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In this work, we explore the application of likelihood ratio as a forensic evidence assessment tool to evaluate the causal mechanism of a bloodstain pattern. It is assumed that there are two competing hypotheses regarding the cause of a bloodstain pattern. The bloodstain patterns are represented as a collection of ellipses with each ellipse characterized by its location, size and orientation. Quantitative measures and features are derived to summarize key aspects of the patterns. A bivariate Gaussian model is chosen to estimate the distribution of features under a given hypothesis and thus approximate the likelihood of a pattern. Published data with 59 impact patterns and 55 gunshot patterns is used to train and evaluate the model. Results demonstrate the feasibility of the likelihood ratio approach for bloodstain pattern analysis. The results also hint at some of the challenges that need to be addressed for future use of the likelihood ratio approach for bloodstain pattern analysis.

Hyperspectral imaging (HSI) collects and processes information from the entire electromagnetic spectrum to obtain the spectrum of each pixel in the image of a scene, with the aim of finding objects and identifying materials. It is a non-contact, non-destructive technology that can be used without modifying or altering the analysed target. Forensic analysis and crime scene investigations are two of the most investigated fields of application, being able to detect and analyse many types of evidence. In this paper we analysed the most commonly reported forensic science applications. The literature indicates that the fields in which HSI appears most promising are the analysis of blood traces, document forgery, gunshot residues and the identification of fingerprints. [ABSTRACT FROM AUTHOR]

Insect artifacts produced by necrophagous adult flies over a variety of depositional surfaces are problematic in crime scene investigations. Assessment of bloodstains through morphological features combined with contextual and presumptive chemical analysis, as traditional practices, are often inconclusive. Absorbance of bloodstains and substantial wicking on fabrics due to underlying texture and repeated stitch loops in the fabric makes the diagnosis even more difficult than stain morphology. Literature related to the application of modern techniques in the diagnosis of fly artifacts was critically reviewed and presented along with working efficacies and challenges. Apart from the traditional morphological comparison of bloodstains, findings on immunoassay, scanning electron microscopy and DNA-based molecular discrimination have been considered more confirmatory differential diagnosis in crime scenes. Scanning electron microscopic methods reveal distinctive features of small crystal-like deposits with fly artifacts absent in bloodstains. The immunodetection method tested positive with antiserum (anti-md3) against defecatory and regurgitate stains. Moreover, mitochondrial DNA cytochrome c oxidase I (CO1) gene-based molecular diagnosis hold promises in discrimination. However, these modern techniques may be applied along with traditional methods to overcome confusion as per suitability of sampling, following conservative and non-conservative approaches that may offer a real help to crime scene investigators.

In the recent past, hyper-spectral imaging has found widespread application in forensic science, performing both geometric characterization of biological traces and trace classification by exploiting their spectral emission. Methods proposed in the literature for blood stain analysis have been shown to be effectively limited to collaborative surfaces. This proves to be restrictive in real-case scenarios. The problem of the substrate material and color is then still an open issue for blood stain analysis. This paper presents a novel method for blood spectra correction when contaminated by the influence of the substrate, exploiting a neural network-based approach. Blood stains hyper-spectral images deposited on 12 different substrates for 12 days at regular intervals were acquired via a hyper-spectral camera. The data collected were used to train and test the developed neural network model. Starting from the spectra of a blood stain deposited in a generic substrate, the algorithm at first recognizes whether it is blood or not, then allows to obtain the spectra that the same blood stain, at the same time, would have on a reference white substrate with a mean absolute percentage error of 1.11%. Uncertainty analysis has also been performed by comparing the ground truth reflectance spectra with the predicted ones by the neural model. [ABSTRACT FROM AUTHOR]

The larval excretions/secretions (ES) of blowflies contain proteolytic enzymes and bacteria that assist with tissue breakdown. Decomposition fluid (DF) contains organic and inorganic waste products from cell death. This study investigated if human DNA recovery from blood was impacted by exposure to ES and DF over time. Lucilia sericata ES were collected daily from 50 larvae, and all available DF was collected from two fetal piglets left to decompose for 2 weeks. Daily for 3-5 days, 28 μL-30 μL of ES, DF, or a 1:1 mixture of the fluids was added to 30 μL of blood on cotton. Three bloodstains per treatment were sampled every 12 h up to 3 days and at 1 and 2 weeks after initial addition of fluid. No PCR inhibition was detected, but DNA degradation increased over time, primarily in samples exposed to ES and ES/DF mixtures. The amount of DNA recovered decreased over time, but generally more DNA was recovered from DF samples than other samples. Full profiles, or partial profiles suitable for routine database searching (14-39 alleles), were generated from all DF and ES samples and at least one mixture sample at all timepoints. Partial profiles of between 1 and 13 alleles were obtained from all other mixture samples, except one mixture sample which generated no profile. These findings indicate bloodstain evidence recovered from maggot-infested and/or decomposing bodies may generate forensically useful DNA evidence and should be analyzed as quickly as possible after collection or stored appropriately to prevent further degradation., (© 2024 The Author(s). Journal of Forensic Sciences published by Wiley Periodicals LLC on behalf of American Academy of Forensic Sciences.)

