Investigation into temporal changes in the human bloodstain lipidome.

Bloodstains are crucial pieces of physical evidences found at violent crime scenes, providing valuable information for reconstructing forensic cases. However, there is limited data on how bloodstain lipidomes change over time after deposition. Hence, we deployed a high-throughput high-performance liquid chromatography-mass spectrometry (HPLC-MS) approach to construct lipidomic atlases of bloodstains, whole blood, plasma, and blood cells from 15 healthy adults. A time-course analysis was also performed on bloodstains deposited for up to 6 months at room temperature (~ 25°C). The molecular levels of 60 out of 400 detected lipid species differed dramatically between bloodstain and whole blood samples, with major disturbances observed in membrane glycerophospholipids. More than half of these lipids were prevalent in the cellular and plasmic fractions; approximately 27% and 10% of the identified lipids were uniquely derived from blood cells and plasma, respectively. Furthermore, a subset of 65 temporally dynamic lipid species arose across the 6-month room-temperature deposition period, with decreased triacylglycerols (TAGs) and increased lysophosphatidylcholines (LPCs) as representatives, accounting for approximately 8% of the total investigated lipids. The instability of lipids increased linearly with time, with the most variability observed in the first 10 days. This study sheds light on the impact of air-drying bloodstains on blood components at room temperature and provides a list of potential bloodstain lipid markers for determining the age of bloodstains.

Forensic stability evaluation of selected miRNA and circRNA markers in human bloodstained samples exposed to different environmental conditions.

Recently, RNA markers have been used to identify tissue origins of different kinds of body fluids. Herein, circRNA and miRNA markers were carried out to examine the presence or absence of peripheral blood (PB) in bloodstained samples exposed to different external environmental conditions, which mimicked PB samples left at the crime scenes. PB samples were placed on sterile swabs and then exposed to different high temperatures (37°C, 55°C and 95°C) and ultraviolet light irradiation for 0 d, 0.5 d, 1 d, 3 d, and 7 d, ultra-low and low temperatures (-80°C, -20°C, and 4°C) for 30 d, 180 d and 365 d and different kinds of disinfectants. Total RNA was extracted from bloodstained samples under the above different conditions, and the expressions of target RNAs (including miR16-5p, miR451a, circ0000095, and two reference genes RNU6b and 18 S rRNA) were detected by the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method. Results showed that these selected RNA markers could be successfully measured at all observation points with their unique degradation rates, which exhibited relative stability in degraded bloodstained samples exposed to different environmental conditions. This study provides insights into the applications of these studied miRNA and circRNA markers in forensic science.

An automated method for the generation of bloodstain pattern metrics from images of blood spatter patterns.

An improved automated bloodstain pattern analysis method has been developed and validated, which utilises computer vision techniques to identify bloodstains on a plain background within a digital image. The method generates metrics relating to the individual stains as well as the overall pattern, including bloodstain pattern specific metrics such as the gamma angle, circularity, solidity, area of convergence, stain density and pattern linearity. This method provides an objective approach to the analysis of bloodstains and bloodstain patterns and can generate a wealth of quantitative data that is currently not obtainable using manual techniques or other image-based programs currently utilised in the discipline. This method will be useful to analysts and researchers investigating the application of quantitative methods to bloodstain pattern analysis.

Bloodstain classification methods: A critical review and a look to the future.

Classifying bloodstains is an essential part of Bloodstain Pattern Analysis. Various experts have developed methods. Each method considers the same basic bloodstain pattern types. These use either terminology based on the observable characteristics or the mechanistic cause of the bloodstain patterns as part of the classification process. This review paper considers ten classification methods from fourteen sources, which are used to classify bloodstain patterns. There are fundamental differences in how the patterns are classified, how differentiated the classification is, and whether the classification process uses clear, unambiguous criteria, and is susceptible to contextual bias. Experts have also reported issues with classifying bloodstains that have indistinguishable features. These differences expose key limitations with current classification methods: mechanistic terminology is too heavily relied on, and the classification process is susceptible to contextual bias. The development of an unambiguous classification method, based on directly observable characteristics within bloodstain patterns is recommended for future work.

Area of origin estimation from multiple arbitrarily oriented surfaces using marker-guided structure from motion.

