Separation of complex biological mixtures using human leukocyte antigen probes and fluorescence activated cell sorting
Creators: name: Dean, Lee
name: Kwon, Ye Jin
name: Seashols-Williams, Sarah J.
name: Sturgill, Jamie
name: Cruz, Tracey Dawson
name: Ehrhardt, Christopher J.
Publisher: name: F1000Research
Year: 2015

description: 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, few can be applied to biological mixtures containing one cell type. Yet, an increasing proportion of samples submitted as evidence are 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.<br><br>To address this problem, a new analytical technique was developed that utilizes the intrinsic immunological variation among individuals to physically separate cells from different sources prior to DNA profiling. For this procedure, fluorescent antibody probes are used to selectively bind to allelic variants of the Human Leukocyte Antigen (HLA) complex on 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 are isolated from the mixture through Fluorescence Activated Cell Sorting (FACS), a high throughput 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 could potentially be generated without any ambiguity or complex pattern interpretation.<br><br>The forensic potential of this technique was tested on a two-person whole blood mixture.  A discriminatory antigen target (HLA-A02) was identified prior to the experiment and the complementary antibody probe was used for all separation procedures. Specificity of HLA-A02 probe to the surface antigens of each contributor was checked prior to mixing samples. First, one microliter of whole blood from each contributor was mixed together and the liquid mixture was hybridized with the HLA-A02 fluorescent probe. Characterization with flow cytometry showed two cell populations each with distinct fluorescence profiles. Optical characteristics of each cell population were consistent with antibody probe binding with cells from one contributor in the mixture only. Isolation of the labeled cell population was conducted by FACS. DNA extraction and STR profiling of the labeled cell population was performed to determine the efficiency of cell separation. Profiles were compared against STR profiles generated for the whole blood mixture and reference samples from each contributor. Results showed that the STR profile of the fluorescently-labeled cell population that was sorted from the mixture was identical to the single source profile for the first contributor across all six CODIS loci tested. <br><br>Overall, these experimental results suggest that HLA-antibody probes can be used to differentiate individual contributors in a complex mixture and that FACS is an efficient way to physically separate antibody-labeled cells for single source STR profiling. This technique 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.