Profiling and imaging of forensic evidence - A pan-European forensic round robin study part 1: Document forgery.

The forensic scenario, on which the round robin study was based, simulated a suspected intentional manipulation of a real estate rental agreement consisting of a total of three pages. The aims of this study were to (i) establish the amount and reliability of information extractable from a single type of evidence and to (ii) provide suggestions on the most suitable combination of compatible techniques for a multi-modal imaging approach to forgery detection. To address these aims, seventeen laboratories from sixteen countries were invited to answer the following tasks questions: (i) which printing technique was used? (ii) were the three pages printed with the same printer? (iii) were the three pages made from the same paper? (iv) were the three pages originally stapled? (v) were the headings and signatures written with the same ink? and (vi) were headings and signatures of the same age on all pages? The methods used were classified into the following categories: Optical spectroscopy, including multispectral imaging, smartphone mapping, UV-luminescence and LIBS; Infrared spectroscopy, including Raman and FTIR (micro-)spectroscopy; X-ray spectroscopy, including SEM-EDX, PIXE and XPS; Mass spectrometry, including ICPMS, SIMS, MALDI and LDIMS; Electrostatic imaging, as well as non-imaging methods, such as non-multimodal visual inspection, (micro-)spectroscopy, physical testing and thin layer chromatography. The performance of the techniques was evaluated as the proportion of discriminated sample pairs to all possible sample pairs. For the undiscriminated sample pairs, a distinction was made between undecidability and false positive claims. It was found that none of the methods used were able to solve all tasks completely and/or correctly and that certain methods were a priori judged unsuitable by the laboratories for some tasks. Correct results were generally achieved for the discrimination of printer toners, whereas incorrect results in the discrimination of inks. For the discrimination of paper, solid state analytical methods proved to be superior to mass spectrometric methods. None of the participating laboratories deemed addressing ink age feasible. It was concluded that correct forensic statements can only be achieved by the complementary application of different methods and that the classical approach of round robin studies to send standardised subsamples to the participants is not feasible for a true multimodal approach if the techniques are not available at one location.

Surface Analysis Techniques in Forensic Science: Successes, Challenges, and Opportunities for Operational Deployment.

Surface analysis techniques have rapidly evolved in the last decade. Some of these are already routinely used in forensics, such as for the detection of gunshot residue or for glass analysis. Some surface analysis approaches are attractive for their portability to the crime scene. Others can be very helpful in forensic laboratories owing to their high spatial resolution, analyte coverage, speed, and specificity. Despite this, many proposed applications of the techniques have not yet led to operational deployment. Here, we explore the application of these techniques to the most important traces commonly found in forensic casework. We highlight where there is potential to add value and outline the progress that is needed to achieve operational deployment. We consider within the scope of this review surface mass spectrometry, surface spectroscopy, and surface X-ray spectrometry. We show how these tools show great promise for the analysis of fingerprints, hair, drugs, explosives, and microtraces.

Visualisation of Amphetamine Contamination in Fingerprints Using TOF-SIMS Technique.

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) was applied to detect traces of amphetamine on fingerprints. In the present study, three different lift tapes and latent powder fingerprints were tested. The obtained results show that it is possible to identify traces of a drug as well as its distribution over the tested fingerprint after its transfer from the primary base onto an adhesive lifter (secondary base). Moreover, images obtained by the TOF-SIMS technique enable the observation of very small areas of the analysed fingerprint as well as the identification of micro-objects (residues of a contaminant) that were left on the fingerprint. The use of the black latent fingerprint powder did not interfere with the TOF-SIMS analysis, which makes it possible to effectively use this technique to study the traces of substances on the revealed fingerprints.

Using case specific experiments to evaluate fingermarks on knives given activity level propositions.

Bayesian networks have shown to be a useful tool for the evaluation of forensic findings given activity level propositions. In this paper, we demonstrate how case specific experiments can be used to assign probabilities to the states of the nodes of a Bayesian network for the evaluation of fingermarks given activity level propositions. The transfer, persistence and recovery of fingermarks on knives is studied in experiments where a knife is either used to stab a victim or to cut food, representing the activities that were disputed in the case of the murder of Meredith Kercher. Two Bayesian networks are constructed, exploring the effect of different uses of the experimental data by assigning the probabilities based on the results of the experiments. The evaluation of the findings using the Bayesian networks demonstrates the potential for fingermarks in addressing activity level propositions.

