Evaluation of the effect of ozone disinfection on forensic identification of blood, saliva, and semen stains.

Good laboratory practice minimizes the biological hazard posed by potentially infectious casework samples. In certain scenarios, when the casework sample is contaminated with highly contagious pathogens, additional safety procedures such as disinfection might be advised. It was previously proven that ozone gas treatment does not hamper STR analysis, but there is no data on how the disinfection affects other steps of the forensic analysis. In this study, we aimed to assess the interference of ozone disinfection with forensic tests used to identify biological stains. A dilution series of blood, saliva, and semen samples were pipetted onto cotton fabric and let completely dry. Half of the samples were subjected to ozone treatment, while the rest served as controls. All the samples were tested with specific lateral flow immunochromatographic assays and for specific RNA markers with quantitative real-time PCR. Additionally, luminol test was carried out on blood spots, Phadebas® Amylase Test on saliva stains, and semen stains were examined with STK Lab kit and light microscope following Christmas Tree or Hematoxylin-Eosin staining. Ozone treatment had no detrimental effect on the microscopic identification of sperm cells. Undiluted blood samples were detected with luminol and immunoassay, but at higher dilution, the sensitivity of the test decreased after disinfection. The same decrease in sensitivity was observed in the detection of semen stains using STK Lab kit from STK® Sperm Tracker, and in the case of the immunoassay specific for prostate-specific antigen (PSA). Ozone treatment almost completely inhibited the enzymatic activity of amylase. The sensitivity of antibody-based detection of amylase was also greatly reduced. RNA markers showed degradation but remained detectable in blood and semen samples after incubation in the presence of ozone. In saliva, the higher Ct values of the mRNA markers were close to the detection limit, even before ozone treatment.

Research Progress on Biological Evidence Identification in Fire Scenes.

Biological evidence is relatively common evidence in criminal cases, and it has strong probative power because it carries DNA information for individual identification. At the scene of fire-related cases, the complex thermal environment, the escape of trapped people, the firefighting and rescue operations, and the deliberate destruction of criminal suspects will all affect the biological evidence in the fire scene. Scholars at home and abroad have explored and studied the effectiveness of biological evidence identification in fire scenes, and found that the blood stains, semen stains, bones, etc. are the main biological evidence which can be easily recovered with DNA in fire scenes. In order to analyze the research status and development trend of biological evidence in fire scenes, this paper systematically sorts out the relevant research, mainly including the soot removal technology, appearance method of typical biological evidence, and possibility of identifying other biological evidence. This paper also prospects the next step of research direction, in order to provide reference for the identification of biological evidence and improve the value of biological evidence in fire scenes.

Empirical investigation of passive blood drop trajectory and first point of contact on inclined surfaces.

The first point of contact between a spherical blood drop and a surface is related to the angle between the trajectory of the blood drop and the surface being struck. This angle is often referred to as the impact angle which can be estimated by knowing the width and length of the resultant elliptical bloodstain. Most software programs dedicated to area of origin analysis indicate the location of the backtracked bloodstain trajectory to be at the geometric centre or at the tip of the bloodstain ellipse. However, it is unknown how the first point of contact and the blood drop trajectory (here defined as the locus of the centre of mass of the drop as it travels) are related empirically. Thus, this study aims to look at how the initial point of contact and the trajectory at the impact of a blood drop relates to the formed bloodstain ellipse. Two volumes of blood (0.013 ml and 0.071 ml) were dropped from a height of 10 cm and 40 cm onto an inclined surface at 0°, 15°, 30°, 45°, 60°, and 75°. The transition from a spherical blood drop to an elliptically shaped bloodstain was recorded using a high-speed camera for all tests. A total of 72 ellipses were analyzed to determine the location of the first point of contact and trajectory point of the blood drop and how they relate to the formed elliptical bloodstain. A relationship was found between the first point of contact and the bloodstain trajectory which was dependent on the impact angle. However, there were clear deviations from theoretical assumptions due to blood drop oscillations, the effects of gravity, and the natural fluid characteristics of blood. The results of this study may assist bloodstain pattern analysts and software developers by more accurately applying the location of the blood drop trajectory based on empirical data.

Assessing iPhone LiDAR & Recon-3D for determining area of origin in bloodstain pattern analysis.

