As submitted by the proposer: Analysis of biological mixtures is a significant problem for forensic laboratories. The presence of cells from multiple individuals in a biologic stain complicates DNA profile interpretation and often leads to loss of evidence. While many analytical techniques have been developed to address complex STR profiles resulting from cell mixtures, most are optimized for separation of sperm from epithelial cells and few can be applied to biological mixtures containing one cell type. Yet, an increasing proportion of samples submitted as evidence are contact epithelial mixtures, in which there are no physical differences between cells contributed by multiple individuals. New techniques are needed to separate individual cell populations from these types of forensic mixtures to generate unambiguous STR profiles. For this project, we will develop a new analytical technique that utilizes the intrinsic immunological variation among individuals to physically separate cells from different sources prior to DNA profiling. Specifically, we will use fluorescent antibody probes that selectively bind to certain Human Leukocyte Antigen (HLA) complexes on epithelial cell surfaces. The natural diversity of antigen alleles and their universal expression on the cell surfaces make them ideal molecular targets for differentiating contributors in a mixture. Once individual cell populations are labeled with HLA-antibody probes, they will be isolated from the mixture through Fluorescence Activated Cell Sorting (FACS), a high through put technique for separating cell populations based on their optical properties. By separating whole cells from a forensic mixture prior to genetic analysis, single source STR profiles can be generated without any ambiguity or complex pattern interpretation. To evaluate the effectiveness of HLA-antibody tagging and FACS separation for profiling individual contributors in a mixture we have developed a two-phased research plan that (1) develops a robust method for differentiating epithelial cell populations, and (2) tests that method on a variety of forensically relevant sample mixtures. This includes low template/touch epithelial samples, epithelial mixtures with varying ratios of cells from each source, and aged/compromised samples. Preliminary results already show that HLA-probes coupled to FACS can be an effective method for generating single source STR profiles from forensic mixtures of two individuals. This research will significantly impact the forensic science community by introducing a new analytical method that can generate robust single source STR profiles for each contributor in a complex epithelial cell mixture. This has the potential to help reduce the analytical bottlenecks, inconclusive results, and loss of evidence that often accompany mixed STR profile interpretation within forensic caseworking units.