A study into evaluating the location of fingermarks on letters given activity level propositions.

A previous paper published in this journal proposed a model for evaluating the location of fingermarks on two-dimensional items (de Ronde, van Aken, de Puit and de Poot (2019)). In this paper, we apply the proposed model to a dataset consisting of letters to test whether the activity of writing a letter can be distinguished from the alternative activity of reading a letter based on the location of the fingermarks on the letters. An experiment was conducted in which participants were asked to read a letter and write a letter as separate activities on A4- and A5-sized papers. The fingermarks on the letters were visualized, and the resulting images were transformed into grid representations. A binary classification model was used to classify the letters into the activities of reading and writing based on the location of the fingermarks in the grid representations. Furthermore, the limitations of the model were studied by testing the influence of the length of the letter, the right- or left-handedness of the donor and the size of the paper with an additional activity of folding the paper. The results show that the model can predict the activities of reading or writing a letter based on the fingermark locations on A4-sized letters of right-handed donors with 98 % accuracy. Additionally, the length of the written letter and the handedness of the donor did not influence the performance of the classification model. Changing the size of the letters and adding an activity of folding the paper after writing on it decreased the model's accuracy. Expanding the training set with part of this new set had a positive influence on the model's accuracy. The results demonstrate that the model proposed by de Ronde, van Aken, de Puit and de Poot (2019) can indeed be applied to other two-dimensional items on which the disputed activities would be expected to lead to different fingermark locations. Moreover, we show that the location of fingermarks on letters provides valuable information about the activity that is carried out.

Imaging-assisted hydrogel formation for single cell isolation.

We report a flexible single-cell isolation method by imaging-assisted hydrogel formation. Our approach consists of imaging-aided selective capture of cells of interest by encasing them into a polymeric hydrogel, followed by removal of unwanted cells and subsequent release of isolated cells by enzymatic hydrogel degradation, thus offering an opportunity for further analysis or cultivation of selected cells. We achieved high sorting efficiency and observed excellent viability rates (>98%) for NIH/3T3 fibroblasts and A549 carcinoma cells isolated using this procedure. The method presented here offers a mask-free, cost-efficient and easy-to-use alternative to many currently existing surface-based cell-sorting techniques, and has the potential to impact the field of cell culturing and isolation, e.g. single cell genomics and proteomics, investigation of cellular heterogeneity and isolation of best performing mutants for developing new cell lines.

Co-deposition of silica and proteins at the interface between two immiscible electrolyte solutions.

In this work, we have simultaneously examined, electrochemically driven deposition of three proteins (haemoglobin, acid phosphatase, and α-amylase) and silica films at a polarized liquid-liquid interface. The interfacial adsorption of the proteins occurs efficiently within the acidic pH range (pH = 2-4). The interfacial charge transfer reactions recorded in the presence of fully positivity charged macromolecules were followed with cyclic voltammetry on the positive side of the potential window. Faradaic currents attributed to the presence of proteins in the aqueous phase appeared for concentrations equal to ca. 0.1 µM for haemoglobin and acid phosphatase and ca. 1 µM for the α-amylase. Concomitant deposition of silica films was achieved via the addition of tetraethoxysilane molecules to the organic phase (1,2-dichloroethane). The hydrolysis and condensation reactions of tetraethoxysilane were controlled via the interfacial transfer of H+ coinciding with the potential for protein adsorption. The effect of tetraethoxysilane concentration - up to 50% by volume - revealed significant shrinkage of the potential window (the region where capacitive currents are recorded). The optimized platform was then used to prepare silica-proteins co-deposits. These could be easily collected from the interface and further analyzed with infrared spectroscopy and transmission electron microscopy.

Positive control tests for fingermark development reagents.