Bloodstain pattern analysis (BPA) has proven to be a useful tool in forensic and criminal investigations for quite some time. Traditionally, documenting a crime scene for a bloodletting event was completed using manual techniques, physical strings, and a tape measure. In more recent years, laser scanners and 3D software programs have become a preferred method to capture accurate data that improves the validity and reliability of BPA. The initial cost of laser scanning equipment is relatively high, rendering these systems inaccessible to some police and smaller agencies. Recon-3D is a newly developed iPhone application that utilizes the iPhone LiDAR sensor in combination with video data to create 3D point clouds of crime scenes. To assess the viability of Recon-3D for area of origin analysis, two tests were performed. One was a series of bloodstain impacts which were analyzed in FARO Zone 3D software, while the second was a series of 6 repeated Recon-3D scans of two 90-degree walls which was then compared to the FARO Focus S350 scanner using CloudCompare software. A total of eight impact patterns were made at three different distances from a wall. The area of origin was measured and compared to the known location of the blood source. The average total 3D error for the area of origin set at 25, 50, and 100 cm from two perpendicular walls was found to be 6.04, 15.16, and 36.59 cm, respectively. These results are similar to past studies where programs such as HemoSpat have been used. The results of the point cloud comparison show that on average, 95% of the points from Recon-3D fall below a threshold of 3.6 mm when compared to a FARO Focus S350 laser scanner. Thus, the results of this test suggest that Recon-3D is an accurate and affordable scanning application for bloodstain patterns at crime scenes and the data provide acceptable results for area of origin analysis in BPA programs which accept laser scanner data.

The effect of the anti-coagulant EDTA on the deposition and adhesion of whole blood deposits on non-porous substrates.

An investigation into whether the addition of a commonly used anti-coagulant agent like ethylenediaminetetraacetic acid (EDTA) has an impact on the adhesion potential of blood to non-porous substrates was conducted. Two non-porous substrates (aluminum and polypropylene) exhibiting six different surface roughness categories (R1-R6) were used as test substrates upon which either whole blood or blood treated with EDTA was deposited. Samples were exposed to different drying periods (24 hours, 48 hours, and 1 week) before undergoing a tapping agitation experiment in order to evaluate the adhesion to the surface. Clear differences in adhesion potential were observed between whole blood and blood treated with EDTA. Blood treated with EDTA displayed a stronger adhesion strength to aluminum after a drying time of 24 h pre-agitation, while whole blood presented with a stronger adhesion strength at the drying time of 48 h and 1 week. Both EDTA-treated and EDTA-untreated blood was shown to dislodge less easily on polypropylene with the only difference observed on smooth surfaces (0.51-1.50 µm surface roughness). Thus, when conducting transfer studies using smooth hydrophobic substrates like polypropylene or considering the likelihood of transfer given specific case scenarios, differences in adhesion strength of blood due to hydrophobic substrate characteristics and a decreased surface area need to be considered. Overall, whole blood displayed a better adhesion strength to aluminum, emphasizing that indirect transfer probability experiments using EDTA blood on substrates like aluminum should take an increased dislodgment tendency into account in their transfer estimations.

Transcriptomic changes and prediction of time since deposition of blood stains.

In forensics, it is important to determine the time since deposition (TSD) of bloodstains, one of the most common types of biological evidence in criminal cases. However, no effective TSD inference methods have been established despite extensive attempts in forensic science. Our study investigated the changes in the blood transcriptome over time, and we found that degradation could be divided into four stages (days 0-2, 4-14, 21-56, and 84-168) at 4 °C. A random forest prediction model based on these transcriptional changes was trained on experimental samples and tested in separate test samples. This model was able to successfully predict TSD (area under the curve [AUC] = 0.995, precision = 1, and recall = 1). Thus, this proof-of-concept pilot study has practical significance for assessing physical evidence. Meanwhile, 11 upregulated and 13 downregulated transcripts were identified as potential time-marker transcripts, laying a foundation for further development of TSD analysis methods in forensic science and crime scene investigation.

Some experiments and remarks regarding the possible formation of blood stains on the Turin Shroud: stains attributed to the crown of thorns, the lance wound and the belt of blood.

