Analysis of the fluorescent properties of vaginal fluid upon ageing.

Detection and identification of body fluids are crucial aspects of forensic investigations, aiding in crime scene reconstructions and providing important leads. Although many methods have been developed for these purposes, no method is currently in use in the forensic field that allows rapid, non-contact detection and identification of vaginal fluids directly at the crime scene. The development of such technique is mainly challenged by the complex chemistry of the constituents, which can differ between donors and exhibits changes based on woman's menstrual cycle. The use of fluorescence spectroscopy has shown promise in this area for other biological fluids. Therefore, the aim of this study was to identify specific fluorescent signatures of vaginal fluid with fluorescence spectroscopy to allow on-site identification. Additionally, the fluorescent properties were monitored over time to gain insight in the temporal changes of the fluorescent spectra of vaginal fluid. The samples were excited at wavelengths ranging from 200 to 600 nm and the induced fluorescence emission was measured from 220 to 700 nm. Excitation and emission maps (EEMs) were constructed for eight donors at seven time points after donation. Four distinctive fluorescence peaks could be identified in the EEMs, indicating the presence of proteins, fluorescent oxidation products (FOX), and an unidentified component as the dominant contributors to the fluorescence. To further asses the fluorescence characteristics of vaginal fluid, the fluorescent signatures of protein and FOX were used to monitor protein and lipid oxidation reactions over time. The results of this study provide insights into the intrinsic fluorescent properties of vaginal fluid over time which could be used for the development of a detection and identification method for vaginal fluids. Furthermore, the observed changes in fluorescence signatures over time could be utilized to establish an accurate ageing model.

DFT Study of Imine-Exchange Reactions in Iron(II)-Coordinated Pincers.

The imine bond is among the most applied motifs in dynamic covalent chemistry. Although its uses are varied and often involve coordination to a transition metal for stability, mechanistic studies on imine exchange reactions so far have not included metal coordination. Herein, we investigated the condensation and transimination reactions of an Fe2+ -coordinated diimine pyridine pincer, employing wB97XD/6-311G(2d,2p) DFT calculations in acetonitrile. We first experimentally confirmed that Fe2+ is strongly coordinated by these pincers, and is thus a justified model ion. When considering a four-membered ring-shaped transition state for proton transfers, the required activation energies for condensation and transimination reaction exceeded the values expected for reactions known to be spontaneous at room temperature. The nature of the incoming and exiting amines and the substituents on the para-position of the pincer had no effect on this. Replacing Fe2+ with Zn2+ or removing it altogether did not reduce it either. However, the addition of two ethylamine molecules lowered the energy barriers to be compatible with experiment (19.4 and 23.2 kcal/mol for condensation and transimination, respectively). Lastly, the energy barrier of condensation of a non-coordinated pincer was significantly higher than found for Fe2+ -coordinating pincers, underlining the catalyzing effect of metal coordination on imine exchange reactions.

Towards Onsite Age Estimation of Semen Stains Using Fluorescence Spectroscopy.

The age estimation of biological traces is one of the holy grails in forensic investigations. We developed a method for the age estimation of semen stains using fluorescence spectroscopy in conjunction with a stoichiometric ageing model. The model describes the degradation and generation rate of proteins and fluorescent oxidation products (FOX) over time. The previously used fluorimeter is a large benchtop device and requires system optimization for forensic applications. In situ applications have the advantage that measurements can be performed directly at the crime scene, without additional sampling or storage steps. Therefore, a portable fiber-based fluorimeter was developed, consisting of two optimized light-emitting diodes (LEDs) and two spectrometers to allow the fluorescence protein and FOX measurements. The handheld fiber can be used without touching the traces, avoiding the destruction or contamination of the trace. In this study, we have measured the ageing kinetics of semen stains over time using both our portable fluorimeter and a laboratory benchtop fluorimeter and compared their accuracies for the age estimation of semen stains. Successful age estimation was possible up to 11 days, with a mean absolute error of 1.0 days and 0.9 days for the portable and the benchtop fluorimeters, respectively. These results demonstrate the potential of using the portable fluorimeter for in situ applications.

Use of Lateral Flow Assays in Forensics.

Already for some decades lateral flow assays (LFAs) are 'common use' devices in our daily life. Also, for forensic use LFAs are developed, such as for the analysis of illicit drugs and DNA, but also for the detection of explosives and body fluid identification. Despite their advantages, including ease-of-use, LFAs are not yet frequently applied at a crime scene. This review describes (academic) developments of LFAs for forensic applications, focusing on biological and chemical applications, whereby the main advantages and disadvantages of LFAs for the different forensic applications are summarized. Additionally, a critical review is provided, discussing why LFAs are not frequently applied within the forensic field and highlighting the steps that are needed to bring LFAs to the forensic market.