At the start of this study, no reliable, consistent, challenging and representative positive control test was commercially available for fingermark visualisation techniques. The goal of this study was to determine the lower limits of detection (LOD) of the fingermark visualisation techniques ninhydrin and 1,2-indanedione-ZnCl and developing positive control tests for these techniques. Spot tests with an amino acid solution were produced to determine the lower LOD's. Secondly, a Dimatix Materials Printer (DMP) was used to print simulated fingermarks using different concentrations of an amino acid solution, to determine the lower LOD of ninhydrin and 1,2-indanedione-ZnCl using the DMP. Concept positive control tests were developed using the lower LOD of ninhydrin and 1,2-indanedione-ZnCl. These were later on adjusted, to form the definitive positive control tests, after a testing period of two months.

Classification of condom lubricants in cyanoacrylate treated fingerprints by desorption electrospray ionization mass spectrometry.

Traces of condom lubricants in fingerprints can be valuable information in cases of sexual assault. Ideally, not only confirmation of the presence of the condom but also determination of the type of condom brand used can be retrieved. Previous studies have shown to be able to retrieve information about the condom brand and type from fingerprints containing lubricants using various analytical techniques. However, in practice fingerprints often appear latent and need to be detected first, which is often achieved by cyanoacrylate fuming. In this study, we developed a desorption electrospray ionization mass spectrometry (DESI-MS) method which, combined with principal component analysis and linear discriminant analysis (PCA-LDA), allows for high accuracy classification of condom brands and types from fingerprints containing condom lubricant traces. The developed method is compatible with cyanoacrylate (CA) fuming. We collected and analyzed a representative dataset for the Netherlands comprising 32 different condoms. Distinctive lubricant components such as polyethylene glycol (PEG), polydimethylsiloxane (PDMS), octoxynol-9 and nonoxynol-9 were readily detected using the DESI-MS method. Based on the analysis of lubricant spots, a 99.0% classification accuracy was achieved. When analyzing lubricant containing fingerprints, an overall accuracy of 90.9% was obtained. Full chemical images could be generated from fingerprints, showing the distribution of lubricant components such as PEG and PDMS throughout the fingerprint, while still allowing for classification. The developed method shows potential for the development of DESI-MS based analyses of CA treated exogenous compounds from fingerprints for use in forensic science.

The evaluation of fingermarks given activity level propositions.

Fingermarks are highly relevant in criminal investigations for individualization purposes. In some cases, the question in court changes from 'Who is the source of the fingermarks?' to 'How did the fingermark end up on the surface?'. In this paper, we explore the evaluation of fingermarks given activity level propositions by using Bayesian networks. The variables that provide information on activity level questions for fingermarks are identified and their current state of knowledge with regards to fingermarks is discussed. We identified the variables transfer, persistency, recovery, background fingermarks, location of the fingermarks, direction of the fingermarks, the area of friction ridge skin that left the mark and pressure distortions as variables that may provide information on how a fingermark ended up on a surface. Using three case examples, we show how Bayesian networks can be used for the evaluation of fingermarks given activity level propositions.

Non-marking Collection of Amino Acids from Fingerprints Using Hydrogels.

The amino acid profile obtained from a fingerprint may provide valuable information on its donor. Unfortunately, the collection of chemicals from the fingerprint is often destructive to the fingerprint ridge detail. Herein we detail the use of cross-linkable solutions of dextran-methacrylate to form hydrogels capable of collecting amino acids from surfaces followed by extraction and quantification with UPLC-MS. This method allows for the amino acid profile analysis of fingerprints while allowing for their increased visualization at a later stage using the standard method of cyanoacrylate fuming followed by basic-yellow dyeing.

Amino Acid Profiling from Fingerprints Using Amide Stationary-Phase UPLC-MS.

UPLC-MS is a commonly used technique to first separate complex samples and subsequently quantify molecules of interest. Herein we describe the use of UPLC-MS using an amide stationary phase to quantify non-derivatized amino acids extracted from fingerprints. As detector either a triple-quadrupole MS/MS or a TOF-MS detector was used. This method allows for a simple and fast sample preparation, which facilitates the analysis of large amounts of samples.

A study into fingermarks at activity level on pillowcases.

