BloodNet: An attention-based deep network for accurate, efficient, and costless bloodstain time since deposition inference.

The time since deposition (TSD) of a bloodstain, i.e., the time of a bloodstain formation is an essential piece of biological evidence in crime scene investigation. The practical usage of some existing microscopic methods (e.g., spectroscopy or RNA analysis technology) is limited, as their performance strongly relies on high-end instrumentation and/or rigorous laboratory conditions. This paper presents a practically applicable deep learning-based method (i.e., BloodNet) for efficient, accurate, and costless TSD inference from a macroscopic view, i.e., by using easily accessible bloodstain photos. To this end, we established a benchmark database containing around 50,000 photos of bloodstains with varying TSDs. Capitalizing on such a large-scale database, BloodNet adopted attention mechanisms to learn from relatively high-resolution input images the localized fine-grained feature representations that were highly discriminative between different TSD periods. Also, the visual analysis of the learned deep networks based on the Smooth Grad-CAM tool demonstrated that our BloodNet can stably capture the unique local patterns of bloodstains with specific TSDs, suggesting the efficacy of the utilized attention mechanism in learning fine-grained representations for TSD inference. As a paired study for BloodNet, we further conducted a microscopic analysis using Raman spectroscopic data and a machine learning method based on Bayesian optimization. Although the experimental results show that such a new microscopic-level approach outperformed the state-of-the-art by a large margin, its inference accuracy is significantly lower than BloodNet, which further justifies the efficacy of deep learning techniques in the challenging task of bloodstain TSD inference. Our code is publically accessible via https://github.com/shenxiaochenn/BloodNet. Our datasets and pre-trained models can be freely accessed via https://figshare.com/articles/dataset/21291825.

Evaluation of reverse transcription yield of RNA standards and forensic samples based on droplet digital PCR.

RNA analysis has shown great value in forensic science, such as body fluids and tissue identification, postmortem interval estimation, biological age prediction, etc. Currently, most RNA follow-up experiments involve reverse transcription (RT) procedures. It has been shown that the RT step is variable and has a greater impact on subsequent data analysis, especially for forensic trace samples. However, the pattern of variation between different RNA template inputs and complementary DNA (cDNA) yield is unclear. In this study, a series of 2-fold gradient dilutions of RNA standards (1 µg/µL - 0.24 ng/µL) and forensic samples (including blood samples, saliva samples, bloodstains, and saliva stains) were reverse-transcribed using EasyQuick RT MasterMix. The obtained cDNA was quantified by droplet digital PCR (ddPCR) to assess the RT yield of the ACTB gene. The results showed that the 125 ng RNA template had the highest RT yield in a 10 µL RT reaction system with the selected kit. For all stain samples, the RT yield improved as the amount of RNA template input increased since RNA quantities were below 125 ng. As many commercialized reverse transcription kits using different kinds of enzymes are available for forensic RNA research, we recommend that systematic experiments should be performed in advance to determine the amount of RNA input at the optimum RT yield when using any kit for reverse transcription experiments.

A novel three-step DNA extraction method for mixed bloodstains.

Mixed DNA samples from at least two contributors can be present at a crime scene, which could be the most crucial piece of genetic evidence. The mixed stains in sexual assault cases are typically separated using differential lysis procedures (a two-step method). Blood mixed stains, however, are usually difficult to separate. In this work, we propose that a mixed stain comprises three layers, that is, (1) the upper layer which is primarily made up of cells from one contributor; (2) the middle layer which is a similar mixture from two contributors; and (3) the lower layer which primarily comprises cells from the other contributor. Based on this concept, a novel three-step DNA extraction method was proposed to solve the challenge involving bloodstains from two contributors. In the experiment, we extracted three layers DNA from mixed bloodstains using three steps. As a result, single-source DNA and approximate single-source DNA were detected from steps 1 and 3, respectively. This study demonstrates that the DNA from some mixed blood stains could be effectively separated following an appropriate extraction strategy, providing valuable insights, and serving as a reference for future examination of blood mixtures.

Further experiments and remarks regarding the possible formation of blood stains on the Turin Shroud: stains attributed to the nailing of the hands.

