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.

Use of hormone-specific antibody probes for differential labeling of contributor cell populations in trace DNA mixtures.

A significant proportion of casework analyzed by forensic science laboratories is often "touch" or trace forensic DNA evidence, which is deposited through physical contact and is comprised of sloughed epidermal cells. These samples can be challenging to analyze due to low DNA concentrations, frequent degradation, and the presence of cells from multiple individuals in the same sample. To address these challenges, we investigated a new approach for characterizing trace evidence prior to DNA profiling that labels epidermal cells with antibody probes targeting hormone molecules testosterone and dihydrotestosterone (DHT). The goal was to test whether cell populations derived from separate individuals showed different binding efficiencies to hormone probes and, thus, could be used to detect the presence of multiple cell populations. Additionally, we investigated whether antibody probes could be used to isolate contributor cell populations from an epidermal cell mixture and facilitate deconvolution of mixed DNA profiles recovered from touch/trace evidence. Results showed that cell populations from some individuals could differentiated in trace samples based on fluorescence histograms following probe labeling. However, certain pairs of contributors showed largely or completely overlapping histogram profiles and could not be resolved. Preliminary efforts to separate cell populations that could be differentiated with hormone probes with fluorescence-activated cell sorting (FACS) coupled to DNA profiling and probabilistic modeling indicated that it is possible to enrich contributor cell populations from touch/trace samples and produce more probative DNA profiles compared to the original mixture sample. The variability in labeling, differentiation, and physical separation of cell populations may be impacted by similarities in biochemical profiles across some contributors as well as imbalance of contributor DNA quantities in certain mixtures as is typical in casework involving touch/trace evidence. Ultimately, screening and separation of trace DNA samples with this approach may be presumptive and constrained by sample-specific parameters of the original mixture.

Atomic force microscopy as a biophysical tool for nanoscale forensic investigations.

The atomic force microscope (AFM) has found its way to the arsenal of tools available to the forensic practitioner for the analysis of samples at the nano and microscales. As a non-destructive probing tool that requires minimal sample preparation, the AFM is very attractive, particularly in the case of minimal or precious sample. To date, the use of the AFM has primarily been in the arena of imaging where it has been complementary to other microscopic examination tools. Forensic applications in the visual examination of evidence such as blood stains, questioned documents, and hair samples have been reported. While a number of reviews have focused on the use of AFM as an imaging tool for forensic analyses, here we not only discuss these works, but also point to a versatile enhancement in the capabilities of this nanoscale tool - namely its use for force spectroscopy. In this mode, the AFM can determine elastic moduli, adhesion forces, energy dissipation, and the interaction forces between cognate ligands, that can be spatially mapped to provide a unique spatial visualization of properties. Our goals in this review are to provide a context for this capability of the AFM, explain its workings, cover some exemplary works pertaining to forensic sciences, and present a critical analysis on the advantages and disadvantages of this modality. Equipped with this high-resolution tool, imaging and biophysical analysis by the AFM can provide a unique complement to other tools available to the researcher for the analysis and characterization of forensic evidence.

3D Printing of Antibacterial Polymer Devices Based on Nitric Oxide Release from Embedded S-Nitrosothiol Crystals.

Controlled release of drugs from medical implants is an effective approach to reducing foreign body reactions and infections. We report here on a one-step 3D printing strategy to create drug-eluting polymer devices with a drug-loaded bulk and a drug-free coating. The spontaneously formed drug-free coating dramatically reduces the surface roughness of the implantable devices and serves as a protective layer to suppress the burst release of drugs. A high viscosity liquid silicone that can be extruded based on its shear-thinning property and quickly vulcanize upon exposure to ambient moisture is used as the ink for 3D printing. S-Nitrosothiol type nitric oxide (NO) donors in their crystalline forms are selected as model drugs because of the potent antimicrobial, antithrombotic, and anti-inflammatory properties of NO. Direct ink writing of the homogenized polymer-drug mixtures generates rough and ill-defined device surfaces because of the exposed S-nitrosothiol microparticles. When a low-viscosity silicone (polydimethylsiloxane) is added into the ink, this silicone diffuses outward upon deposition to form a drug-free outermost layer without compromising the integrity of the printed structures. S-Nitrosoglutathione (GSNO) or S-nitroso-N-acetylpenicillamine (SNAP) embedded in the printed silicone matrix releases NO under physiological conditions from days to about one month. The microsized drug crystals are well-preserved in the ink preparation and printing processes, which is one reason for the sustained NO release. Biofilm and cytotoxicity experiments confirmed the antibacterial property and safety of the printed NO-releasing devices. This additive manufacturing platform does not require dissolution of drugs and involves no thermal or UV processes and, therefore, offers unique opportunities to produce drug-eluting silicone devices in a customized manner.

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.