Bloodstain pattern analysis plays a crucial role in forensic investigations. Projected patterns can offer valuable insights into the dynamics of crime scenes. In this paper, we propose and validate a novel approach that extends existing software, HemoVision, to analyze impact patterns that are distributed across multiple arbitrarily oriented surfaces. The proposed method integrates HemoVision's marker-based system with structure from motion (SfM) techniques to reconstruct the three-dimensional geometry of impact patterns using only two-dimensional photographs. Controlled experiments were used to validate the proposed approach, demonstrating robustness in reconstruction accuracy with median translation errors below 3 mm and median angular errors below 0.2°, irrespective of imaging device or image resolution. Comparing the estimated areas origin to their known ground truth, the proposed method achieved an average total error of 8.12 cm, with the primary source of error being the vertical dimension. Despite this, the overall error remains well within the ranges of error reported in prior work. This study demonstrates that HemoVision can be used to analyze complex impact patterns using only two-dimensional photographs, providing forensic experts with an efficient and accessible tool for investigating intricate crime scenes involving multi-surface impact patterns.

Raman Spectroscopy for the Time since Deposition Estimation of a Menstrual Bloodstain.

Forensic chemistry plays a crucial role in aiding law enforcement investigations by applying analytical techniques for the analysis of evidence. While bloodstains are frequently encountered at crime scenes, distinguishing between peripheral and menstrual bloodstains presents a challenge. This is due to their similar appearance post-drying. Raman spectroscopy has emerged as a promising technique capable of discriminating between the two types of bloodstains, offering invaluable probative information. Moreover, estimating the time since deposition (TSD) of bloodstains aids in crime scene reconstruction and prioritizing what evidence to collect. Despite extensive research focusing on TSD estimations, primarily in peripheral bloodstains, a crucial gap exists in determining the TSD of menstrual bloodstains. This study demonstrates how Raman spectroscopy effectively analyzes biological samples like menstrual blood, showing similar aging patterns to those of peripheral blood and provides proof-of-concept models for determining the TSD of menstrual blood. While this work shows promising results for creating a universal model for bloodstain age determination, further testing with more donors needs to be conducted before the implementation of this method into forensic practice.

Study on the methods of visualizing bloodstains after thermal exposure.

To establish the correlation between thermal conditions imposed on bloodstains and visualizing effect of enhancement techniques, infrared photography and four chemical enhancement reagents were used to visualize bloodstains following thermal exposure. A black tile was selected as the substrate to intensify the visualization challenge, with a Cone Calorimeter serving as the standardized heating source to control thermal conditions. Compared with standard photography, infrared photography is proven to be a valuable complement to chemical reagents, showing significant advantages in visualizing bloodstains after thermal exposure. However, it is worth noting that infrared image fell short of standard image when bloodstains displayed raised, embossed morphology or when bloodstains almost disappeared under specific conditions. The enhancement effectiveness was found to be strongly correlated with thermal conditions imposed on bloodstains, and the morphology evolution of bloodstains during heating affected the chemical enhancement effect additionally, especially when the bulge morphology was formed, and it was observed that reagents were more effective after removing the dense shell of the bulge. Among the four selected chemical enhancement reagents, fluorescein performed exceptionally well, maintaining its effectiveness even for bloodstains heated at 641°C for 10 min. TMB demonstrated its visualizing ability for bloodstains heated at 396°C for 5 min and heated at 310°C for 20 min. BLUESTAR® followed afterwards, while luminol performed worst. The correlation between thermal conditions imposed on bloodstains and the corresponding visualizing effectiveness of enhancement techniques provides important references for detecting bloodstains at fire scenes.

Identification of Bloodstains by Species Using Extreme Learning Machine and Hyperspectral Imaging Technology.

How to identify bloodstains and obtain some potential evidence is of great significance for solving criminal cases. First, the spectral data of different species of bloodstain samples (human blood and animal blood) were acquired by using a hyperspectral imager. Then, an extreme learning machine (ELM) algorithm was used to build the training models of different species of bloodstain samples. Meanwhile, two traditional support vector machine and random forest classification algorithms were also compared with the ELM algorithm. The prediction results showed that the precision, sensitivity, specificity, and F1 score of the ELM algorithm were the highest. This indicates that hyperspectral technology, together with an ELM algorithm, could identify bloodstain species rapidly, non-destructively, and accurately. It has provided a new technical reference for bloodstain detection and identification.