The formation of red discolorations ('blood stains') on the Turin Shroud (TS), a Christian relic believed to be the burial cloth of Jesus of Nazareth, is controversially discussed. We performed experiments to identify possible explanations for the formation of the stains on the hands and forearms of the Turin Shroud Man (TSM). In preliminary non-standardised experiments, after applying blood to the dorsal and palmar side of the probands' wrists, they moved their arms around at their own discretion to provoke blood flows as similar as possible to those on the TS. A blood stain pattern similar to that on the left wrist could be provoked by slowly turning the wrist to the ulnar side. In contrast, a branched pattern of multiple streaks, as depicted on the forearms, was difficult to reproduce. In a standardised test setup, the probands moved their dry, dirtied, or oiled arms jerkily in a predetermined sequence of movements. More body hair only slightly facilitated the formation of a branched pattern. On oiled skin, however, the formation of branches was significantly facilitated. This may support the hypothesis that the blood stains on the forearms were formed by moving the body between the unnailing and the burial. The formation of a branched pattern seems feasible if the arms were moved jerkily and were possibly exposed to water and oils postmortem (e.g. transporting the washed and oiled body). Nevertheless, the well-defined blood stains with multiple branchings are difficult to explain. Additionally, the blood stains on the forearms may have originated from deep scourging wounds, where dried blood was again mobilised by water (and oil). We are aware that no reliable conclusions about the formation of the 'blood stains' on the TS can be drawn from our findings. However, they may contribute to the discussion on this topic.

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.

New alginate-gelatine method for casting of staining inside firearm barrels.

Contact shots to the head often leave behind biological traces inside firearm barrels, a phenomenon of great forensic interest. Until now, the visualization and preservation of these traces presented a significant challenge, lacking a reliable method. This study addresses this gap by searching for a suitable method to extract the traces within a casting. Using alginate or gelatine as suitable materials, the results were hampered by serious adhesion issues and their extraction out of the firearm barrel was impeded. Finally, the combination of 11% gelatine with 1% alginate, introduced into the barrel around a 'central spine', succeeded to consistently produce replicable castings. Experimental contact shots displayed a distinct staining gradient from the muzzle to the rear of the barrel, as revealed through endoscopy and proved in the macroscopic casting. The technique proved effective for various common handgun barrels and successfully preserved blood and gunshot residue (GSR) patterns within the barrel. This method offers the dual benefits of visually mapping staining patterns and securing localized samples for targeted molecular genetic analysis in forensic investigations.

Longitudinal Analysis of the Microbial Community on the Surface of Bloodstains Under Different Environmental Conditions in Areas with Minimal Human Interference.

The association between human metabolites and the environmental microbiome has primarily been investigated in relation to disease. In this study, the associations between environmental conditions and microbial communities on the surface of bloodstains were analyzed from a forensic science approach. The composition of microbial communities can be affected by numerous variables. After exposing bloodstains to two different environments with limited airflow and human interference, the microbial communities of the bloodstain surfaces were subjected to longitudinal analysis. Various microbes showed increasing or decreasing trends at the phylum and species level. The microbes identified in this study are usually found in soil, freshwater, and seawater and are known to exhibit unique properties, such as sporulation. Longitudinal variation in temperature and humidity were associated with various changes and correlations with the blood surface microbial community. Understanding these changes could introduce a new perspective to forensic science and could be used to develop a forensic tool used at crime scenes to analyze blood stains in more detail.

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.

The Role of Molecular Investigations in Estimating the Time since Deposition (TSD) of Bloodstains: A Systematic Review of the Literature.

At many crime scenes, investigators are able to trace and find traces of blood. For many years, it was believed that such traces could only be subjected to genetic investigations, such as those aimed at comparing DNA profiling with a suspect to verify his identity, and that it was therefore not possible to backdate the traces. In recent years, various works have used experimental models to investigate the possibility of identifying markers and methodologies for estimating the time since deposition (TSD) of bloodstains. Despite the results, these methods are still not part of standard procedures, and there is no univocal analysis methodology. In this work we carried out a systematic literature review of all the papers published in the last ten years on this topic, comparing the experimental models created. This review demonstrates the potential that different molecular approaches, such as transcriptomics, metabolomics, proteomics, and spectrometry, can have in the analysis of TSD, with notable sensitivity and specificity. This paper also analyzes the intrinsic and extrinsic limits of these models and emphasizes the need to continue research work on this topic, considering the importance that this parameter can assume in forensic investigations against a suspect.