Specific fluorescent signatures for body fluid identification using fluorescence spectroscopy.

Non-invasive, rapid, on-site detection and identification of body fluids is highly desired in forensic investigations. The use of fluorescence-based methods for body fluid identification, have so far remain relatively unexplored. As such, the fluorescent properties of semen, serum, urine, saliva and fingermarks over time were investigated, by means of fluorescence spectroscopy, to identify specific fluorescent signatures for body fluid identification. The samples were excited at 81 different excitation wavelengths ranging from 200 to 600 nm and for each excitation wavelength the emission was recorded between 220 and 700 nm. Subsequently, the total emitted fluorescence intensities of specific fluorescent signatures in the UV-visible range were summed and principal component analysis was performed to cluster the body fluids. Three combinations of four principal components allowed specific clustering of the body fluids, except for fingermarks. Blind testing showed that 71.4% of the unknown samples could be correctly identified. This pilot study shows that the fluorescent behavior of ageing body fluids can be used as a new non-invasive tool for body fluid identification, which can improve the current guidelines for the detection of body fluids in forensic practice and provide the robustness of methods that rely on fluorescence.

Characterising extracellular vesicles from individual low volume cerebrospinal fluid samples, isolated by SmartSEC.

Extracellular vesicles (EVs) are suggested to have a role in the progression of neurodegeneration, and are able to transmit pathological proteins from one cell to another. One of the biofluids from which EVs can be isolated is cerebrospinal fluid (CSF). However, so far, few studies have been performed on small volumes of CSF. Since pooling of patient samples possibly leads to the loss of essential individual patient information, and CSF samples are precious, it is important to have efficient techniques for the isolation of EVs from smaller volumes. In this study, the SmartSEC HT isolation kit from System Biosciences has been evaluated for this purpose. The SmartSEC HT isolation kit was used for isolation of EVs from 500 µL starting volumes of CSF, resulting in two possible EV fractions of 500 µL. Both fractions were characterised and compared to one another using a whole range of characterisation techniques. Results indicated the presence of EVs in both fractions, albeit fraction 1 showed more reproducible results over the different characterisation methods. For example, CMG (CellMask Green membrane stain) fluorescence nanotracking analysis (NTA), ExoView, and the particles/µg ratio demonstrated a clear difference between fraction 1 and 2, where fraction 1 came out as the one where most EVs were eluted with the least contamination. In the other methods, this difference was less noticeable. We successfully performed complementary characterisation tests using only 500 µL of CSF starting volume, and, conclude that fraction 1 consisted of sufficiently pure EVs for further biomarker studies. This means that future EV extractions may be based upon smaller CSF quantities, such as from individual patients. In that way, patient samples do not have to be pooled and individual patient information can be included in forthcoming studies, potentially linking EV content, size and distribution to individualised neurological diagnoses.

Improving the visualization of fingermarks using multi-target immunolabeling.

The development of fingermarks is an important step in visualizing ridge patterns for individualization purposes. Immunolabeling can be applied to fingermarks to selectively and sensitively detect antigens in fingermarks, and can be used as a developing method to visualize fingermarks. In this study we investigated single (the detection of one antigen) and multiple targeting approaches (the detection of multiple antigens simultaneously) to improve fingermark development. The detection of dermcidin, an antimicrobial peptide, was used as the gold standard to compare single and multi-target detection of keratins, albumin and/or dermcidin. Single detection of dermcidin and albumin mostly resulted in clear ridge details and/or pore detection, whereas the single keratin detection resulted in a poor visualization of the fingermarks. The multi-target approach in which both dermcidin and albumin were targeted, resulted in improved fingermark development compared to single dermcidin detection. Therefore, we recommend the use of multi-target detection consisting of anti-dermcidin and anti-albumin when using immunolabeling as fingermark development technique. Additionally, the optimized multi-target approach was tested as a pre- and post-development technique in combination with powder dusting and cyanoacrylate fuming. Immunolabeling has not been implemented yet in forensic case work, however we expect that immunolabeling can be used to redevelop poorly developed and/or smudged fingermarks in the nearby future. Currently, an ongoing pilot-study is being conducted in collaboration with the Dutch police.

The compatibility of immunolabeling with STR profiling.