Determination of angle of impact and directionality of drip stains on various fabrics.

Bloodstain pattern analysis (BPA) on absorbent surfaces, such as fabrics, is far more complex compared to its application on smooth, hard, non-porous surfaces. Angle of impact and directionality are commonly interpreted from bloodstains but may be adversely affected by porous surfaces. In fact, there is a lack of evidence that traditional approaches to BPA are even applicable when blood impacts absorbent materials such as clothing and other fabrics. Hence, there is a critical need for research focusing on the validity and reliability of methods for bloodstain pattern analysis on textiles. Here, human blood drops were deposited on six different fabric types (cotton, satin polyester, rayon, blended polyester/spandex, blended nylon/spandex, and blended modal/polyester/spandex) at two known impact angles: 30° and 10°. Bloodstain morphology was found to be unique for each fabric. Calculated angles of impact for cotton and satin polyester were not statistically different from the known angle of impact while blended polyester/spandex, blended nylon/spandex, and blended modal/polyester/spandex significantly underestimated the known angle of impact. Even when stain morphology on fabric resembled those on a glass control, the angle of impact significantly underestimated the known. The ability to assign directionality based upon bloodstain morphology was dependent on the fabric type. These findings support the need for further research and the development of guidelines for bloodstain pattern interpretation on fabric materials.

Circadian metabolites for evaluating the timing of bloodstain deposition: A preliminary study.

Metabolites, as products of cellular metabolism, can provide a wealth of biological information and are less susceptible to degradation than other biomarkers due to their low molecular weight. Due to these properties, metabolites can be used as valuable biomarkers for forensic investigations. Knowing the timing of deposition of bloodstain could help to reconstruct crime scenes, draw conclusions about the time of the crime, and narrow down the circle of possible suspects. Previous studies have indicated that the concentration of some metabolites in blood is subject to circadian changes. However, the circadian metabolites of bloodstains have been still unclear. A total of sixty-four bloodstain samples were prepared under real conditions in three time categories (morning/noon (09:00 h ∼ 17:00 h), afternoon/evening (18:00 h ∼ 23:00 h) and night/early morning (24:00 h ∼ 08:00 h)). Fifty metabolites of bloodstains with significant differences were identified in the three time categories. Twenty-eight of these metabolites exhibited significant circadian changes. Finally, three independently contributing circadian metabolites were selected to build the logistic regression model, with an area under the curve of 0.91, 0.84 and 0.87 for the prediction of bloodstain deposition time in the morning/noon, afternoon/evening and night/early morning, respectively. The study indicated that circadian metabolites can be used for evaluating the timing of bloodstain deposition. This would provide a valuable perspective for analyzing the deposition time of biological traces in forensic investigations.

3D scanning a crime scene to enhance juror understanding of Bloodstain Pattern Analysis evidence.

There are numerous crime scene investigation applications of 3D scanning that have been previously documented. This paper documents the application of a 3D point cloud in the presentation of Bloodstain Pattern Analysis evidence to mock jurors. 150 mock jurors viewed a presentation of Bloodstain Pattern Analysis evidence from a murder trial in the UK. After viewing the evidence, the participants were tested on their knowledge of the evidence and repeated the test again 2 weeks later; to simulate criminal trial conditions; whereby there is a time lapse between the initial viewing of evidential material and deliberation. This paper found that the mock jurors who additionally viewed a 3D flythrough of a point cloud of the crime scene, better retained knowledge of the evidence over time, reported a greater ability to visualise the crime scene and had higher levels of interest in the evidence. Crucially, the 3D flythrough group did not report different levels of confidence in the accuracy of their memories of the evidence, nor different levels of emotional arousal to the group that viewed the evidence without the 3D presentation. Together, these findings suggest that 3D scanning of crime scenes, and the resultant point cloud's presentation to jurors, could add further value to the justice system when spatial information, such as Bloodstain Pattern Analysis evidence, is presented.

Estimating bloodstain age in the short term based on DNA fragment length using nanopore sequencer.