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.

Alginate/xanthan gum hydrogels as forensic blood substitutes for bloodstain formation and analysis.

Understanding the behaviour of human blood outside of the body has important implications in forensic research, especially related to bloodstain pattern analysis (BPA). The design of forensic blood substitutes (FBSs) can provide many advantages, including the incorporation of multiple physiological components for use as safe and reliable materials for forensic applications. In this work, we present the design of synthetic alginate and xanthan gum-based hydrogels that contain electrosprayed microparticles (MPs) with and without crosslinked DNA. In addition to the MPs, the alginate/xanthan gum FBS materials include fillers to alter the physical appearance and fluid properties of the material. The optimized FBS consisted of alginate (1% w/v) and xanthan gum (5.0 × 10-3% w/v), 2 mM CaCl2, ferric citrate (0.5% w/v), magnesium silicate (0.25% w/v), Allura Red dye (2% w/v), 0.025% v/v Tween 20 and 9.5% v/v MPs. The FBS was tested in passive dripping experiments relevant to BPA scenarios at various impact angles. The spreading ratio (Ds/D0) was determined for 90° stains made on a paper surface and compared to bovine blood where the FBS was shown to simulate accurate and predictable spreading behaviour. In addition, we simulated other common BPA scenarios (e.g., impact patterns) and evidence processing potential. The FBS could be swabbed, and the DNA could be extracted, amplified, and genotyped analogous to human blood evidence. A stability test was also conducted which revealed a shelf-life of over 4 weeks where the material remains relevant to human blood at physiological temperature.

Identity of the numerous bloodstains at the murder scene: molecular identification of fly artifacts and fly species by CO1 analysis.

Crime scenes may contain insect artifacts as well as samples of human origin. While the presence of insects can be important evidence in forensic medicine and forensic entomology, the insect artifacts sometimes interfere with the interpretation of bloodstain pattern analysis (BPA) which can be critical for accurate crime reconstruction. Fly artifacts are especially complicated to distinguish from true bloodstains. Indeed, we encountered a murder scene with numerous bloodstains inconsistent with the cause of death and had trouble interpreting them. The morphological method has been developed to distinguish them, but this method has to rely on the analyst's experience and opinion. This study aims not only to distinguish fly artifacts from true bloodstains but also to identify fly species by detecting fly DNA in small amounts of bloodstains at the scenes. Melt curve analysis of real-time PCR (qPCR) targeting cytochrome c oxidase subunit 1 (CO1) of mitochondrial DNA (mtDNA) was able to detect fly DNA in bloodstains from a murder scene. The fly DNA was sequenced from the qPCR product, and the fly species were identified by BLAST search. Fluorescence-labeled specific primers for four species of necrophagous flies were designed based on the sequences of the CO1 region, and differences in the length of the amplification products were used to identify fly species from trace amounts of fly DNA in the artifacts.

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.

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.

Effect of environmental conditions on bloodstain metabolite analysis.

Establishing a correlation between environmental variables and chemical change can significantly improve the quality of research in multiple fields. Among various environmental variables, temperature and humidity are closely related to the rate of chemical reactions. This study aimed to confirm changes in metabolite markers that were previously discovered in other temperature and humidity environment conditions and to confirm the possibility that they could act as markers. After blood collection from the subjects and bloodstain preparation, the quantitative values of the bloodstain metabolites were confirmed (when the age of the bloodstain was within a month) under eight environmental conditions (4 °C/30%, 4 °C/60%, 25 °C/30%, 25 °C/60%, 25 °C/90%, 40 °C/30%, 40 °C/60%, and 40 °C/90%). Age-of-bloodstain estimation models were constructed to confirm the applicability of bloodstain metabolites as markers for bloodstain age in various environments. The average concentration of metabolite markers exhibited a decreasing trend with the age of the bloodstain, which transformed into an increasing trend from day 7 onwards. In terms of temperature and humidity, 25 °C and 90%, respectively, showed the most dissimilar metabolite change pattern compared to other conditions. The age-of-bloodstain estimation models developed here have an R-square value of up to 0.92 for each condition and an R-square value of 0.71 when all environmental conditions were combined. The findings herein highlight the immense potential of blood metabolites for field application, confirming the possibility of predicting metabolite changes from the rates of their chemical reactions and validating the importance of metabolites as age-of-bloodstain markers under various environmental conditions.