Mitochondrial DNA (mtDNA) is an important genetic marker for degraded biological sample identification, maternal pedigree tracing, and population genetic structure study owing to its characteristics of high copy number, anti-degradable ring structure, and maternal inheritance. Whole mtDNA genome sequencing is an optimal method for the analysis of mtDNA polymorphism and heterogeneity because it allows for the comprehensive use of maternal genetic information. However, because of lacking quantitative evaluations for sequencing data, the scientific interpretation standards for mtDNA sequencing results of the previously used sequencing systems are often different, and false positive or false negative results are prone to occur when faced with the interference of nuclear genomic DNA, or the heterogeneities of mtDNA sequence and structure. In this study, we evaluated a novel mtDNA whole genome sequencing system using long fragment amplification strategy on the DNA nanoball (DNB) sequencing platform. This system demonstrated high sequencing quality and specific mtDNA sequencing efficiencies on positive control DNA and FTA bloodstain samples, as the average Q20 and Q30 values of the corresponding samples were 97.17 % and 91.93 %; 97.37 % and 92.48 %, respectively. The mean mapping percentages for the reference sequences of whole genome DNA (wgDNA), mtDNA, and nuclear genomic DNA (ngDNA) in the corresponding samples were 99.98 %, 99.97 %, 0.03 %, and 99.91 %, 99.40 %, 0.60 %; respectively. The average error calling rates for the bases A, C, G, and T of the whole mtDNA genome were 0.2519 %, 0.2550 %, 0.2906 %; and 0.2392 %, respectively. The efficacy of heteroplasmy identification was assessed using a set of theoretical sites with predetermined rates. These sites were created by combining the samples with known mtDNA haplotypes in certain proportions. The absolute errors between observed and theoretical heteroplasmy values were 89.59 %, 74.68 %, 50.20 %, 12.65 %, 8.31 %, and 4.85 %, while the theoretical heteroplasmy values were 5 %, 10 %, 20 %, 80 %, 90 %, and 95 %, respectively. The absolute error exhibited relative stability when the mtDNA sequencing depth exceeded 500×. Furthermore, the system sequencing efficiency was also confirmed among different kinds of samples, and these samples included natural samples (e.g., peripheral blood samples preserved on FTA cards for 2 and 11 years, and on filter paper for 6 and 9 years), degraded samples, sensitivity samples, samples derived from various bodily fluids, and maternal pedigree samples. In summary, the whole mtDNA genome sequencing system used for forensic identification demonstrated high performance in analyzing mtDNA sequence information, and showed significant prospects for forensic application and maternal genetic research., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Cases of cruelty can occur in wild animal, livestock, and pet animal. Cruelty to cats choose to be the background of this research because many cases of cruelty to cats have not been reported although the cases are still high., Aim: The aim of this research was to know how much dilution of cat blood can still be detected by Leucomalachite Green (LMG) and Takayama reagent., Methods: Three samples of domestic cat blood were diluted with ratios 1:10; 1:100; 1:1,000; 1:10,000; and 1:100,000. Bloodstain is made by dropping each blood dilution on the filter paper for the LMG test and on the object glass for the Takayama test. Bloodstain for the LMG test was done with ten repetitions from each sample and Duplo for the Takayama test. Positive results from the LMG test are presented as a bluish-green discoloration in stains. A positive result from the Takayama test is the formation of hemochromogen crystals under a microscope with 400× magnification., Results: Based on this research, cat bloodstain can be detected with LMG reagent until 1:40,000 dilution, while Takayama reagent only can detect cat bloodstain until 1:1,000., Conclusion: LMG and Takayama reagents are reagents that are often used in human blood spot testing. If there is a case of violence against cats and other animals, these two reagents can be relied on to help with the proof process., Competing Interests: The authors declare that there is no conflict of interest.

