Fingermarks in wildlife forensics: A review.

Wildlife forensics is defined as providing forensic evidence to support legal investigations involving wildlife crime, such as the trafficking and poaching of animals and/ or their goods. While wildlife forensics is an underexplored field of science, the ramifications of poaching can be catastrophic. The consequences of wildlife crime include disease spread, species and habitat loss, human injury, and cultural loss. Efforts to use forensic science to combat poaching are currently limited to DNA-based techniques. However, fingermark analysis for the identification of perpetrators of wildlife crimes has not been explored to the same extent, despite being a cost-effective, simple-to-use forensic method that is easy to deploy in-field. This review covers literature that has explored fingermark examination techniques used on wildlife-related samples, such as pangolin scales, ivory-based substances, bone, and eggs, as well as feathers and skins, among more obscure trafficked items. Useful preliminary work has been conducted in this subject area, demonstrating that commonly used fingermark analysis techniques can be applied to wildlife-based items. However, many of these studies suffer from limitations in terms of experimental design. More work should be done on creating studies with larger sample sizes and novel approaches should be validated under environmental conditions that mimic real crime scenes. Further research into determining the forensic fingermark analysis techniques that perform the most efficiently in the environmental conditions of the countries where they are needed would therefore benefit legal investigations and help to reduce instances of poaching.

Technical Note: Lysis and purification methods for increased recovery of degraded DNA from touch deposit swabs.

Touch deposits are a routine yet challenging sample type in forensic casework and research. Recent work investigating their contents has indicated corneocytes to be the major cellular constituent while cell-free DNA is present at significant levels. Prolonged incubation including a reducing agent such as DTT has been shown to lyse corneocytes; a plasma cfDNA recovery kit which targets shorter DNA fragments has been demonstrated to improve cfDNA recovery from hand rinses. Herein these methods are combined and tested on mock casework touch deposit swabs from communal surface areas. Both fluorescence- and qPCR-based quantification methods are used and their results compared to query DNA degradation levels. Both proposed lysis and purification methods demonstrate increased recovery of DNA detectable with fluorescence quantification and some additional alleles at short loci, indicating high levels of fragmented DNA in these samples.

Accumulation of endogenous and exogenous nucleic acids in "Touch DNA" components on hands.

Successful forensic DNA profiling from handled items is increasingly routine in casework. This "touch DNA" is thought to contain both cellular and acellular nucleic acid sources. However, there is little clarity on the origins or characteristics of this material. The cellular component consists of anucleate, terminally differentiated corneocytes (assumed to lack DNA), and the occasional nucleated cell. The acellular DNA source is fragmentary, presumably cell breakdown products. This study examines the relative contributions each component makes to the hand-secretions (endogenous) and hand-accumulations (exogenous) by recovering rinses from the inside and outside of worn gloves. Additionally, cellular and acellular DNA was measured at timepoints up to 2 h after hand washing, both with and without interim contact. Microscopic examination confirmed cell morphology and presence of nucleic acids. Following the novel application of a hair keratinocyte lysis method and plasma-DNA fragment purification to hand rinse samples, DNA profiles were generated from both fractions. Exogenous cell-free DNA is shown to be a significant source of touch DNA, which reaccumulates quickly, although its amplifiable nuclear alleles are limited. Endogenous DNA is mostly cellular in origin and provides more allelic information consistently over time.

Corneocyte lysis and fragmented DNA considerations for the cellular component of forensic touch DNA.

DNA deposited by individuals' hands is a routine part of forensic analysis, yet little is understood about the precise cellular contents left by handling. "Dead" skin cells known as corneocytes make up the majority of the cellular material left in touch deposits by people's hands but are known to lack nuclei, making their DNA content ambiguous. Here we measure DNA released from anucleate corneocytes following various lysis methods to determine how much DNA may be present in these cells and how best to recover it from inside the cornified envelope. We demonstrate that enhanced lysis methods using a reducing agent and longer incubation may be valuable for hand deposit samples. Corneocyte DNA can be characterized as highly degraded based on the quantification, STR profiling and fluorescence microscopy of the cells from freshly washed hands. Purification to target shorter DNA fragments is demonstrated. DNA from the washed corneocyte cells is shown to constitute the majority of recoverable DNA with these methods. We consider the use of new methods adapted to cornified cells and fragmented DNA for future research into this sample type.

