrinsic fluorescence intensities of keratinized cells from different donors have been observed in the far red wavelengths, which suggests that environmental factors may affect the autofluorescence of their cells. Shedder status may facilitate mixture separation through area side-scatter and forward-scatter profiles. Corneocytes on the surface of our skin may be more abundant as fragments or as large intact cells, depending on the individual, and their membrane permeability status along with different DNA content may provide new avenues for discriminating contributors in a mixture.