Fingermark impressions in blood are commonly encountered at violent crime scenes and represent a critical trace that can link an individual to the scene. A range of techniques are available for detecting and enhancing bloody impressions; however, many chemical methods involve using hazardous solvents or require alternative light sources to visualise fluorescence. This is particularly challenging for bloody impressions on dark substrates. An alternative treatment is the protein dye known as acid fuchsin (commonly known as 'Hungarian Red'), which can be visualised under both white light and fluorescence lighting. However, there is limited research available on this method, especially concerning its use in detecting bloody fingermarks on dark surfaces and its fluorescence qualities. To address these knowledge gaps, this study broadly aimed to evaluate the effectiveness of acid fuchsin for enhancing bloody fingermarks on a range of common substrates, along with comparing the performance of a formulation made from base components against a commercially-available Hungarian Red reagent. Through a multi-phased experimental approach, results supported an all-in-one treatment that contained 2 % SSA, 0.2 % acid fuchsin, and deionised water as the most effective. This formulation performed as well or better than commercial Hungarian Red, amido black and acid yellow in the validation trial. Enhanced impressions could be visualised under white light on light and dark surfaces, whilst 530 nm excitation provided improved detection via both fluorescence and absorption modes depending on substrate background interference. Moreover, the reagent was applied by spraying directly onto substrates placed at near-vertical angles, with no evidence of any fingermarks being affected by running or inadequate fixing. The ability to enhance and visualise bloody impressions on light and dark surfaces, under white light or excitation, using a single, water-based treatment is highly advantageous to operational crime scene examiners and forensic scientists., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The use of protein stains to enhance fingermark ridge detail in blood is a common technique used by forensic practitioners around the world. Amido Black is one of the most favoured protein stains due to its strong staining ability. The most common formulation of Amido Black is methanol based, with an ability to simultaneously fix and stain the blood impression, however methanol is toxic and can disrupt some surfaces, potentially compromising fingermark detail. If the surface is suspected of being a material that is impacted by methanol, there is an alternative aqueous formulation, which requires a fixative step to set the blood prior to staining so as not to wash away potential ridge detail. The multi-step process of aqueous protein stains is tedious and numerous studies have been conducted to improve the formula to achieve a combined fixing/staining solution that performs like the methanolic reagent. A combined fixative and stain formulation of aqueous based Amido Black was compared to a multi-step formulation with a separate sulfosalicylic acid fixative. Of the 243 split fingermark impressions analysed the majority (63.5 %) showed no preference to either treatment, with a marginally greater proportion of the remaining marks slightly favouring the combined fixative and stain formulation. Given that the new combined formulation performed broadly similarly to the existing multi step formulation, the potential time savings of this simpler approach may be beneficial to implement into operational use., Competing Interests: Declaration of Competing Interest None., (Crown Copyright © 2024. Published by Elsevier B.V. All rights reserved.)