In this paper, we describe a promising method to evaluate the location of fingermarks on two-dimensional objects, which provides valuable information for the evaluation of fingermarks at activity level. For this purpose, an experiment with pillowcases was conducted at the Dutch music festival Lowlands, to test whether the activity 'smothering' can be distinguished from an alternative activity like 'changing a pillowcase' based on the touch traces on pillowcases left by the activities. Participants performed two activities with paint on their hands: smothering a victim with the use of a pillow and changing a pillowcase of a pillow. The pillowcases were photographed and translated into grid representations. A binary classification model was used to classify the pillowcases into one of the two classes of smothering and changing, based on the distance between the grid representations. After applying the fitted model to a test set, we obtained an accuracy of 98.8%. The model showed that the pillowcases could be well separated into the two classes of smothering and changing, based on the location of the fingermarks. The proposed method can be applied to fingermark traces on all two-dimensional items for which we expect that different activities will lead to different fingermark locations.

Proteomics as a new tool to study fingermark ageing in forensics.

Fingermarks are trace evidence of great forensic importance, and their omnipresence makes them pivotal in crime investigation. Police and law enforcement authorities have exploited fingermarks primarily for personal identification, but crucial knowledge on when fingermarks were deposited is often lacking, thereby hindering crime reconstruction. Biomolecular constituents of fingermark residue, such as amino acids, lipids and proteins, may provide excellent means for fingermark age determination, however robust methodologies or detailed knowledge on molecular mechanisms in time are currently not available. Here, we address fingermark age assessment by: (i) drafting a first protein map of fingermark residue, (ii) differential studies of fresh and aged fingermarks and (iii), to mimic real-world scenarios, estimating the effects of donor contact with bodily fluids on the identification of potential age biomarkers. Using a high-resolution mass spectrometry-based proteomics approach, we drafted a characteristic fingermark proteome, of which five proteins were identified as promising candidates for fingermark age estimation. This study additionally demonstrates successful identification of both endogenous and contaminant proteins from donors that have been in contact with various bodily fluids. In summary, we introduce state-of-the-art proteomics as a sensitive tool to monitor fingermark aging on the protein level with sufficient selectivity to differentiate potential age markers from body fluid contaminants.

Electrified Soft Interface as a Selective Sensor for Cocaine Detection in Street Samples.

A straightforward, direct, and selective method is presented for electrochemical cocaine identification in street samples. The sensing mechanism is based on a simple ion transfer reaction across the polarized liquid-liquid interface. The interfacial behavior of a number of cutting agents is also reported. Interfacial miniaturization has led to improved electroanalytical properties of the liquid-liquid interface based sensor as compared with the macroscopic analogue. The reported method holds great potential to replace colorimetric tests with poor selectivity for on-site street sample analysis.

Noninvasive Detection of Cocaine and Heroin Use with Single Fingerprints: Determination of an Environmental Cutoff.

BACKGROUND: Recent publications have explored the possibility of using fingerprints to confirm drug use, but none has yet dealt with environmental contamination from fingertips. Here we explored the possibility of establishing an environmental cutoff for drug testing from a single fingerprint. METHODS: Fingerprint samples (n = 100) were collected from the hands of 50 nondrug users before and after handwashing to establish separate environmental cutoff values and testing protocols for cocaine, benzoylecgonine, heroin, and 6-monoacetylmorphine. The cutoff was challenged by testing the fingerprints of drug-free volunteers after shaking hands with drug users. Fingerprints from patients who testified to taking cocaine (n = 32) and heroin (n = 24) were also collected and analyzed. RESULTS: A different cutoff value needed to be applied, depending on whether the fingerprints were collected as presented or after handwashing. Applying these cutoffs gave a 0% false-positive rate from the drug-free volunteers. After application of the cutoff, the detection rate (compared to patient testimony) for washed hands of patients was 87.5% for cocaine use and 100% for heroin use. CONCLUSIONS: Fingerprints show enhanced levels of cocaine, heroin, and their respective metabolites in patients who testified to taking the substances, compared with the population of naïve drug users surveyed, and a cutoff (decision level) can be established. The cutoff is robust enough to account for small increases in analyte observed after secondary transfer.

Collection of amino acids and DNA from fingerprints using hydrogels.