We used a nanopore sequencer to quantify DNA fragments > 10,000 bp in size and then evaluated their relationship with short-term bloodstain age. Moreover, DNA degradation was investigated after bloodstains were wetted once with water. Bloodstain samples on cotton gauze were stored at room temperature and low humidity for up to 6 months. Bloodstains stored for 1 day were wetted with nuclease-free water, allowed to dry, and stored at room temperature and low humidity for up to 1 week. The proportion of fragments > 20,000 bp in dry bloodstains tended to decrease over time, particularly for fragments > 50,000 bp in size. This trend was modeled using a power approximation curve, with the highest R2 value (0.6475) noted for fragments > 50,000 bp in size; lower values were recorded for shorter fragments. The proportion of longer fragments was significantly reduced in bloodstains that were dried after being wetted once, and there was significant difference in fragments > 50,000 bp between dry conditions and once-wetted. This result suggests that even temporary exposure to water causes significant DNA fragmentation, but not extensive degradation. Thus, bloodstains that appear fresh but have a low proportion of long DNA fragments may have been wetted previously. Our results indicate that evaluating the proportion of long DNA fragments yields information on both bloodstain age and the environment in which they were stored.

A study on the estimation of area of origin of swing cast-off pattern.

In bloodstain pattern analysis (BPA), a field of forensic science, there has been active discussion on the estimation of the area of origin of impact spatter. However, there is no established methodology to quantitatively analyze the area of origin of a swing cast-off pattern. To quantitatively analyze the methodology of previous research on estimation of area of origin, a device for generating uniform swing cast-off patterns was produced. Using artificial blood, 10 swing cast-off patterns were generated on porous paper; in each, 10 blood drops were selected for the calculation of the impact angle. Hemospat software was used for individual bloodstain analysis, and an open source code was used for estimation of area of origin. Under the same conditions, an additional 10 swing cast-off patterns were generated, and quantitative analysis was performed using trigonometric functions and an adjustment formula that minimized errors in calculating the impact angle. The adjustment formula was corrected to calculate the impact angle for the bloodstains on the porous surface. As uncertainty decreases, the error increases, and the point at which both uncertainty and error can be minimized is calculated as 75%. The existing formula included the trajectory in the estimated likelihood range in 75% of samples. When the adjustment formula was applied, the accuracy was improved, with the trajectory included in the area with a 90% likelihood.

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.

Accidental Cut-Throat Injury by a Broken Glass: Bloodstain Pattern Analyses and Autopsy Findings.

Injuries in the neck region are rarely observed in forensic practice, especially of accidental origin. Primarily, such cases are associated with homicide or suicide. The neck region comprises different and vital anatomical structures, and even minor trauma could be lethal. In the absence of witnesses to the accident, each finding is of utmost importance, from the death/crime scene investigation - bloodstain patterns and trace evidence - to careful examination of the deceased body. The forensic pathologist has the challenging task of analyzing all the findings to make a statement concerning the cause and manner of death and, if there is something suspicious about the current case, to inform the relevant authorities.

DNA degradation of bloodstains on cotton fabric caused by different washing procedures.

DNA degradation in biological material needs to be better understood. Bloodstains on washed clothing are disturbed by washing procedures, sometimes transferred to other fabrics, often with latent bloodstains and usually with significantly degraded DNA. The samples (cotton fabric with bloodstains) are divided into six main groups, depending on the washing method regarding water temperature (95, 60, and 30 °C) and the detergent use. After completing the washing process, samples were stored for a certain period (1 day to 6 months) and subsequently analyzed. Analyses were performed using standard protocols and commercial kits to measure the remaining DNA quantity (concentration) and DNA degradation index in the processed samples. Our results revealed that the high washing temperature (60 and 95 °C) and the application of detergent have a synergic action on DNA degradation, while at 30 °C this effect is absent. Furthermore, the effect of detergent on accelerated DNA degradation is observed about a month after the washing. This delayed effect of detergent has no explanation in current literature data. To obtain optimal results from the bloodstains, we recommended that the period from the crime event and attempted cleaning by a perpetrator to the laboratory analysis should be less than 1 month.

Differences of circular RNA expression profiles between monozygotic twins' blood, with the forensic application in bloodstain and saliva.