Exploration of cell-free DNA (cfDNA) recovery for touch deposits.

Although touch deposit DNA is widely used in forensic casework, its cellular and acellular contents and their biological origins are poorly understood. There is evidence that the cell-free component of DNA deposited by handling may contribute substantial genetic information; however, most research into touch DNA recovery does not separate cellular and cell-free fractions or seek to characterize their contents. This work is an important early step in developing methods to isolate the cfDNA from biological material deposited by handling. Size-filtration as a separation technique was determined to be prone to DNA loss, even on optimized control samples of pure ladder DNA. Centrifugal separation was optimized to determine minimum speed and time required to reliably remove all cellular debris from the material collected by rinsing donor hands. To determine if the centrifugal force risked rupturing shed corneocyte cells and releasing cellular DNA into the supernatant, DNA levels were measured, and cells were visualized microscopically before and after centrifugation of hand rinses. Heated buccal cells were used as a positive control to demonstrate cell rupture would be detected with these methods. Following the determination of a suitable separation technique, an investigation into purification methods for cfDNA was conducted. DNA recovery using three kits for plasma cfDNA, one for PCR clean-up and one for genomic DNA were assessed on both ladder DNA to simulate cfDNA fragments and on collected hand deposit supernatants from both unwashed and washed hands. Purification methods designed for recovery of short DNA fragments from plasma yielded the highest recovery percentage across sample types, with BioChain cfPure performing the best. Donors' hands were shown to shed high levels of cfDNA, which were better recovered with a method for short fragments than with a traditional genomic technique often used on touch DNA samples.

Illuminating touch deposits through cellular characterization of hand rinses and body fluids with nucleic acid fluorescence.

Forensic DNA typing from touched or handled items in routine casework is increasing as the sensitivity of detection techniques improves. Our understanding of the cellular/acellular content of touch deposits and the origins of the DNA therein is still limited. This work explores the cellular content of rinses from washed and unwashed hands, as well as saliva, nasal and eye washes which could be sources of transferred DNA onto hands. Flow cytometry and microscopic examination were used to detect granularity, size and nucleic acid fluorescence data. Cellular content did not vary significantly within an individual, although some differences were observed between donors. Saliva contained populations of nucleated epithelia as well as smaller cells and debris, all positive for DNA. Hand rinses consisted almost entirely of anucleate corneocytes, many of which also stained positive for nucleic acids. These data raise questions about shed corneocyte DNA content previously assumed to be negligible.

DNA Profiles from Fingerprint Lifts-Enhancing the Evidential Value of Fingermarks Through Successful DNA Typing.

This study evaluated the compatibility of the most common enhancement methods and lifting techniques with DNA profiling. Emphasis is placed on modern lifting techniques (i.e., gelatin lifters and Isomark™) and historical fingerprint lifts for which limited research has been previously conducted. A total of 180 fingerprints were deposited on a glass surface, enhanced, lifted, and processed for DNA typing. DNA could be extracted and profiled for all the powders and lifts tested and from both groomed fingerprints and natural prints with no significant difference in the percentage of profile recovered. DNA profiles could also be obtained from historical fingerprint lifts (79.2% of 72 lifts) with one or more alleles detected. These results demonstrate the compatibility between different powder/lift combinations and DNA profiling therefore augmenting the evidential value of fingerprints in forensic casework.

A review of trace "Touch DNA" deposits: Variability factors and an exploration of cellular composition.

The use in courtrooms of forensic DNA typing results from presumably touched or handled items is increasing as the sensitivity of detection techniques improves. Research investigating how much DNA can be recovered from handled items, whether trace DNA can be detected under certain scenarios including varying degrees of indirect transfer, and factors which may influence these results is summarized here. Fundamentally, our current understanding of the cellular content of touch deposits and the origins of the potential trace DNA therein is extremely limited. Possible origins include anucleate corneocytes, fragmentary cells/nuclei, nucleated epithelial cells from hands, transferred nucleated cells, and cell-free DNA. Here we review the existing evidence for each possible source and consider remaining knowledge gaps regarding forensically relevant touch depositions.

The use of the M-Vac® wet-vacuum system as a method for DNA recovery.