Immunolabeling is a technique, which has recently been introduced to enhance the quality of developed fingermarks and subsequently strengthen the evidential value. The effect of this method on subsequent DNA analysis, however, has not been explored yet. Therefore, the current pilot study aimed to determine whether STR profiling is possible after immunolabeling. Since immunolabeling involves washing steps which could reduce DNA quantities, the use of different fixatives including methanol, formaldehyde and universal molecular fixative (UMFIX) were investigated. STR profiles from the (immunolabeled) fingermarks were generated after four days and four weeks by a direct PCR method to enable comparison of relatively fresh and old fingermarks. The fingermarks were deposited on diverse forensically relevant substrates, including glass, metal and tile. STR profiles could be recovered for all tested fixatives with no significant difference in performance. However, the mean number of detected alleles was the highest when methanol was used for fixation. Furthermore, immunolabeling on aged fingermarks (4 weeks) was also possible, but the number of detected alleles showed a non-significant decrease. DNA could be recovered from deposits on all substrates, of which glass showed the highest mean number of detected alleles followed by metal and tile.

Human milk triggers coagulation via tissue factor-exposing extracellular vesicles.

Almost a century ago, it was discovered that human milk activates the coagulation system, but the milk component that triggers coagulation had until now been unidentified. In the present study, we identify this component and demonstrate that extracellular vesicles (EVs) present in normal human milk expose coagulant tissue factor (TF). This coagulant activity withstands digestive conditions, mimicking those of breastfed infants, but is sensitive to pasteurization of pooled donor milk, which is routinely used in neonatal intensive care units. In contrast to human milk, bovine milk, the basis of most infant formulas, lacks coagulant activity. Currently, the physiological function of TF-exposing vesicles in human milk is unknown, but we speculate that these vesicles may be protective for infants. Another explanation could be nipple skin damage, which occurs in most breastfeeding women. Milk-derived TF-exposing EVs may seal the wound and thereby reduce bleeding and breast inflammation.

Detection of extracellular vesicles in plasma and urine of prostate cancer patients by flow cytometry and surface plasmon resonance imaging.

Large (> 1 µm) tumor-derived extracellular vesicles (tdEVs) enriched from the cell fraction of centrifuged whole blood are prognostic in metastatic castration-resistant prostate cancer (mCRPC) patients. However, the highest concentration of tdEVs is expected in the cell-free plasma fraction. In this pilot study, we determine whether mCRPC patients can be discriminated from healthy controls based on detection of tdEVs (< 1µm, EpCAM+) and/or other EVs, in cell-free plasma and/or urine. The presence of marker+ EVs in plasma and urine samples from mCRPC patients (n = 5) and healthy controls (n = 5) was determined by flow cytometry (FCM) and surface plasmon resonance imaging (SPRi) using an antibody panel and lactadherin. For FCM, the concentrations of marker positive (+) particles and EVs (refractive index <1.42) were determined. Only the lactadherin+ particle and EV concentration in plasma measured by FCM differed significantly between patients and controls (p = 0.017). All other markers did not result in signals exceeding the background on both FCM and SPRi, or did not differ significantly between patients and controls. In conclusion, no difference was found between patients and controls based on the detection of tdEVs. For FCM, the measured sample volumes are too small to detect tdEVs. For SPRi, the concentration of tdEVs is probably too low to be detected. Thus, to detect tdEVs in cell-free plasma and/or urine, EV enrichment and/or concentration is required. Furthermore, we recommend testing other markers and/or a combination of markers to discriminate mCRPC patients from healthy controls.

The use of crime scene detection dogs to locate semen stains on different types of fabric.

In sexual assault cases, the detection and identification of semen is extremely important as this type of evidence can be used as a source for investigative leads and contributes to case evidence. However, the detection of semen stains is often difficult, even indoors, because of different (environmental) factors, such as substrate type, coloured items and large search areas. In 2015, a project was initiated by the Dutch police to evaluate the feasibility of the use of detection dogs to locate semen stains in forensic practise. Since promising results were obtained, here, a double-blind study was designed to investigate how these detection dogs can optimally be implemented in the current work flow of crime scene investigators and to compare the dog's sensitivity and specificity with current detection methods. The performance of the detection dogs was compared to three commonly used detection methods for semen, (i) forensic light sources (FLS), (ii) the RSID semen field kit and (iii) the enzymatic Acid Phosphatase (AP)-test on semen deposited at different types of fabrics. A 100% sensitivity and specificity for the detection of semen stains using the detection dogs was obtained, compared to an overall sensitivity and specificity of 76.3% and 100% for FLS, 81.6% and 100% for RSID-test, and 92.1% and 100% for AP-test, respectively. Especially, on fabrics with a pattern or interfering fluorescent properties, detection dogs demonstrated to be of additional value to locate the semen stains. We recommend to use the following order of testing, FLS, detection dog, AP-test and RSID test in a forensic workflow.