Open source software tool for the automated detection and characterization of epithelial cells from trace biological samples.

This paper presents a strategy for an unsupervised workflow for identifying epithelial cells in microscopic images and characterizing their morphological and/or optical properties. The proposed method can be used on cells that have been stained with fluorescent dyes and imaged using conventional optical microscopes. The workflow was tested on cell populations that were imaged directly on touch/contact surfaces and stained with nucleic acid dyes to visualize genetic content. Our results show that this approach could be a useful strategy for characterizing differences in staining efficiency and/or morphological properties of individual cells or aggregate populations within a biological sample. Further, they can potentially reduce the laborious nature of microscopic analysis and increase throughput and reproducibility of similar studies.

Chitosan Stabilized Silver Nanoparticles for the Electrochemical Detection of Lipopolysaccharide: A Facile Biosensing Approach for Gram-Negative Bacteria.

Negatively charged lipopolysaccharide (LPS), a major endotoxin and component of the outer membrane of several Gram-negative bacteria, provides a useful biomarker for the indirect detection of these pathogens. For instance, Escherichia coli (E. coli) is a pathogenic bacterium that causes infections in almost all age groups, and has been implicated in food and water contamination. Current diagnostic and detection methods tend to be labor-intensive or expensive, necessitating the need for an easy, sensitive, rapid, and low-cost method. We report on the synthesis and use of positively charged chitosan stabilized silver nanoparticles (Chi-AgNPs) as a sensitive electrochemical nanobiosensor for the detection of LPS. Chi-AgNPs were synthesized through a facile, single step protocol, and characterized for size, charge, and morphology. Glassy carbon electrodes modified with Chi-AgNPs resulted in an enhancement of signal in the presence of both LPS and E. coli. Detection was accomplished over a large concentration range (several orders of magnitude) of 0.001-100 ng/mL and 10-107 CFU/mL. The biosensors can reliably detect LPS and E. coli at very low concentrations. Chi-AgNPs have potential as low cost, sensitive nanobiosensors for Gram-negative bacteria due to strong electrostatic interaction with LPS present in their outer membranes.

Nanoscale Phenotypic Textures of Yersinia pestis Across Environmentally-Relevant Matrices.

The persistence of bacterial pathogens within environmental matrices plays an important role in the epidemiology of diseases, as well as impacts biosurveillance strategies. However, the adaptation potentials, mechanisms for survival, and ecological interactions of pathogenic bacteria such as Yersinia pestis are largely uncharacterized owing to the difficulty of profiling their phenotypic signatures. In this report, we describe studies on Y. pestis organisms cultured within soil matrices, which are among the most important reservoirs for their propagation. Morphological (nanoscale) and phenotypic analysis are presented at the single cell level conducted using Atomic Force Microscopy (AFM), coupled with biochemical profiles of bulk populations using Fatty Acid Methyl Ester Profiling (FAME). These studies are facilitated by a novel, customizable, 3D printed diffusion chamber that allows for control of the external environment and easy harvesting of cells. The results show that incubation within soil matrices lead to reduction of cell size and an increase in surface hydrophobicity. FAME profiles indicate shifts in unsaturated fatty acid compositions, while other fatty acid components of the phospholipid membrane or surface lipids remained consistent across culturing conditions, suggesting that phenotypic shifts may be driven by non-lipid components of Y. pestis.

Nanoscale visualization of extracellular DNA on cell surfaces.

Nanoscale analysis of extracellular DNA (eDNA) that is present on the surface of cells in trace biological samples can provide insight into the understanding of DNA transfer through touch, and thereby, the role of eDNA is a biologically and forensically relevant phenomenon. While various bulk scale tools and DNA analysis can be used to quantitatively obtain this information, obtaining a three dimensional (3D) visualization of the eDNA can provide a unique look into the spatial and temporal dynamics at the cellular level. In this study, we show how atomic force microscopy (AFM) can be integrated with optical microscopy to visualize the distribution of surface associate eDNA at a single cell level. Using a nucleic acid fluorophore such as Diamond™ Dye, the surface eDNA can be observed and quantified using fluorescence microscopy. This informational channel can then be overlaid with surface topography and cellular elasticity to provide structural visualization. Finally, chemical force spectroscopy can be used to obtain the distribution of surface-associated eDNA on the cell surface at the molecular level. Such integrated techniques can enhance understanding of the biological role of eDNA, and can also be potentially valuable for investigating challenging trace samples, containing very few cells for various analyses.

Synthesis of chitosan coated metal organic frameworks (MOFs) for increasing vancomycin bactericidal potentials against resistant S. aureus strain.