Collecting sufficient template DNA from a crime scene sample is often challenging, especially with low quantity samples such as touch DNA (tDNA). Traditional DNA collection methods such as double swabbing have limitations, in particular when used on certain substrates which can be found at crime scenes, thus a better collection method is advantageous. Here, the effectiveness of the M-Vac® Wet-Vacuum System is evaluated as a method for DNA recovery on tiles and bricks. It was found that the M-Vac® recovered 75% more DNA than double swabbing on bricks. However, double swabbing collected significantly more DNA than the M-Vac® on tiles. Additionally, it was found that cell-free DNA is lost in the filtration step of M-Vac® collection. In terms of peak height and number of true alleles detected, no significant difference was found between the DNA profiles obtained through M-Vac® collection versus double swabbing of tDNA depositions from 12 volunteers on bricks. The results demonstrate that the M-Vac® has potential for DNA collection from porous surfaces such as bricks, but that alterations to the filter apparatus would be beneficial to increase the amount of genetic material collected for subsequent DNA profiling. These results are anticipated to be a starting point to validate the M-Vac® as a DNA collection device, providing an alternative method when DNA is present on a difficult substrate, or if traditional DNA collection methods have failed.

Touch DNA-The prospect of DNA profiles from cables.

Metal theft in the railroad industry poses significant challenges to transport investigators. Cable sheaths left behind at crime scenes, if appropriately analysed, could provide valuable evidence in a forensic investigation, but attempts at recovering DNA are not routinely made. Experiments were set up to ascertain the success in DNA recovery from the surface of cable sheaths after deposition of (a) sweat, (b) extracted DNA and (c) fingermarks. Since investigators try to collect fingermarks and often treat the cables with cyanoacrylate fuming (CNA fuming) or wet powder suspensions (WPS) to enhance the marks this study investigated the recovery of DNA from fingermarks pre- and post-enhancement. The double-swab technique and mini-taping were compared as options to recover DNA from the cable sheaths. Results demonstrate that generally, there is no significant difference between using swabs or mini-tapes to recover the DNA from the non-porous cables (p>0.05). It was also illustrated that CNA fuming performed better than WPS in terms of subsequent recovery and profiling of DNA. CNA fuming resulted in an average increase in DNA recovered via swabbing and taping (more than 4× and 8×, respectively), as compared to no treatment, with 50% of the DNA recovered after CNA fuming generating full DNA profiles.

Monitoring Criminal Activity through Invisible Fluorescent "Peptide Coding" Taggants.

Complementing the demand for effective crime reduction measures are the increasing availability of commercial forensic "taggants", which may be used to physically mark an object in order to make it uniquely identifiable. This study explores the use of a novel "peptide coding" reagents to establish evidence of contact transfer during criminal activity. The reagent, containing a fluorophore dispersed within an oil-based medium, also includes a unique synthetic peptide sequence that acts as a traceable "code" to identify the origin of the taggant. The reagent is detectable through its fluorescent properties, which then allows the peptide to be recovered by swabbing and extracted for electrospray ionization-mass spectrometry (ESI-MS) analysis via a simple liquid-liquid extraction procedure. The performance of the reagent in variable conditions that mimic the limits of a real world use are investigated.

Establishing evidence of contact transfer in criminal investigation by a novel 'peptide coding' reagent.

Forensic investigators are often faced with the challenge of forming a logical association between a suspect, object or location and a particular crime. This article documents the development of a novel reagent that may be used to establish evidence of physical contact between items and individuals as a result of criminal activity. Consisting of a fluorescent compound suspended within an oil-based medium, this reagent utilises the addition of short customisable peptide molecules of a specific known sequence as unique owner-registered 'codes'. This product may be applied onto goods or premises of criminal interest and subsequently transferred onto objects that contact target surfaces. Visualisation of the reagent is then achieved via fluorophore excitation, subsequently allowing rapid peptide recovery and analysis. Simple liquid-liquid extraction methods were devised to rapidly isolate the peptide from other reagent components prior to analysis by ESI-MS.

A novel application of real-time RT-LAMP for body fluid identification: using HBB detection as the model.

