Front End Separation of Compromised Blood Mixtures for Single Source DNA Profiling

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. One promising strategy for analyzing complex mixtures is labelling cell populations from individual contributors using allele-specific antibody probes and physically separating them from the mixture with Fluorescence Activated Cell Sorting (FACS) prior to DNA extraction. Thus far, this approach has been demonstrated on a variety of biological mixture systems but has had limited success on aged/degraded samples that are typically encountered during forensic casework. This is likely due to various molecular processes associated with cellular decomposition that reduce the efficiency and selectivity of antigen-specific probe binding on the cell surface. Although there are a number of well-established methods for improving the sensitivity of antibody hybridization and cell sorting in compromised samples, they have not been explicitly tested or optimized for forensically relevant biological mixtures. The goal of this project is to test new methods that increase the efficiency of antibody tagging and cell sorting on degraded blood cell populations with the ultimate goal of developing a robust approach for obtaining single source STR profiles from compromised forensic mixtures. We have developed a two-phased research plan that first characterizes the biochemical effects of cellular degradation on antibody binding on white blood cells and then tests specific molecular strategies to maximize the efficiency of both probe hybridization and cell sorting on challenged mixture samples. In addition, we will examine the effectiveness of these same techniques on other types of compromised samples including blood-epithelial and blood-saliva mixtures. Preliminary results already indicate that antibody-labelling and FACS can effectively differentiate individual contributors in aged/degraded blood mixture samples. We will build off these results by testing mixtures that vary in the number of target cells, the number and ratio of contributors, and the degree of degradation. This research will have a significant impact on the forensic community by optimizing a front-end cell separation technique to the particular demands of forensic casework. 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. This project contains a research and/or development component, as defined in applicable law. ca/ncf