Time elapsed since bloodstain deposition is a crucial aspect in forensic investigations, where non-destructive spectroscopic methods play a pivotal role. While extensive research has been conducted by UV-Vis spectroscopy, showcasing its utility in specific cases, there is still a paucity of studies based on NIR spectroscopy, which has the potential to overcome the limitations of the UV-Vis-based methods. To compensate for this disequilibrium, the present study aimed to evaluate the NIR applicability for estimating the age of forensic bloodstains and develop a performance comparison with UV-Vis spectroscopy methods. Capillary blood was sampled and subjected to a 16-day aging, during which it was repeatedly analyzed using both spectroscopic methods. Subsequently, chemometric analysis was applied to process the spectral data and independently assess the methods' performance. Classical preprocessing transforms (i.e., Savitzky-Golay derivatives and SNV transform) were used together with more targeted strategies, such as class centering, whose benefit was highlighted by PCA. Lastly, PLS regression models were computed to evaluate the effectiveness of both spectroscopic methods in estimating the time elapsed since blood trace deposition. Comparable root mean square errors in prediction (RMSEP) - 40 and 55 h for UV-Vis and NIR spectroscopy, respectively - were observed for both techniques, featuring an improvement with respect to the existing literature for NIR spectroscopy. Data fusion strategies for a multi-instrumental platform were also explored, evaluating advantages and disadvantages of low-level and mid-level approaches. The results indicated that NIR spectroscopy integrated with adequate chemometric strategies deserves increased appreciation in forensic bloodstain dating., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Previous literature has established that recovering heat damaged body fluids is possible, however with little investigation into the effect of accelerants used in initiating arson fires. This study therefore aimed to determine whether presumptive blood detection was affected by heat damage resulting from accelerant facilitated fires. Another objective was to examine various techniques for removing soot, which is a noted barrier to blood detection. The study focused on blood deposited on household flooring materials, one porous and one nonporous surface: carpet and tile respectively. Samples were burned with butane, petrol, and kerosene then presumptively tested using the Kastle Meyer colourimetric blood detection test. Testing was then repeated following soot removal by either wiping, scraping, or using liquid latex. The "strength" of positive detections was evaluated using a scale based on reaction speed and colour intensity. Results demonstrated that accelerants weakened detection strength, although nearly all samples tested positive overall, and the impact of each accelerant on both surface types was largely similar. It was also discovered that soot removal improved the strength of blood detection results in approximately 69% of carpet and 47% of tile samples, with wiping being the superior method on both surface types. Consequently, introducing this investigative step may be critical to maximizing blood evidence recovery in arson casework. These findings indicate the worth in recovering severely burned items, particularly for evidence as crucial as blood., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of Conflicting InterestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Biological trace samples consisting of very few cells pose a challenge to conventional forensic genetic DNA analysis. RNA may be an alternative to DNA when handling low template samples. Whereas each cell only contains two copies of an autosomal DNA segment, the transcriptome retains much of the genomic variation replicated in abundant RNA fragments. In this study, we describe the development of a prototype RNA-based SNP selection set for forensic human identification from low template samples (50 pg gDNA). Whole blood from a subset of the Danish population (41 individuals) and blood stains subjected to degradation at room temperature for up to two weeks were analysed by whole transcriptome shotgun sequencing. Concordance was determined by DNA genotyping with the Infinium Omni5-4 SNP chip. In the 100 protein-coding genes with the most reads, 5214 bi-allelic SNPs with gnomAD minor allele frequencies > 0.1 in the African/African American, East Asian, and (non-Finnish) European populations were identified. Of these, 24 SNPs in 21 genes passed screening in whole blood and degraded blood stains, with a resulting mean match probability of 4.5 ∙ 10 -9 . Additionally, ancestry informative SNPs and SNPs in genes useful for body fluid identification were identified in the transcriptome. Consequently, shotgun sequencing of RNA from low template samples may be used for a vast host of forensic genetics purposes, including simultaneous human and body fluid identification, leading to direct donor identification in the identified body fluid., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Abstract Background Among the physical evidence, bloodstain is one of the most common biological pieces of evidence at the crime scene, especially in violent crimes. Bloodstains are frequently seen at the crime scene and can be easily detected due to their color and structure. Because blood and bloodstains can potentially be evidence of a crime, offenders often tend to clean or wash them to get rid of relevant evidence. Some researchers think that washed bloodstains cannot obtain enough quality DNA for DNA profiling. However, some studies have shown that bloodstains on a piece of fabric can be used for DNA profiling even after washing. This study, it is aimed to determine whether a sufficient amount of DNA can be obtained for DNA profiling used for forensic purposes from blood-stained nylon and cotton fabrics washed at different temperatures such as 40 °C, 60 °C and 90 °C. Results In this study, bloodstains were created on cotton and nylon fabrics in a representative crime scene. After washing with laundry detergent at 40 °C, 60 °C and 90 °C was performed, studies were conducted in order to make bloodstains visible and obtain DNA for genetic profiling. The result is that more DNA is lost due to easy exposure to external factors in the non-absorbent nylon fabric type compared to the absorbent cotton fabric. Moreover, the rates of obtaining DNA from bloodstains on different types of fabrics washed at several temperatures are shown in this study. Conclusions It was determined that while a sufficient amount of DNA for the forensic genetic profiling can be obtained from cotton and nylon fabrics washed at 40 and 60 °C, a sufficient amount could not be obtained from the ones washed at 90 °C. And that shows even if bloodstained fabrics are washed at 60 °C, the fabrics still can be considered as physical evidence of the crime and can be associated with the scene, perpetrator and victim triangle.