Multiple drug resistant (MDR) has become a major issue in developing countries. MDR bacterial infections lead to significant increase in morbidity, mortality and cost of prolonged treatments. Therefore, designing of strategies for improving the antimicrobial potential of the therapeutic agents are highly required. Metal organic frameworks (MOFs) are highly tunable hybrid material, consist of metal ions linked together by organic bridging ligands have been used as an efficient drug delivery carrier because of their biodegradability, low toxicity and structure integrity upon loading and functionalizing process. Current study was based on the synthesis of chitosan coated MOFs with enhanced contact with S. aureus cell surface. Chitosan is deacetylated derivative of chitin and capable for non-bonding interaction with negatively charged bacterial cell leading to enhanced contact of MOFs with S. aureus. Chitosan coated MOFs were characterized with various techniques such as atomic force microscopy, scanning electron microscopy, DLS, FT-IR, TGA, DSC and Powder X-ray diffraction. They were also studied for their efficacy on resistant S. aureus, results revealed that Vancomycin bactericidal activity significantly increased upon loading in chitosan coated MOFs and caused increased inhibition of resistant S. aureus. AFM analysis of S. aureus strains clearly revealed complete distortion of morphology by treating with chitosan modified drug loaded MOFs. Findings of the current study suggest the potential of chitosan coated MOFs for reversing bacterial resistance against Vancomycin and provide new perspectives for improved antibiotic therapy of infections associated with MDR.

Customizable 3D printed diffusion chambers for studies of bacterial pathogen phenotypes in complex environments.

Gaps in our understanding of the natural ecology and survival mechanisms of pathogenic bacteria in complex microenvironments such as soil typically occur due to the difficulty in characterizing biochemical profiles and morphological characteristics as they exist in environmental samples. Conversely, accurate simulation of the abiotic and biotic chemistries of soil habitats within the laboratory is often a significant challenge. Herein, we present the fabrication of customizable and precisely engineered 3D printed diffusion chambers that can be used to incubate bacterial cultures directly in soil matrices within a controlled laboratory experiment, and study the dynamics between bacterial cells and soil components. As part of the design process, different types of 3D printing materials were evaluated for ease of sterilization, structural integrity throughout the experiment, as well as cost/ease of production. To demonstrate potential applications for environmental studies, the diffusion chamber was used to incubate cultures of Bacillus cereus T-strain and Escherichia coli strain O157 directly in soil matrices. We show that the chamber facilitates diffusion of abiotic/biotic components of the soil with target cells without contamination from in situ microbial communities, while allowing for single cell and ensemble level phenotypic analyses of bacteria cultured with and without soil matrices.

Evaluation of Bluestar® Forensic Magnum and Other Traditional Blood Detection Methods on Bloodstained Wood Subjected to a Variety of Burn Conditions.

Accurate blood detection is a primary concern for forensic scientists, especially in highly compromised situations. In this study, blood was added to wood blocks and subjected to a variety of fire treatments: the absence or presence of accelerant, burn time (1, 3, or 5 min), and extinguishment method (smothering or dousing with water). Burned blocks were given a qualitative burn score, followed by removal of half of the char from each block and subsequent testing of each half for blood using luminol (13% positive; n = 96), Bluestar® Forensic Magnum (5.2% positive; n = 96), and combined phenolphthalein tetramethylbenzidine test (0% positive; n = 192). Luminol and Bluestar® Forensic Magnum performed similarly, both outperforming PTMB. Additionally, positive results were more likely from samples that were smothered, had a low burn score, and had more concentrated blood solutions (neat or 1:2). Overall, it is extremely unlikely that blood would be detected on combustible substrates exposed to direct fire.

Open-Source Tools for Dense Facial Tissue Depth Mapping of Computed Tomography Models.

Computed tomography (CT) scans provide anthropologists with a resource to generate three-dimensional (3D) digital skeletal material to expand quantification methods and build more standardized reference collections. The ability to visualize and manipulate the bone and skin of the face simultaneously in a 3D digital environment introduces a new way for forensic facial approximation practitioners to access and study the face. Craniofacial relationships can be quantified with landmarks or with surface-processing software that can quantify the geometric properties of the entire 3D facial surface. This article describes tools for the generation of dense facial tissue depth maps (FTDMs) using deidentified head CT scans of modern Americans from the Cancer Imaging Archive public repository and the open-source program Meshlab. CT scans of 43 females and 63 males from the archive were segmented and converted to 3D skull and face models using Mimics and exported as stereolithography files. All subsequent processing steps were performed in Meshlab. Heads were transformed to a common orientation and coordinate system using the coordinates of nasion, left orbitale, and left and right porion. Dense FTDMs were generated on hollowed, cropped face shells using the Hausdorff sampling filter. Two new point clouds consisting of the 3D coordinates for both skull and face were colorized on an RGB (red-green-blue) scale from 0.0 (red) to 40.0-mm (blue) depth values and exported as polygon (PLY) file format models with tissue depth values saved in the "vertex quality" field. FTDMs were also split into 1.0-mm increments to facilitate viewing of common depths across all faces. In total, 112 FTDMs were generated for 106 individuals. Minimum depth values ranged from 1.2 mm to 3.4 mm, indicating a common range of starting depths for most faces regardless of weight, as well as common locations for these values over the nasal bones, lateral orbital margins, and forehead superior to the supraorbital border. Maximum depths were found in the buccal region and neck, excluding the nose. Individuals with multiple scans at visibly different weights presented the greatest differences within larger depth areas such as the cheeks and neck, with little to no difference in the thinnest areas. A few individuals with minimum tissue depths at the lateral orbital margins and thicker tissues over the nasal bones (>3.0 mm) suggested the potential influence of nasal bone morphology on tissue depths. This study produced visual quantitative representations of the face and skull for forensic facial approximation research and practice that can be further analyzed or interacted with using free software. The presented tools can be applied to preexisting CT scans, traditional or cone beam, adult or subadult individuals, with or without landmarks, and regardless of head orientation, for forensic applications as well as for studies of facial variation and facial growth. In contrast with other facial mapping studies, this method produced both skull and face points based on replicable geometric relationships, producing multiple data outputs that are easily readable with software that is openly accessible.