Monozygotic twins (MZTs) possess identical genomic DNA sequences and are usually indistinguishable through routine forensic DNA typing methods, which can be relevant in criminal and paternity cases. Recently, novel epigenetic methods involving DNA methylation and microRNA analysis have been introduced to differentiate MZTs. In this study, we explore the potential of using epigenetic markers, specifically circular RNAs (circRNAs), a type of non-coding RNA (ncRNA), to identify MZTs, and investigate the unique expression patterns of circRNAs within pairs of MZTs, enabling effective differentiation. Epigenetics regulates gene expression at the post-transcriptional level and plays a crucial role in cell growth and aging. CircRNAs, a recently characterized subclass of ncRNA, have a distinct covalent loop structure without the typical 5' cap or 3' tail. They have been reported to modulate various cellular processes and play roles in embryogenesis and eukaryotic development. To achieve this, we conducted a comprehensive circRNA sequencing analysis (circRNA-seq) using total RNA extracted from the blood samples of five pairs of MZTs. We identified a total of 15,257 circRNAs in all MZTs using circRNA-seq. Among them, 3, 21, 338, and 2967 differentially expressed circRNAs (DEcircRNAs) were shared among five, four, three, and two pairs of MZTs, respectively. Subsequently, we validated twelve selected DEcircRNAs using real-time quantitative polymerase chain reaction (RT-qPCR) assays, which included hsa_circ_0004724, hsa_circ_0054196, hsa_circ_004964, hsa_circ_0000591, hsa_circ_0005077, hsa_circ_0054853, hsa_circ_0054716, hsa_circ_0002302, hsa_circ_0004482, hsa_circ_0001103, novel_circ_0030288 and novel_circ_0056831. Among them, hsa_circ_0005077 and hsa_circ_0004482 exhibited the best performance, showing differences in 7 out of 10 pairs of MZTs. These twelve differentially expressed circRNAs also demonstrated strong discriminative power when tested on saliva samples from 10 pairs of MZTs. Notably, hsa_circ_0004724 displayed differential expression in 8 out of 10 pairs of MZTs in their saliva. Additionally, we evaluated the detection sensitivity, longitudinal temporal stability, and suitability for aged bloodstains of these twelve DEcircRNAs in forensic scenarios. Our findings highlight the potential of circRNAs as molecular markers for distinguishing MZTs, emphasizing their suitability for forensic application.

[Fatal injury to the neck while working with an angle grinder]. / Smertel'noe ranenie shei pri vypolnenii montazhnykh rabot s ispol'zovaniem uglovoi shlifoval'noi mashiny.

A fatal accident using an angle grinder is presented. During the forensic medical examination of the corpse, a soft tissue wound of the neck was found, localized in the region of its anterior and right lateral surfaces, with damage to the common carotid artery, internal jugular vein, sternocleidomastoid and sternothyroid muscles, thyroid lobe, incomplete transection of the trachea and esophagus caused by the rotating disc of an angle grinder. This observation supplements the existing ideas about injuries caused by power tools, as well as the possibility of reconstructing an event based on a comprehensive assessment of data from the scene.

Influence of the relative humidity on the blood drip stain formation on jeans fabrics.

The impact of whole human blood drops on jeans fabrics is studied as a function of the impact velocity U0, room relative humidity RH, and spacing S between the fabric and backing substrate. Experiments are performed with blood drops of the same initial diameter of D0 = 3 ± 0.08 mm and temperature of 37∘C. Whole human blood is collected from the same donor. The impact dynamics of blood drops is described as a function of U0 and S, and it is shown that the spacing has an effect on the splashing limit and the surface area of the drip stains. At RH = 30%, the drip stains (parent stains) after impact do not wick the fabrics. Moreover, the area of the drip stains increases with the impact velocity until a critical value (U0 = 3.3 ± 0.1 m∕s), where it becomes constant. A modified correlation is proposed to predict the drip stains area. At RH = 70%, the drip stains after impact wick the jeans fabrics. The area of the drip stains after impact is dependent on the impact velocity while the final area after wicking is not. Further, the contribution of the wicking, to the formation of the drip stains, decreases with the impact velocity. These findings show the importance of taking into account RH in future research work and in the analysis of the drip stains at crime scenes.