To establish the time of death, it focuses on the detection and identification of body fluids at the crime scene, which /is a very important forensic model. There are many methods for estimating the time of death, which in medicine is called Livor Mortis. This paper is mainly focused on forensic medicine by determining the approximate time of death by collecting various bodily fluids. There are number of different approaches that affect the post-mortem appearance of the body. Here, as a first step in the investigation, photographs are taken to ascertain the change in skin color after death caused by the accumulation of blood. It has been noticed that skin color is due to predetermined hemoglobin and red chromospheres present in red blood cells called melanin. They are separated and grouped by an outstanding digital technology called 3D Filter Block Similarity Clustering (3-DFBSC), which is a very good approach for the proposed system where clustering is very easy. With these digital methods, cluster analysis plays an important role in the analysis of image matching methods with which it can establish the time of death with these digital methods. [ABSTRACT FROM AUTHOR]

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., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors have no relevant financial or non-financial interests to disclose., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Species and sex confirmation of the biological specimen play a crucial role in crime investigation. However, the specimen found in the scene is always trace quantity, which is hard to be analyzed by current methods. Moreover, the time-consuming DNA extraction, sophisticated apparatus, and complex data processing make it difficult to satisfy the demand of speediness and convenience for point-of-care tests. In this study, we first exhibit a phosphate-based visual system for field-based species and sex identification derived from trace bloodstain. By introducing phosphate ion-based colorimetry into loop-mediated isothermal amplification (LAMP) for result interpretation, not only the bloodstain can be directly submitted to mitochondrial variant amplification owing to the enhanced amplification efficiency by pyrophosphate ion hydrolyzation, but also the colorimetric signal can be recognized by the naked eye for result output within 30 min through molybdophosphate generation. Aerosol contamination, the major conflict of LAMP, has been solved once and for all by integrating uracil-DNA glycosylase into this system that still holds on a constant temperature. As a demonstration, cytochrome b and Y-chromosomal amelogenin are employed to identify species and sex respectively, which has achieved a highly sensitive and specific distinguishability under a strong interferential background. Accurate results can be obtained from both the simulative degraded and dated specimen, which indicates that this novel system may serve as a promising tool in forensic practice., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This study aimed to identify if biological material could be detected on the opposite side to deposition on fabric by commonly used presumptive and/or secondary tests. Additionally, this study aimed to ascertain if there is a difference in the DNA quantity and quality from samples obtained from both sides of the same substrate: cotton, polyester, denim, or combined viscose and polyester swatches. Blood, semen, or saliva (25 μL) was deposited on one side of 5 replicates of each fabric type and left for 24 h. Blood swatches were tested using Hemastix® and the ABACard® HemaTrace® immunoassay, semen swatches were tested using acid phosphatase (AP) reagent, the ABACard® p30® immunoassay and hematoxylin and eosin staining, and saliva swatches were tested using Phadebas® paper and the RSID-Saliva™ immunoassay. Both sides of each swatch were separately wet/dry swabbed and subjected to DNA analysis. Blood was able to be detected on the underside of all fabrics using both tests. Semen was able to be detected on the underside of swatches using the presumptive AP test but not p30®, and sperm was rarely observed. Saliva was able to be detected by RSID-Saliva™ but not Phadebas® paper when the underside of swatches were tested. Across all biological materials, DNA was able to be recovered from the top side of all 60 swatches. For the underside, DNA was able to be recovered from 54 swatches. Of the 6 swatches that DNA was unable to be recovered from, one sample was from semen and the rest were from saliva. This study has demonstrated that DNA and components of interest in forensically relevant biological material can be recovered from the opposite side to where it was originally deposited, and that observing biological material and/or DNA on one side of fabric does not definitively indicate direct deposition on that side., Competing Interests: Declaration of Competing Interest None., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

The aim of this study was to investigate the effect of sunlight on the degradation of DNA samples taken from blood stains from different types of surfaces. A blood sample obtained from a single male donor was placed on seven different surfaces (galvanized sheet, iron rod, newspaper, white printer paper, glass, soil, and ceramic panel). Samples were kept, during a 4-week summer period, in a room, but next to an open window. Every 7 days, 1 mm 2 of blood sample was collected from each substrate and stored in labeled tube for later analysis. DNA was extracted with the Chelex method, amplified using AmpFISTR TM Minifiler TM Plus Amplification Kit, and quantified using a Quantifiler TM Human DNA Quantification kit. After 7 days of sun exposure, the highest DNA concentration was determined to be from the sample from a galvanized sheet stain, followed by, in order of decreasing concentration, the ceramic panel, glass, newspaper, iron rod, and white printer paper surface. As expected, the DNA concentration from all samples decreased as the sunlight exposure time progressed. The results obtained after the amplification in the MiniFiler TM system were in correlation with the DNA concentrations measured by the qPCR method for all samples, except for the glass, soil, and white printer paper samples. The obtained data show that DNA degradation is correlated to the length of sunlight exposure and to the type of surface the samples are collected from. A negative qPCR result does not mean negative PCR amplification in the STR system; therefore, both methods should be applied when analyzing forensic samples collected from trace evidence.