Simplification of complex DNA profiles using front end cell separation and probabilistic modeling.

Forensic samples comprised of cell populations from multiple contributors often yield DNA profiles that can be extremely challenging to interpret. This frequently results in decreased statistical strength of an individual's association to the mixture and the loss of probative data. The purpose of this study was to test a front-end cell separation workflow on complex mixtures containing as many as five contributors. Our approach involved selectively labelling certain cell populations in dried whole blood mixture samples with fluorescently labeled antibody probe targeting the HLA-A*02 allele, separating the mixture using Fluorescence Activated Cell Sorting (FACS) into two fractions that are enriched in A*02 positive and A*02 negative cells, and then generating DNA profiles for each fraction. We then tested whether antibody labelling and cell sorting effectively reduced the complexity of the original cell mixture by analyzing STR profiles quantitatively using the probabilistic modeling software, TrueAllele® Casework. Results showed that antibody labelling and FACS separation of target populations yielded simplified STR profiles that could be more easily interpreted using conventional procedures. Additionally, TrueAllele® analysis of STR profiles from sorted cell fractions increased statistical strength for the association of most of the original contributors interpreted from the original mixtures.

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.

Nanoscale characterization of forensically relevant epithelial cells and surface associated extracellular DNA.

Atomic force microscopy provides a novel morphological and physico-chemical perspective to analyze epithelial cell samples in forensic investigations. As a nanoscale, single cell tool, it allows the investigation of scarce samples in a non-destructive fashion. Using chemical force spectroscopy, it permits the identification of specific functional groups or surface molecules. Of specific interest is the presence of extracellular DNA (eDNA) on the surface of epithelial cells that line the exterior skin and interior cavities of human bodies, and can transfer onto surfaces through contact with skin and saliva. To date, this eDNA has only been measured a bulk level. Here, using nanoscale imaging, we first describe the unique differences between keratinized epithelial cells and non-keratinized buccal cells. Then via a force mapping technique, we show how eDNA can be spatially located and quantified on the cell surface. Our results suggest that presence and relative quantity of surface-associated, extracellular DNA signatures can be analyzed on individual epithelial cells from different tissue sources, providing a new tool in the forensic analysis of touch samples.

Analysis of cellular 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 distinguish and then sort cell populations from separate contributors in a trace biological mixture. Although we were not able to detect meaningful biochemical variation in touch samples deposited by different contributors through preliminary antibody surveys, we did observe distinct differences in red autofluorescence emissions (650-670 nm), with as much as a tenfold difference in mean fluorescence intensities observed between certain pairs of donors. Results indicate that the level of red autofluorescence in touch samples can be influenced by a donor's contact with specific material prior to handling the substrate from which cells were collected. In particular, we observed increased red autofluorescence in cells deposited subsequent to handling laboratory gloves, plant material, and certain types of marker ink, which could be easily visualized microscopically or using flow cytometry, and persisted after hand washing. 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 650-670 nm emissions and then subjected to DNA analysis. Genetic analysis of the sorted fractions provided partial DNA profiles that were consistent with separation of individual contributors from the mixture suggesting that variation in autofluorescence signatures, even if driven by extrinsic factors, may nonetheless be a useful means of isolating contributors to some touch mixtures. Graphical Abstract Conceptual workflow diagram. Trace biological mixtures containing cells from multiple individuals are analyzed by flow cytometry. Cells are then physically separated into two populations based on intensity of red autofluorescence using Fluorescence Activated Cell Sorting. Each isolated cell fraction is subjected to DNA analysis resulting in a DNA profile for each contributor.

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.