Estimating the Time of Deposition of Semen Traces using Fluorescence Protein-Lipid Oxidation Signatures.

In the forensic field, knowledge about the time of deposition of semen traces is extremely valuable to law enforcement agencies to assess the relevance of the traces and the validity of witness testimonies. However, currently, no method exists that is able to estimate the time of deposition of semen stains, due to the complex chemistry of the constituents and variation in degradation patterns. Here, we demonstrate a non-contact age estimation method to assess the time of deposition of semen stains using fluorescence spectroscopy. Protein-lipid oxidation reactions were monitored in semen stains over time using protein fluorescence and fluorescent oxidation product signatures to reveal distinctive aging patterns. On the basis of the relative amounts of these fluorescent products and the rate at which they are converted, successful age estimation was achieved up to a value of 16 days, with a median absolute error of 1.7 days. We demonstrate here a new tool that can fill the current gap in intelligence about the age of semen traces that has been challenging the forensic community worldwide.

Prediction of DNA concentration in fingermarks using autofluorescence properties.

During criminal investigations trace DNA samples, including fingermarks, are submitted to laboratories for short tandem repeat (STR) analysis. For most common STR analysis systems a minimum amount of input DNA is required. Upon intake by the forensic laboratory the DNA concentration is estimated using quantitative polymerase chain reaction (qPCR) analysis after which most fingermarks are excluded. To tackle the problem of unnecessary processing in the lab, our study aimed to develop a method, which is able to predict the DNA content in fingermarks directly at the crime scene. Upon excitation with a UV Crime-lite, fingermark residues have autofluorescent properties. We hypothesize that the intensity of the autofluorescence signal of the fingermark content correlates to the DNA concentration in fingermarks. In this study, 164 fingermarks were examined on their autofluorescence intensity when excited at 365nm, the number of nucleated cells, their DNA concentration and the completeness of the STR profiles. No significant correlation was observed between the DNA concentration in fingermarks and the autofluorescence signal, indicating that a high amount of autofluorescence, thus a high amount of biomaterial, does not necessarily guarantee a higher amount of DNA. In addition, the completeness of the STR profiles did not correlate to the autofluorescence signal of fingermarks. A moderate correlation was found between the predicted DNA quantity, based on the number of nucleated cells and the DNA quantity. In summary, the autofluorescence signal of fingermarks cannot directly be used as a guide to select fingermarks for DNA analysis directly at the crime scene. However, predicting the amount of DNA using a sensitive and specific DNA staining method can probably be used to estimate the DNA concentration in touch samples.

Identification and detection of protein markers to differentiate between forensically relevant body fluids.

The identification of body fluids at a crime scene is an important aspect of forensic casework analysis, being a source for investigative leads and contributing to case evidence. Yet, current methods for the forensic identification of body fluids suffer from several limitations, ranging from poor sensitivity and specificity, to sample destruction and interference with subsequent DNA analysis. Moreover, current identification assays target only one body fluid at the time. Besides being inefficient in terms of time, money and sample consumption, poor identification methods can also negatively influence the outcome of a (court) case. In this study, eleven potential protein biomarkers and antibodies were selected and assessed on their suitability for serving as identification markers, as a first step towards the development of a new multiplex protein-based body fluid identification assay relying on antigen-antibody interactions. Performing antibody-based dot blot assays, the specificity of the biomarkers for their target body fluids was evaluated, and biomarker detection was studied in diluted, mixed, aged and simulated casework samples. Hereby, nine out of eleven markers were identified as promising biomarkers to identify blood, semen, saliva, urine and sweat. With the identification of these targets and detection antibodies, a major step forward has been taken towards the development of a highly sensitive and specific, fast and non-labour-intensive protein-based body fluid identification assay, suitable for on-site analysis and able to test for multiple body fluids in a single reaction.

Techniques that acquire donor profiling information from fingermarks - A review.

