Separation of uncompromised whole blood mixtures for single source STR profiling using fluorescently-labeled human leukocyte antigen (HLA) probes and fluorescence activated cell sorting (FACS)

Analysis of biological mixtures is a significant problem for forensic laboratories, particularly when the mixture contains only one cell type. Contributions from multiple individuals to biologic evidence can complicate DNA profile interpretation and often lead to a reduction in the probative value of DNA evidence or worse, its total loss. To address this, we have utilized an analytical technique that exploits the intrinsic immunological variation among individuals to physically separate cells from different sources in a mixture prior to DNA profiling. Specifically, we applied a fluorescently labeled antibody probe to selectively bind to one contributor in a mixture through allele-specific interactions with human leukocyte antigen (HLA) proteins that are expressed on the surfaces of most nucleated cells. Once the contributor's cells were bound to the probe, they were isolated from the mixture using fluorescence activated cell sorting (FACS) - a high throughput technique for separating cell populations based on their optical properties - and then subjected to STR analysis. We tested this approach on two-person and four-person whole blood mixtures where one contributor possessed an HLA allele (A∗02) that was not shared by other contributors to the mixture. Results showed that hybridization of the mixture with a fluorescently-labeled antibody probe complimentary to the A∗02 allele's protein product created a cell population with a distinct optical profile that could be easily differentiated from other cells in the mixture. After sorting the cells with FACS, genetic analysis showed that the STR profile of this cell population was consistent with that of the contributor who possessed the A∗02 allele. Minor peaks from the A∗02 negative contributor(s) were observed but could be easily distinguished from the profile generated from A∗02 positive cells. Overall, this indicates that HLA antibody probes coupled to FACS may be an effective approach for generating STR profiles of individual contributors from forensic mixtures.