We report on a novel application of real-time reverse transcription-loop-mediated isothermal amplification (real-time RT-LAMP) to identify the presence of a specific body fluid using blood as a proof-of-concept model. By comparison with recently developed methods of body fluid identification, the RT-LAMP assay is rapid and requires only one simple heating-block maintained at a single temperature, circumventing the need for dedicated equipment. RNA was extracted from different body fluids (blood, semen, saliva, menstrual blood, sweat, and urine) for use in real-time RT-LAMP reaction. The 18S rRNA locus was used as the internal control and hemoglobin beta (HBB) as the blood-specific marker. Reverse transcription and LAMP reaction were performed in the same tube using a turbidimeter for real-time monitoring the reaction products within a threshold of 60 min. HBB LAMP products were only detected in blood and not in any of the other body fluid, but products from the 18S rRNA gene were detected in all the tested body fluids as expected. The limit of detection was a minimum of 10(-5) ng total RNA for detection of both 18S rRNA and HBB. Augmenting the detection of RT-LAMP products was performed by separation of the products using gel electrophoresis and collecting the fluorescence of calcein. The data collected indicated complete concordance with the body fluid tested regardless of the method of detection used. This is the first application of real-time RT-LAMP to detect body fluid specific RNA and indicates the use of this method in forensic biology.

Assessing psychosocial vulnerability and care needs of pretransplant patients by means of the INTERMED.

OBJECTIVE: We investigated whether the INTERMED, a generic instrument for assessing biopsychosocial case complexity and direct care, identifies organ transplant patients at risk of unfavourable post-transplant development by comparing it to the Transplant Evaluation Rating Scale (TERS), the established measure for pretransplant psychosocial evaluation. METHOD: One hundred nineteen kidney, liver, and heart transplant candidates were evaluated using the INTERMED, TERS, SF-36, EuroQol, Montgomery-Åsberg Depression Rating Scale (MADRS), and Hospital Anxiety & Depression Scale (HADS). RESULTS: We found significant relationships between the INTERMED and the TERS scores. The INTERMED highly correlated with the HADS,MADRS, and mental and physical health scores of the SF-36 Health Survey. CONCLUSIONS: The results demonstrate the validity and usefulness of the INTERMED instrument for pretransplant evaluation. Furthermore, our findings demonstrate the different qualities of INTERMED and TERS in clinical practice. The advantages of the psychiatric focus of the TERS and the biopsychosocial perspective of the INTERMED are discussed in the context of current literature on integrated care.

Application of fluorescent substrates to the in situ detection of prostate specific antigen.

The forensic identification of body fluids frequently presents an important source of genetic material and investigative interpretation. However, presumptive testing techniques presently employed in the discrimination of biological fluids are subject to criticism for poor specificity, lack of fluid localisation ability and detrimental effects on DNA recovery rates. The recognition of fluid-specific biomarkers by fluorogenic substrates may provide a novel resolution to these issues but research has yet to establish any pertinent in situ fluid detection applicability. This study therefore utilises a fluorogenic substrate (Mu-HSSKLQ-AFC) specific to the seminal protein prostate specific antigen in an effort to detect human semen deposited on a number of surfaces typical to criminal investigation. The ability of fluorescent fluorogenic substrates to simultaneously identify and visualise biological fluids in situ is demonstrated for the first time, whilst the production of complete STR profiles from fluid sources is also confirmed to be completely unaffected by substrate application.

Enabling fluorescent biosensors for the forensic identification of body fluids.

The search for body fluids often forms a crucial element of many forensic investigations. Confirming fluid presence at a scene can not only support or refute the circumstantial claims of a victim, suspect or witness, but may additionally provide a valuable source of DNA for further identification purposes. However, current biological fluid testing techniques are impaired by a number of well-characterised limitations; they often give false positives, cannot be used simultaneously, are sample destructive and lack the ability to visually locate fluid depositions. These disadvantages can negatively affect the outcome of a case through missed or misinterpreted evidence. Biosensors are devices able to transduce a biological recognition event into a measurable signal, resulting in real-time analyte detection. The use of innovative optical sensing technology may enable the highly specific and non-destructive detection of biological fluid depositions through interaction with several fluid-endogenous biomarkers. Despite considerable impact in a variety of analytical disciplines, biosensor application within forensic analyses may be considered extremely limited. This article aims to explore a number of prospective biosensing mechanisms and to outline the challenges associated with their adaptation towards detection of fluid-specific analytes.