The Turin Shroud (TS) is a Christian relic interpreted to be the burial cloth of Jesus of Nazareth. It exhibits red discolorations that have been interpreted as blood stains and that are the subjects of a highly controversial discussion. We conducted experiments to identify theoretically possible explanations for the stains attributed to the crown of thorns, the lance wound and the belt of blood. In the experiments with a focus on the stains attributed to the crown of thorns, a very similar stain pattern as on the TS could be provoked by simulating the following sequence of events: blood from antemortem scalp wounds is covering hair and face; blood is coagulating and/or drying; blood components are mobilised by postmortem washing and oiling. A stain pattern very similar to the belt of blood on the TS was successfully provoked by simulating the following sequence of events: The body is lying in a supine position, blood or bloodied water flowing from a wound at the right lateral chest wall; the body is rotated to the left side; the Shroud is tucked under the back; the body is rotated back to a supine position and laid onto the Shroud. The so-called serum ring surrounding the stain attributed to the lance wound could be reproduced by sequential application of serum and whole blood samples or of pleural effusion and whole blood samples onto cotton cloth. It is obvious that any attempt to interpret the assumed blood stain pattern on the TS has serious limitations. Nevertheless, it seems remarkable that we were able to reproduce findings that appear to be very similar to stains on the TS.

Characterization of insect stains produced by Dermestes maculatus De Greer (Coleoptera: Dermestidae) resulting from interactions with human blood.

Insect stains produced by adult Dermestes maculatus were characterized during interactions with human blood. Beetles were offered wet or dried blood positioned on ceramic tiles under laboratory conditions. Despite a life history strategy geared toward consumption of dried food stuffs, adult beetles interacted with wet blood more frequently than dried and produced more insect stains after ingesting wet blood. Most (> 95%) of the insect stains produced were the result of fecal elimination. These stains varied in morphologies but were consistently tan/light, black/grey, or red in color; were round to amorphous in shape; and frequently possessed tails. Tailed stains typically were tadpole-shaped or long and tapering from the stain body, yielding Ltl/Lb ratios greater than 1. Tails were the result of beetle locomotion while defecating. Human blood was detected in defecatory stains when using ABA Hematrace® lateral flow assays. When beetles interacted with dried blood, the bloodstains were most often modified due to physical disruption rather than feeding activity. This yielded flaking or dislodgement of the original stains. Within a forensic context, it is unknown whether D. maculatus interacts with any type of bloodstains at a crime scene.

Classification of bloodstains deposited at different times on floor tiles using hierarchical modelling and a handheld NIR spectrometer.

Bloodstains are commonly encountered at crime scenes, especially on floor tiles, and can be deposited over different periods and intervals. Therefore, it is crucial to develop techniques that can accurately identify bloodstains deposited at different times. This study builds upon a previous investigation and aims to enhance the performance of three distinct hierarchical models (HMs) designed to differentiate and identify stains of human blood (HB), animal blood (AB), and common false positives (CFPs) on nine different types of floor tiles. Soft Independent Modeling Class Analogies (SIMCA), and Partial Least Squares-Discriminant Analysis (PLS-DA) were employed as decision rules in this process. The originally published model was constructed using a training set that included samples with a known time of deposit of six days. This model was then tested to predict samples with various deposition times, including human blood samples aged for 0, 1, 9, 20, 30, and 162 days, as well as animal blood samples aged for 0, 1, 10, 13, 20, 29, 105, and 176 days. To improve the identification of human blood, the models were modified by adding zero-day and one-day-old bloodstains to the original training set. All models showed improvement when fresher samples were included in the training set. The best results were achieved with the hierarchical model that used partial least squares-discriminant analysis as the second decision rule and incorporated one-day-old samples in the training set. This model yielded sensitivity values above 0.92 and specificity values above 0.7 for samples aged between zero and 30 days.

TrACES of time: Transcriptomic analyses for the contextualization of evidential stains - Identification of RNA markers for estimating time-of-day of bloodstain deposition.