Fingermarks are among the most important types of evidence that can be encountered at the scene of a crime since the unique ridge pattern of a fingerprint can be used for individualization. But fingermarks contain more than the characteristic pattern of ridges and furrows, they are composed of a wide variety of different components that originate from endogenous and exogenous sources. The chemical composition can be used to obtain additional information from the donor of the fingermark, which in turn can be used to create a donor profile. Donor profiling can serve at least two purposes i) to enhance the evidential value of fingermarks and ii) to provide valuable tactical information during the crime scene investigation. Retrieving this additional information is not limited to fingermarks that have been used for individualization, but can also be applied on partial and/or distorted fingermarks. In this review we have summarized the types of information that can be obtained from fingermarks. Additionally, an overview is given of the techniques that are available addressing their unique characteristics and limitations. We expect that in the nearby future, donor profiling from contact traces, including fingermarks will be possible.

On the autofluorescence of aged fingermarks.

Fingermark autofluorescence changes with time, both spectrally and in total intensity. In this study we investigate which components in the aged fingermarks cause this change in autofluorescent signal. Thin layer chromatography combined with fluorescence spectroscopy was used to identify fluorescent aging products. Based on our results, tryptophan derivatives, including indoleacetic acid, (nor)harman and xanthurenic acid are indicated as important contributors to the autofluorescence of aged fingermarks. Knowledge about which fluorescent aging products are present in fingermarks might be useful in the development of fingermark age estimation methods. This work is part of a larger project of which the major goal is to develop a method to estimate the time of deposition of fingermarks. Additionally, by selective targeting of aging products the development of aged fingermarks might be improved.

Immunolabeling and the compatibility with a variety of fingermark development techniques.

Much information can be obtained from the chemical composition of a fingermark, which can be helpful in crime scene investigation. Immunolabeling can be used to extract information about the donor of the fingermark and it can also act as a fingermark development tool in sequence with the standard fingermark development techniques. However, before immunolabeling can be used in forensic practice more information on the possibilities and limitations of this technique is required. In this study, our aim was to investigate if immunolabeling is compatible with standard development protocols (indanedione-zinc, indanedione-zinc followed by ninhydrin spraying, physical developer, cyanoacrylate fuming, cyanoacrylate followed by basic yellow staining, lumicyanoacrylate fuming and polycyanoacrylate fuming). Immunolabeling was carried out successfully on all developed fingermarks, whereby dermcidin was selected as antigen of interest. We can conclude that immunolabeling is compatible with a wide variety of different fingermark developers. This finding in combination with previous findings, makes immunolabeling an interesting technique, which can be of great value in the forensic field.

Oxidation monitoring by fluorescence spectroscopy reveals the age of fingermarks.

No forensic method exists that can reliably estimate the age of fingermarks found at a crime scene. Information on time passed since fingermark deposition is desired as it can be used to distinguish between crime related and unrelated fingermarks and to support or refute statements made by the fingermark donors. We introduce a non-contact method that can estimate the age of fingermarks. Fingermarks were approached as protein-lipid mixtures and an age-estimation model was build based on the expected protein and lipid oxidation reactions. Two measures of oxidation are required from the fingermark to estimate its age: 1) the relative amount of fluorescent oxidation products 2) the rate at which these products are formed. Fluorescence spectroscopy was used to obtain these measures. We tested the method on 44 fingermarks and were able to estimate the age of 55% of the male fingermarks, up to three weeks old with an uncertainty of 1.9 days.

Simultaneous labeling of multiple components in a single fingermark.

A fingermark contains important forensic information of the donor, not only in its ridge pattern, but also in the chemical composition of its secretion. Detection and identification of these secretions can be done by immunolabeling. In this study, we describe for the first time a reproducible immunolabeling method that allows the simultaneous detection of multiple components of interest. This method not only reduces the manipulation of fingermarks, but also different types of information can be obtained about the donor in one labeling session. To prove the concept of this technique, we selected two general components as antigens of interest, dermcidin and the human serum albumin. Conjugation of both antibodies to two different synthetic fluorophores, followed by simultaneous incubation of both conjugated antibodies, resulted in successful multiple immunolabeling of fingermarks left on a porous nitrocellulose membrane and on a non-porous glass slide surface. In order to minimize false positives to prevent non-specific binding of antibodies to fingermarks and surface carriers, careful blocking and washing steps were found crucial. With this reproducible protocol, high quality images could be obtained from the multiple labeled fingermarks. In conclusion, simultaneous multiple immunolabeling of antibodies in fingermarks can identify specific components in the secretion of the fingermark, including components related to hygiene, diet, time of day, contacts gender and drug use. Multiple immunolabeling therefore has the potential to make a major impact in the forensic field.