Preliminary assessment of three quantitative approaches for estimating time-since-deposition from autofluorescence and morphological profiles of cell populations from forensic biological samples.

Determining when DNA recovered from a crime scene transferred from its biological source, i.e., a sample's 'time-since-deposition' (TSD), can provide critical context for biological evidence. Yet, there remains no analytical techniques for TSD that are validated for forensic casework. In this study, we investigate whether morphological and autofluorescence measurements of forensically-relevant cell populations generated with Imaging Flow Cytometry (IFC) can be used to predict the TSD of 'touch' or trace biological samples. To this end, three different prediction frameworks for estimating the number of day(s) for TSD were evaluated: the elastic net, gradient boosting machines (GBM), and generalized linear mixed model (GLMM) LASSO. Additionally, we transformed these continuous predictions into a series of binary classifiers to evaluate the potential utility for forensic casework. Results showed that GBM and GLMM-LASSO showed the highest accuracy, with mean absolute error estimates in a hold-out test set of 29 and 21 days, respectively. Binary classifiers for these models correctly binned 94-96% and 98-99% of the age estimates as over/under 7 or 180 days, respectively. This suggests that predicted TSD using IFC measurements coupled to one or, possibly, a combination binary classification decision rules, may provide probative information for trace biological samples encountered during forensic casework.

Differentiation of vaginal cells from epidermal cells using morphological and autofluorescence properties: Implications for sexual assault casework involving digital penetration.

Analysis of DNA mixtures from sexual assault evidence is an ongoing challenge for DNA casework laboratories. To assist the forensic scientist address source and activity level propositions there is a significant need for new techniques that can provide information as to the source of DNA, particularly for sexual assault samples that do not involve semen. The goal of this study was to develop a new biological signature system that provides additional probative value to samples comprised of mixtures of epidermal and vaginal cells, as may be observed in cases involving digital penetration. Signatures were based on morphological and autofluorescence properties of individual cells collected through Imaging Flow Cytometry (IFC). Comparisons to reference cell populations from vaginal tissue and epidermal cells collected from hands showed strong multivariate differences across > 80 cellular measurements. These differences were used to build a predictive framework for classifying unknown cell populations as originating from epithelial cells associated with digital penetration or epidermal tissue. As part of the classification scheme, posterior probabilities of specific tissue group membership were calculated for each cell, along with multivariate similarity to that tissue type. We tested this approach on cell populations from reference tissue as well as mock casework samples involving hand swabbings following digital vaginal penetration. Many more cells classifying as non-epidermal tissue were detected in digital penetration hand swab samples than control hand swabbings. Minimum interpretation thresholds were developed to minimize false positives; these thresholds were also effective when screening licked hands, indicating the potential utility of this method for a variety of biological mixture types and depositional events relevant to forensic casework. Results showed that samples collected subsequent to digital penetration possessed markedly higher numbers of cells classifying as vaginal tissue as well as higher posterior probabilities for vaginal tissue (≥ 0.90) compared to cell populations collected from hands without prior contact with vaginal tissue. Additionally, digital penetration cell populations may be resolved from saliva cell populations and other non-target tissue types.

Differentiation of vaginal cells from epidermal cells using morphological and autofluorescence properties: Implications for sexual assault casework involving digital penetration.

Analysis of DNA mixtures from sexual assault evidence is an ongoing challenge for DNA casework laboratories. There is a significant need for new techniques that can provide information as to the source of DNA, particularly for sexual assault samples that do not involve semen. The goal of this study was to develop a new biological signature system that provides additional probative value to samples comprised of mixtures of epidermal and vaginal cells, as may be observed in cases involving digital penetration. Signatures were based on morphological and autofluorescence properties of individual cells collected through Imaging Flow Cytometry (IFC). Comparisons to reference cell populations from vaginal tissue and epidermal cells collected from hands showed strong multivariate differences across >80 cellular measurements. These differences were used to build a predictive framework for classifying unknown cell populations as originating from epithelial cells associated with digital penetration or epidermal tissue. As part of the classification scheme, posterior probabilities of specific tissue group membership were calculated for each cell, along with multivariate similarity to that tissue type. We tested this approach on cell populations from reference tissue as well as mock casework samples involving digital penetration. Many more cells classifying as non-epidermal tissue were detected in digital penetration samples than control hand swabbings. Minimum interpretation thresholds were developed to minimize false positives; these thresholds were also effective when screening licked hands, indicating the potential utility of this method for a variety of biological mixture types and depositional events relevant to forensic casework. Results showed that samples collected subsequent to digital penetration possessed markedly higher numbers of cells classifying as vaginal tissue as well as higher posterior probabilities for vaginal tissue (≥ 0.90) compared to cell populations collected from hands without prior contact with vaginal tissue. Additionally, digital penetration cell populations may be resolved from saliva cell populations and other non-target tissue types.

Technical note: Survey of extracellular and cell-pellet-associated DNA from 'touch'/trace samples.

The goal of this study was to characterize the reproducibility of extracellular and cell pellet associated DNA yields recovered from handled substrates. Results showed that extracellular DNA yields were extremely variable between contributors-ranging between 0 and >10ng-and tended to dwarf cell pellet yields, which varied between 0 and ∼230pg. DNA yields across multiple samples from the same contributor on different days showed similar levels of variability in both DNA fractions, indicating that extracellular DNA yield is largely influenced by extrinsic and/or environmental factors and is not a contributor-specific attribute. Microscopic surveys of cells from the pellet fraction as well as fingerprints from the same contributor samples were conducted following treatment with fluorescent DNA stain. Nearly all imaged cells exhibited diffuse fluorescence across the cell without discernable evidence of nuclei. This is consistent with the limited nature of DNA recovery from the pellet fraction and the prevalence of extracellular DNA in these samples.

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.

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.

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.

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.

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.