Obtaining forensically relevant information beyond who deposited a biological stain on how and under which circumstances it was deposited is a question of increasing importance in forensic molecular biology. In the past few years, several studies have been produced on the potential of gene expression analysis to deliver relevant contextualizing information, e.g. on nature and condition of a stain as well as aspects of stain deposition timing. However, previous attempts to predict the time-of-day of sample deposition were all based on and thus limited by previously described diurnal oscillators. Herein, we newly approached this goal by applying current sequencing technologies and statistical methods to identify novel candidate markers for forensic time-of-day predictions from whole transcriptome analyses. To this purpose, we collected whole blood samples from ten individuals at eight different time points throughout the day, performed whole transcriptome sequencing and applied biostatistical algorithms to identify 81 mRNA markers with significantly differential expression as candidates to predict the time of day. In addition, we performed qPCR analysis to assess the characteristics of a subset of 13 candidate predictors in dried and aged blood stains. While we demonstrated the general possibility of using the selected candidate markers to predict time-of-day of sample deposition, we also observed notable variation between different donors and storage conditions, highlighting the relevance of employing accurate quantification methods in combination with robust normalization procedures.This study's results are foundational and may be built upon when developing a targeted assay for time-of-day predictions from forensic blood samples in the future.

Discovery and validation of metabolite markers in bloodstains for bloodstain age estimation.

Bloodstain age estimation involves measuring time-dependent changes in the levels of biomolecules in bloodstains. Although several studies have identified bloodstain metabolites as markers for estimating bloodstain age, none have considered sex, age-related metabolomic differences, or long-time bloodstain age. Therefore, we aimed to identify metabolite markers for estimating the age of bloodstains at weekly intervals within 28 days and validate them through multiple reaction monitoring. Adenosine 5'-monophosphate, choline, and pyroglutamic acid were selected as markers. Seven metabolites were validated, including five previously reported metabolites, ergothioneine, hypoxanthine, L-isoleucine, L-tryptophan, and pyroglutamic acid. Choline and hypoxanthine can be used to differentiate bloodstains between days 0 and 14 after deposition at weekly intervals, whereas L-isoleucine and L-tryptophan can help distinguish bloodstains between 7 days before and 14 days after deposition. Evaluation of the changes in metabolite levels according to sex and age revealed that the average levels of all seven metabolites were higher in women on day 0. Moreover, the level of ergothioneine was significantly higher in elderly individuals than in young individuals at all time points. In this study, we confirmed the potential effectiveness of metabolites in bloodstains as forensic markers and provided a new perspective on metabolomic approaches linked to forensic science.

Quantitative PCR analysis of bloodstains of different ages.

An accurate method to estimate the age of a stain or the time since deposition (TsD) would represent an important tool in police investigations for evaluating the true relevance of a stain. In this study, two laboratories reproduced an mRNA-based method for TsD estimation published by another group. The qPCR-based assay includes four transcripts (B2M, LGALS2, CLC, and S100A12) and showed preferential degradation of the 5' end over the 3' end. In this study, the blood-specific marker ALAS2 was added to examine whether it would show the same degradation pattern. Based on our qPCR data several elastic net models with different penalty combinations were created, using training data from the two laboratories separately and combined. Each model was then used to estimate the age of bloodstains from two independent test sets each laboratory had prepared. The elastic net model built on both datasets with training samples up to 320 days old displayed the best prediction performance across all test samples (MAD=18.9 days). There was a substantial difference in the prediction performance for the two laboratories: Restricting TsD to up to 100 days for test data, one laboratory obtained an MAD of 2.0 days when trained on its own data, whereas the other laboratory obtained an MAD of 15 days.

Distinguishing between stamping in blood from walking through blood using blood pattern analysis.

Bloodstains are typically encountered in violent incidents involving the use of a weapon or physical actions, such as punching, kicking, or stamping. Bloodstain pattern analysis can provide inceptive evidence or intelligence about what happened in an alleged incident, the sequence of events, along with indicating possible suspects if blood is analysed through DNA profiling. This research project focused on the differences in patterns created on footwear during a violent action, such as stamping on a person, and a non-violent action, such as walking through a pool of blood. In this project, several experiments were designed to simulate the stamping and walking actions on a surface wet with blood: carpet, lino flooring, and belly pork meat. Two volunteers with varying body weights were recruited to perform the two actions, using a pair of trainers and a pair of Wellington boots. Defibrinated horse blood was used to simulate real human blood. It was found that the patterns created from stamping and walking through blood differed by the type of pattern and the number and size of stains. The footwear used in the stamping action was characterised by a larger contact stain on the sole than those used in the walking actions: ∼209 mm in length by ∼92 mm in width versus ∼65 mm in length by ∼60 mm in width. The stamping action produced a large number of impact spatters (∼435) on the sides of the footwear versus no impact spatters in the walking actions. The presence of impact spatters was found to be the most prominent feature that differentiated between the two actions. The findings were statistically significant (p < 0.05) and could assist in evaluating whether a defendant was actively involved in a stamping action, or the evidence found was due to innocent reasons.

