FSC-A SSC-A 0 65536 131072 196608 26214 101 102 103 104 105 Propidium Iodide: Permeabilized Cells Propidium Iodide: Non-permeabilized Cells Abstract Abstract Separation of Epithelial Cell Mixtures Using Fluorescently Labeled Antibodies and Flow Cytometry Cristina E. Stanciu, BS*, Ye Jin Kwon, MS, Sarah R. Ingram, BS, Eduardo E. Bustamante, BS, Jamie Sturgill, PhD, Sarah J. Seashols, PhD, Tracey Dawson Cruz, PhD, Christopher J. Ehrhardt, PhD, Virginia Commonwealth University, Department of Forensic Science, 1015 Floyd Avenue, Rm 2015, Richmond, VA 23284 During this presentation, attendees will be introduced to a novel molecular technique that utilizes fluorescent antibody probes that target antigens within the Major Histocompatability Complex (MHC) on epithelial cell surfaces. Attendees will also learn how antibody hybridization can be coupled to flow cytometry to provide rapid separation of targeted cell populations from a forensic mixture. This research will impact the forensic science community by introducing a high throughput, nondestructive method for generating single source DNA profiles corresponding to each contributor of an epithelial cell mixture. The technique can assist forensic caseworking units by providing an alternative to complex profile interpretation procedures, thereby reducing analytical bottlenecks and loss of evidence. Previous research has shown the application of flow cytometry coupled to antibody probes targeting the Human Leukocyte Antigen system for selectively isolating individual cell populations from a blood mixture to generate single source STR profiles. Our goal with this research was to test this technique against various types of epithelial cell mixtures for resolving individual contributor profiles. Due to its efficacy in earlier studies, we chose an antibody probe complementary to the A*02 allele for all experiments. Hybridization experiments were conducted on both buccal cell mixtures and contact 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 A02 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 A02 probe was tested against contact epithelial cells. Cells were sampled from the stratum corneum of the epidermal layer from 4 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. Key words: epithelial, mixture, FACS Flow Cytometry: Buccal and Contact Epithelial Cells Flow Cytometry: Buccal and Contact Epithelial