Forensic DNA methylation profiling--potential opportunities and challenges.

Investigating the DNA sequence is the most powerful tool that can be employed in forensic genetics for the identification of an individual, or to determine specific ethnic and phenotypic characteristics. However, there are also other heritable changes in gene function or cellular phenotype which are caused by mechanisms other than differences in the DNA sequence itself. Over the last decade it has become evident that epigenetic markers can be of substantial forensic significance. The determination of possible alterations in DNA methylation patterns could aid various forensic investigations, such as differentiating monozygotic twins, identifying the tissue source or determining the age of tissue donors. This review aims to give a brief overview of the possible advantages of forensic DNA methylation profiling and sheds light on the limitations of this approach.

Development of a biosensor for human blood: new routes to body fluid identification.

The identification of human blood at a crime scene can provide crucial information to an investigation whilst also providing a source of nuclear material which can be targeted for DNA profiling. Here, we report on the development of an immunofluorescent biosensor for the identification of human blood which has the potential to overcome the drawbacks of the current body fluid identification techniques. An antibody (Ab) raised against human erythrocytes was conjugated to fluorescent semiconductor quantum dots (QDs) by sulfhydryl chemistry. The conjugation was verified by agarose gel electrophoresis and immunohistochemistry. Incubation of liquid blood samples with the conjugated nanocrystals was shown to quench the fluorescence emission spectra in a concentration-dependent manner. A different effect was observed with unconjugated QDs incubated in blood. Full profiles were obtained from blood samples previously treated with the Ab-QDs, demonstrating that the method does not interfere with DNA profiling. To our knowledge, this is the first example of a hybrid Ab-QD sensor that has the potential to be employed for the identification of human blood. The results of this study are expected to open up a new research direction in the field of body fluid detection.

Development of an immunoassay for the differentiation of menstrual blood from peripheral blood.

Forensic identification of body fluids is a crucial aspect of the discipline. To date, there are no robust tests to make a distinction between menstrual blood and peripheral blood. Past techniques have fallen short of producing clear and consistent results while present mRNA techniques are still in their infancy. The aim of this study was to develop of an accurate and rapid immunoassay based identification method. Three different targets were evaluated: matrix metalloproteinase 14 (MMP14), oestrogen receptor α (ERα), and fibrinogen. Cellular or membrane bound antigens were analyzed using immunocytochemical staining while soluble antigens were analyzed by indirect ELISA. Results showed that ERα and MMP14 are present in the endometrial cells of menstrual blood but absent in peripheral blood. At a total protein concentration of 10 µg/mL or lower, fibrinogen was detected in menstrual blood but absent in peripheral. If evaluated on a larger scale, immunoassays may prove to be beneficial in discriminating menstruum and peripheral blood.

Detection and identification of body fluid stains using antibody-nanoparticle conjugates.

Body fluids are considered one of the most important evidence types in forensic casework. The presence and location of blood, semen and saliva can provide crucial information to investigators. Current practice relies on an accurate visual examination followed by the use of presumptive tests to determine the identity of the body fluid type. Further laboratory based tests are required to unequivocally confirm the identity of a stain. Body fluid stains can be difficult to detect with the naked eye, particularly on dark backgrounds and hence vital evidence may be overlooked. Current methods are fluid-type specific, with a separate, and different, test required for each body fluid. The laborious nature of such analysis and the impossibility of being carried out at the crime scene, leads to a delay in the investigation process that could prove detrimental to the solving of the case. Hence, there is a need for sensitive, specific and direct methods which can simultaneously detect, differentiate, and locate human fluids on items of forensic evidence. Here, we describe the preparation of functionalized iron oxide nanoparticles conjugated to antibodies specific to blood and saliva components and their use in detecting small traces against non-contrasting substrates including glass, ceramic tile, paper and black fabric. The advantage of our technique is that it can simultaneously detect blood and saliva and can spatially locate and differentiate these body fluid types. Most importantly, our technology, which exploits the superparamagnetic properties of iron oxide nanoparticles, works in situ with no need to remove the body fluid stains for testing and with no washing steps and does not interfere with downstream DNA profiling. Thus, our technology represents a novel and effective alternative to existing methods.