The amino acid profile obtained from a fingerprint may provide valuable information on its donor. For forensic scientists, recovering evidence relating to the amino acid profile of a suspect can potentially be valuable for identification and exclusion purposes. Herein we detail the use of cross-linkable solutions of dextran-methacrylate to form hydrogels capable of collecting amino acids from surfaces followed by extraction and quantification with UPLC-MS. This method allows for the amino acid profile analysis of fingerprints while allowing for their increased visualisation at a later stage using the standard method of cyanoacrylation. We will demonstrate this method to also be capable of collecting DNA from fingerprints with a 20-60% yield in comparison to using a conventional cotton swab.

Rapid, Secure Drug Testing Using Fingerprint Development and Paper Spray Mass Spectrometry.

BACKGROUND: Paper spray mass spectrometry (PS-MS) is a technique that has recently emerged and has shown excellent analytical sensitivity to a number of drugs in blood. As an alternative to blood, fingerprints have been shown to provide a noninvasive and traceable sampling matrix. Our goal was to validate the use of fingerprint samples to detect cocaine use. METHODS: Samples were collected on triangular pieces (168 mm2) of washed Whatman Grade I chromatography paper. Following application of internal standard, spray solvent and a voltage were applied to the paper before mass spectrometry detection. A fingerprint visualization step was incorporated into the analysis procedure by addition of silver nitrate solution and exposing the sample to ultraviolet light. RESULTS: Limits of detection for cocaine, benzoylecgonine, and methylecgonine were 1, 2, and 31 ng/mL respectively, with relative standard deviations < 33%. No matrix effects were observed. Analysis of 239 fingerprint samples yielded a 99% true-positive rate and a 2.5% false-positive rate, based on the detection of cocaine, benzoylecgonine, or methylecgonine with use of a single fingerprint. CONCLUSIONS: The method offers a qualitative and noninvasive screening test for cocaine use. The analysis method developed is rapid (4 min/sample) and requires no sample preparation.

Analysis of Urine, Oral fluid and Fingerprints by Liquid Extraction Surface Analysis Coupled to High Resolution MS and MS/MS - Opportunities for Forensic and Biomedical Science.

Liquid Extraction Surface Analysis (LESA) is a new, high throughput tool for ambient mass spectrometry. A solvent droplet is deposited from a pipette tip onto a surface and maintains contact with both the surface and the pipette tip for a few seconds before being re-aspirated. The technique is particularly suited to the analysis of trace materials on surfaces due to its high sensitivity and low volume of sample removal. In this work, we assess the suitability of LESA for obtaining detailed chemical profiles of fingerprints, oral fluid and urine, which may be used in future for rapid medical diagnostics or metabolomics studies. We further show how LESA can be used to detect illicit drugs and their metabolites in urine, oral fluid and fingerprints. This makes LESA a potentially useful tool in the growing field of fingerprint chemical analysis, which is relevant not only to forensics but also to medical diagnostics. Finally, we show how LESA can be used to detect the explosive material RDX in contaminated artificial fingermarks.

Rapid detection of cocaine, benzoylecgonine and methylecgonine in fingerprints using surface mass spectrometry.

Latent fingerprints provide a potential route to the secure, high throughput and non-invasive detection of drugs of abuse. In this study we show for the first time that the excreted metabolites of drugs of abuse can be detected in fingerprints using ambient mass spectrometry. Fingerprints and oral fluid were taken from patients attending a drug and alcohol treatment service. Gas chromatography mass spectrometry (GC-MS) was used to test the oral fluid of patients for the presence of cocaine and benzoylecgonine. The corresponding fingerprints were analysed using Desorption Electrospray Ionization (DESI) which operates under ambient conditions and Ion Mobility Tandem Mass Spectrometry Matrix Assisted Laser Desorption Ionization (MALDI-IMS-MS/MS) and Secondary Ion Mass Spectrometry (SIMS). The detection of cocaine, benzoylecgonine (BZE) and methylecgonine (EME) in latent fingerprints using both DESI and MALDI showed good correlation with oral fluid testing. The sensitivity of SIMS was found to be insufficient for this application. These results provide exciting opportunities for the use of fingerprints as a new sampling medium for secure, non-invasive drug detection. The mass spectrometry techniques used here offer a high level of selectivity and consume only a small area of a single fingerprint, allowing repeat and high throughput analyses of a single sample.