[The distinc features of a blood drip stain on a surface covered with house dust]. / Osobennosti sleda kapli krovi na poverkhnosti, pokrytoi bytovoi pyl'yu.

The purpose of the investigation is to establish the characteristics of a blood drip stains on a surface covered with house dust. In most cases, objects at the crime scene are covered in some degree of dust deposits. Accordingly, in external bleeding wounds, blood drops on dusty surfaces. The experiment established the peculiarities of drip stains on glass covered with dust. Compared with a clean surface, there was a greater number of spines at the edge of the drip stain. There was pronounced widespread and sectoral splashing. This can be explained by the fact that the dust particles are obstacles which, when the droplet spreads across the surface, form an irregular edge and cause splashing.

Estimation of bloodstain deposition time within a 24-h day-night cycle with rhythmic mRNA based on a machine learning algorithm.

Estimating the time that bloodstains are left at a crime scene can provide invaluable evidence for law enforcement investigations, including determining the time of the crime, linking the perpetrator to the crime scene, narrowing the pool of possible suspects, and verifying witness statements. There have been some attempts to estimate the time since deposition of bloodstains, i.e., how much time has passed since the bloodstain was left at a crime scene. However, most studies focus on the time interval of days. As far as we know, previous study have been conducted to estimate the deposition time of blood within a 24-h day-night cycle. To date, there is a lack of studies on whether rhythmic mRNA of blood is suitable for bloodstain samples. In this study, we estimated the bloodstain deposition time within a 24-h day-night cycle based on the expression of messenger RNAs (mRNAs) by real-time quantitative polymerase chain reaction. Bloodstain samples were prepared from eight individuals at eight time points under real and uncontrolled conditions. Four mRNAs expressed rhythmically and were used to construct a regression model using the k-nearest neighbor (KNN) algorithm, resulting in a mean absolute error of 3.92 h. Overall, using the rhythmic mRNAs, a machine learning model was developed which has allowed us to predict the deposition time of bloodstains within the 24-h day-night cycle in East Asian populations. This study demonstrates that mRNA biomarkers can be used to estimate the bloodstain deposition time within a 24-h period. Furthermore, rhythmic mRNA biomarkers provide a potential method and perspective for estimating the deposition time of forensic traces in forensic investigation. Case samples in forensic analysis are usually limited or degraded, so the stability and sensitivity of rhythmic biomarkers need to be further investigated.

Impinging blood droplets on different wettable surfaces: Impact phenomena, contact line motion, post-impact oscillation and dried stains.

Understanding the underlying hydrodynamics of impinging blood droplets and finding out the physical parameters determining the bloodstain characteristics are of great importance in blood related forensic investigations. In this work, the impact of non-Newtonian blood droplets on solid surfaces ranging from lyophilic to superlyophobic was systematically investigated and compared to that of Newtonian droplets with a similar dynamic shear viscosity. We show that impinging blood droplets behave as low-viscosity Newtonian droplets in the short-time spreading, which is dominated by capillary and inertial forces, but their non-Newtonian viscoelasticity would notably affect the droplet retraction and post-impact oscillation occurring in large timescales. Whereas the strong liquid-solid adhesion and the non-Newtonian elongational viscosity hinder droplet recoiling and thus alter the impact phenomena on lyophobic and superlyophobic surfaces, the shear and elongational viscosities are coupled to result in higher damping coefficients of oscillating blood droplets after deposition, in comparison to that of impinging Newtonian droplets. The size of the dried bloodstain was found to be different from both the maximum spreading radius of the droplet that can reach during impact and the final radius of the deposited droplet after oscillation, and their correlations are highly dependent on the impact velocity and surface wettability. Moreover, the morphologic characteristics of the bloodstains would also be changed by varying either the impact velocity or the surface wettability. We envision that these findings can not only find applications in the bloodstain pattern analysis, but also provide useful information for medical diagnosis based on blood droplet test.

Bloodstain pattern analysis & Bayes: A case report.

