M. Katherine Philpott

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 contributors 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 alleles 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.

Optical characterization of epidermal cells and their relationship to DNA recovery from touch samples

The goal of this study was to investigate the relative contributions of different cellular and genetic components to biological samples created by touch or contact with a surface – one of the most challenging forms of forensic evidence. Touch samples were generated by having individuals hold an object for five minutes and analyzed for quantity of intact epidermal cells, extracellular DNA, and DNA from pelleted cell material after elution from the collection swab. Comparisons were made between samples where individuals had washed their hands immediately prior to handling and those where hand washing was not controlled. The vast majority (84-100%) of DNA detected in these touch samples was extracellular and was uncorrelated to the number of epidermal cells detected. Although little to no extracellular or cell pellet-associated DNA was detected when individuals washed their hands prior to substrate handling, we found that a significant number of epidermal cells (between ~5x10 3 and ~1x10 5) could still be recovered from these samples, suggesting that other types of biological information may be present even when no amplifiable nuclear DNA is present. These results help to elucidate the biological context for touch samples and characterize factors that may contribute to patterns of transfer and persistence of genetic material in forensic evidence.

Rapid differentiation of epithelial cell types in aged biological samples using autofluorescence and morphological signatures

Establishing the tissue source of epithelial cells within a biological sample is an important capability for forensic laboratories. In this study we used Imaging Flow Cytometry (IFC) to analyze individual cells recovered from buccal, epidermal, and vaginal samples that had been dried between 24 hours and more than eight weeks. Measurements capturing the size, shape, and fluorescent properties of cells were collected in an automated manner and then used to build a multivariate statistical framework for differentiating cells based on tissue type. Results showed that epidermal cells could be distinguished from vaginal and buccal cells using a discriminant function analysis of IFC measurements with an average classification accuracy of ~94%. Ultimately, cellular measurements such as these, which can be obtained non-destructively, may provide probative information for many types of biological samples and complement results from standard genetic profiling techniques.

Analysis of red autofluorescence (650-670nm) in epidermal cell populations and its potential for distinguishing contributors to 'touch' biological samples

Interpretation of touch DNA mixtures poses a significant challenge for forensic caseworking laboratories. Front end techniques that facilitate separation of contributor cell populations before DNA extraction are a way to circumvent this problem. The goal of this study was to survey intrinsic fluorescence of epidermal cells collected from touch surfaces and investigate whether this property could potentially be used to discriminate between contributor cell populations in a biological mixture. Analysis of red autofluorescence (650-670nm) showed that some contributors could be distinguished on this basis. Variation was also observed between autofluorescence profiles of epidermal cell populations from a single contributor sampled on different days. This dataset suggests that red autofluorescence may be a useful marker for identifying distinct cell populations in some mixtures. Future efforts should continue to investigate the extrinsic or intrinsic factors contributing to this signature, and to identify additional biomarkers that could complement this system.

Rapid Identification of Epithelial Cell Types in Forensic Samples Using Intrinsic Fluorescence and Morphological Signatures

Establishing the tissue source of epithelial cells within a biological sample is an important capability for forensic laboratories. In this study we used Imaging Flow Cytometry (IFC) to analyze individual cells recovered from buccal, contact epithelial, and vaginal samples that had been dried between 24 hours and more than eight weeks. Measurements capturing the size, shape, and fluorescent properties of cells were collected in an automated manner and then used to build a multivariate statistical framework for differentiating cells based on tissue type. Results showed that cell populations from the three tissue types could be differentiated using a Discriminant Function plot of IFC measurements. Epidermal cells were distinguished from vaginal and buccal cells with an average classification accuracy of ~94%. Ultimately, cellular measurements such as these, which can be obtained non-destructively, may provide probative information for many types of biological samples and complement results from standard genetic profiling techniques.

Analysis of Antibody Hybridization and Autofluorescence in Touch Samples by Flow Cytometry: Implications for Front End Separation of Trace Mixture Evidence

The goal of this study was to survey optical and biochemical variation in cell populations deposited onto a surface through touch or contact and identify specific features that may be used to differentially label and then sort cell populations from separate contributors in a trace biological mixture. Cell characterizations initially focused on two different protein systems, Human Leukocyte Antigen (HLA) complex and cytokeratin (CK) filaments. Hybridization experiments using pan and allele-specific HLA antibody probes showed that surface antigens on cells transferred from the palmar surface of volunteers are largely unreactive, suggesting that they cannot be used to differentiate cell populations in a touch mixture. Touch samples were also hybridized with the pan-CK probe AE1, which targets CK proteins 10, 14, 15, 16 and 19. Fluorescence levels of AE1 hybridized cells were observed to vary across donors, although these differences were not consistent across all sampling days. We then investigated variations in red autofluorescence profiles (650-670nm) as a potential signature for distinguishing contributor cell populations. Although distinct differences in red autofluorescence profiles were observed ‐‐ with one donor consistently exhibiting higher levels of fluorescence than others ‐‐ some variation was also observed in touch samples collected from the same individual on different days. While this suggests that contributor touch samples cannot be defined by a discrete level of autofluorescence, this attribute may still be a useful means of isolating contributors to some touch mixtures. To test whether these observed optical differences could potentially be used as the basis for a cell separation workflow, a controlled two person touch mixture was separated into two fractions via Fluorescence Activated Cell Sorting (FACS) using gating criteria based on intensity of 650-670nm emissions, and then subjected to DNA analysis. STR typing of the sorted fractions provided partial profiles that were consistent with separation of individual contributors from the mixture.

Flow cytometry dataset for cells collected from touched surfaces

‘Touch’ or trace cell mixtures submitted as evidence are a significant problem for forensic laboratories as they can render resulting genetic profiles difficult or even impossible to interpret. Optical signatures that distinguish epidermal cell populations from different contributors could facilitate the physical separation of mixture components prior to genetic analysis, and potentially the downstream production of single source profiles and/or simplified mixtures. This dataset comprises the results from antibody hybridization surveys using Human Leukocyte Antigen (HLA) and Cytokeratin (CK) probes, as well as surveys of optical properties of deposited cells, including forward scatter (FSC), side scatter (SSC), and fluorescence emissions in the Allophycocyanin (APC) channel. All analyses were performed on “touch” samples deposited by several different contributors on multiple days to assess inter- and intra-contributor variability.