Proteomic Difference Analysis of Whole Blood and Bloodstains.

OBJECTIVES: To study the changes of protein levels in peripheral blood after it dried. METHODS: The proteins from whole blood and bloodstains were detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and normalized by the label-free quantification (LFQ) method. The differential proteins were analyzed by using R 4.2.1 software, limma and edgeR package. The analysis of biological function, signaling pathway and subcellular localization for the differential proteins was then performed. RESULTS: A total of 623 and 596 proteins were detected in whole blood and bloodstains, respectively, of which 31 were statistically significant in the quantitative results, including 10 up-regulated and 21 down-regulated proteins in bloodstains. CONCLUSIONS: The protein abundances in whole blood and bloodstains are highly correlated, and the variation of protein abundances may be related to the changes of endogenous and structural proteins in cells. The application of proteomics technology can assist the screening and identification of protein biomarkers, thereby introducing new biomarkers for forensic research.

[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.

Determination of the Time since Deposition of Blood Traces Utilizing a Liquid Chromatography-Mass Spectrometry-Based Proteomics Approach.

Knowledge about when a bloodstain was deposited at a crime scene can be of critical value in forensic investigation. A donor of a genetically identified bloodstain could be linked to a suspected time frame and the crime scene itself. Determination of the time since deposition (TsD) has been extensively studied before but has yet to reach maturity. We therefore conducted a proof-of-principle study to study time- and storage-dependent changes of the proteomes of dried blood stains. A bottom-up proteomics approach was employed, and high-resolution liquid-chromatography-mass-spectrometry (HR-LC-MS) and data-independent acquisition (DIA) were used to analyze samples aged over a 2 month period and two different storage conditions. In multivariate analysis, samples showed distinct clustering according to their TsD in both principal component analysis (PCA) and in partial least square discriminant analysis (PLS DA). The storage condition alters sample aging and yields different separation-driving peptides in hierarchical clustering and in TsD marker peptide selection. Certain peptides and amino acid modifications were identified and further assessed for their applicability in assessing passed TsD. A prediction model based on data resampling (Jackknife) was applied, and prediction values for selected peptide ratios were created. Depending on storage conditions and actual sample age, mean prediction performances ranges in between 70 and 130% for the majority of peptides and time points. This places this study as a first in investigating LC-MS based bottom-up proteomics approaches for TsD determination.

Bloodstain Metabolite Markers: Discovery and Validation for Estimating Age of Bloodstain within 7 Days.

Metabolomic research using analytical chemistry methods has been carried out in a wide range of research fields. However, research combining forensic science and metabolomics is rare. Determining the age of bloodstains could provide key information regarding when a crime was committed. Currently, validated methods for estimating the age of bloodstains are unavailable. Metabolites are intermediate and final products of chemical reactions. Therefore, they are less likely to be degraded than other components of blood under field conditions. In this study, metabolites in bloodstains were analyzed using liquid chromatography-mass spectrometry to discover and validate metabolic markers for determining the age of bloodstains within a week post-bleeding. Nontargeted analysis of bloodstain metabolites revealed statistically significant differences over time. Quantitative analysis of identified candidates via multiple reaction monitoring confirmed the statistical significance according to the age of bloodstain. Pyroglutamic acid, l-glutamine, acetylcarnitine, and adenosine 5'-monophosphate were selected as the final markers. The content of each marker exhibited a statistically significant and consistent tendency to decrease with the age of bloodstain. Furthermore, the effect of hemolysis was considered according to the blood fraction spots of the four markers. This study is the first to identify and validate metabolite markers that may help determine the age of bloodstains within a week post-bleeding. If applied to crime scenes as indicators of the age of bloodstains, they can be used as innovative and important tools for reconstructing crime scenes, suggesting initial investigative direction. This study highlights the forensic utility of blood metabolites ex vivo.