The findings from a bloodstain pattern analysis (BPA) may assist in formulating or falsifying scenarios that are considered in the investigative stages of a criminal investigation. When a case proceeds to trial the bloodstain pattern expert may be asked about the relevance of their findings given scenarios that are proposed by the prosecution and defense counsel. Such opinions provided by an expert are highly relevant to police investigation or legal proceedings, but the reasoning behind the opinion or implicit assumptions made by the expert may not be transparent. A proper framework for the evaluation of forensic findings has been developed since the late twentieth century, based on the hierarchy of propositions, Bayesian reasoning and a model for case assessment and interpretation. This framework, when implemented in casework, mitigates some of the risks of cognitive biases, and makes the reasoning and scientific basis for the opinion transparent. This framework is broadly used across forensic science disciplines. In this paper we describe its application to the field of BPA using a case example from the Netherlands Forensic Institute (NFI).

Breaking with trends in forensic dating: A likelihood ratio-based comparison approach.

Further steps toward understanding the time-related information contained within bloodstains found at the crime scene are rightly considered a top priority in forensic science. Contrary to widely held assumptions, the reason for the delayed exploitation of bloodstains dating methods in practice is not the lack of suitable analytical techniques for monitoring degradation processes. The problem lies in the variability of the environmental and circumstantial conditions, playing a vital role in the degradation kinetics of blood deposits. The present article demonstrates the possibility of breaking with current approaches based on absolute age estimations to finally answer time-centered questions in real forensic scenarios. The proposed novel framework for situating forensic traces in time is based on the likelihood ratio assessment of the (dis)similarity between the evidence decomposition and sets of reference materials obtained through supervised aging. In such a strategy, every dating procedure is constructed on a case-by-case basis to fit examined blood traces, thereby limiting the adverse influence of external factors on the validity of age estimations and providing a way for future crime scene implementation.

Development of a novel forensic age estimation strategy for aged blood samples by combining piRNA and miRNA markers.

In forensic investigations, age estimation is vital for determining whether a suspect is under or over the legally defined adult age. With breakthroughs in RNA sequencing technology, small noncoding RNAs have provided new ways to solve problems related to the age estimation of trace or aged samples, owing to their small molecular weight and better stability. In our previous study, we had applied miRNAs for the age estimation of bloodstains; however, further improvement of the existing model is needed. PIWI-interacting RNAs (PiRNAs), which are 24-32 nt noncoding small RNA molecules involved in the PIWI-piRNA pathway, play an important role in the aging process. In this study, we explored the possibility of simultaneously analyzing piRNAs and miRNAs for better age estimation purpose. Through massively parallel sequencing, five age-related piRNAs were identified in blood samples that had been stored for eight years. Further real-time PCR analysis revealed that two piRNAs (piR-000753 and piR-020548) showed relatively higher efficiency in age estimation. Additionally, two age-related miRNAs (miR-324-3p and miR-330-5p) were used to build the estimation model. Among all algorithms tested, gradient boosting showed the lowest mean absolute error (MAE) and root mean square error (RMSE) values (3.171 and 4.403 years, respectively) for the validation dataset (n = 110). The errors of the model were less than 5 years and 10 years for 81.82% and 96.36% of the samples, respectively. The results suggest that the combined use of piRNA and miRNA markers may increase the accuracy of age estimation, and our new model has great potential for application in forensic casework.

Hyperspectral imaging in forensic science: An overview of major application areas.

Analysis of evidence is a challenge. Crime scene materials are complex, diverse, sometimes of an unknown nature. Forensic science provides the most critical applications for their examination. Chemical tests, analytical methods, and techniques to process the evidence must be carefully selected by the forensic scientist. Ideally, it may be interpreted, analyzed, and judged in the original context of the crime scene. In this sense, hyperspectral imaging (HSI) has been employed as an analytical tool that maintains the integrity of the samples/objects for multiple and sequential analysis and for counter-proof exams. This paper is an overview of forensic science trends for the application of HSI techniques in the last ten years (2011-2021). The examination of documents was the main area of exploration, followed by bloodstain analysis aging process; trace analysis of explosives and gunshot residue. Chemometric tools were also addressed since they are crucial to obtain the most important information from the samples. There are great challenges in applying HSI in forensic science, but there have been clear technological and scientific advances, and a solid foundation has been built for the use of HSI in real-life cases.