Background: Bloodstain Pattern Analysis (BPA) is a forensic scientific discipline used to resolve criminal events. By studying the size, shape, and distribution of the bloodstains that constitute one or more bloodstain patterns, it is possible to determine the physical events responsible for their creation, as well as the positions and movements of the victim and, in cases of homicide, the perpetrator during the act., Materials and Methods: BPA analysis was applied as a support in the reconstruction of the event dynamics in four forensic cases, in addition to the data collected during on-site inspections and instrumental investigations including PMCT, autopsies, histological, and toxicological analyses. Particularly laborious was its application in a case involving a decomposed body. In all cases, a thorough photographic analysis of the bloodstains found on the clothing worn as well as in the areas surrounding the location of the corpse was conducted., Conclusions: The combination of investigations, together with the data derived from the application of BPA, allowed events to be attributed to: homicide by blunt force trauma; homicide-suicide using a bladed weapon; homicide using firearms; unplanned complex suicide. The analysis of the cases presented highlights the importance of a multidisciplinary approach through the use of additional instrumental and specialist investigations such as the study of bloodstains present at the crime scene for the reconstruction of criminal events.

Bloodstain age estimation is important in forensic science. Although several studies have used spectroscopy to estimate bloodstain ages, this method has not yet been practically applied due to the need for expensive equipment and low reproducibility. Thus, we aimed to develop a bloodstain age estimation model that can be easily performed using a spectrophotometric colorimeter. First, bloodstains were prepared by placing blood obtained from five healthy volunteers on a plastic plate. The bloodstains were kept on conditions with various brightness and temperatures. Then, each bloodstain was dissolved in saline every 24 h to a final concentration of 1%, measured with a spectrophotometric colorimeter, and subjected to machine learning to generate a random forest regression (RFR) model, and finally, the prediction accuracy of the bloodstain age was verified. We also elucidated the mechanism of the color changes utilizing aminoguanidine, which is an inhibitor of Maillard reaction. Finally, we measured the time-dependent color changes of the blood fluids obtained from healthy volunteers and examined if the method could be potentially applied to estimate postmortem interval (PMI). Our results showed that the RFR model estimated the bloodstain age with no substantial assessment, and it was applicable to bloodstains, regardless of the brightness or temperature. The color changes were affected by the addition of aminoguanidine. Furthermore, the method could be applied to blood fluids, suggesting its potential usefulness for PMI estimation. Considering its feasibility, the present method could potentially be introduced to practical forensic sciences in the near future., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

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., (© 2024 American Academy of Forensic Sciences.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

It is a common practice in forensic casework to use presumptive tests for blood stains before DNA extraction and testing. Stains are usually swabbed and then the swabs are sent for analysis. The Kastle-Meyer (KM) and Leucomalachite green (LMG) presumptive tests for blood are widely used, and their sensitivities have been thoroughly tested in the literature in solution and directly on stains, but not on swabbed stains to mimic casework. In this study, the sensitivity of the KM and LMG tests was tested on eight blood dilutions on cotton fabric and ceramic tile that were stained and subsequently swabbed. Both tests showed sensitivity up to 1:5000, which is slightly lower than reported values in solution or directly on stain but still highly effective in most cases. Stains were also cleaned with common agents, then swabbed and re-tested. Stained ceramic tiles cleaned with soap/water or bleach gave mixed positive and negative results for the 1:10 dilution, presumably due to variance in how thoroughly each investigator cleaned the stain, and other dilutions were undetectable after cleaning. The LMG test gave false positives for bleach cleaned stains, due to reagent reactivity with bleach. Surprisingly, blood was detectible up to the 1:100 dilution with both tests on stained cotton fabric that was cleaned in a washing machine with detergent and dried. Ultimately the KM and LMG presumptive tests remain effective tools for swabbed blood stains, and their practicality for cleaned stains is dependent on material containing the stain, cleaning agent and processing., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Blood is the most encountered type of biological evidence in violent crimes and contains pertinent information to a forensic investigation. The false presumption that blood encountered at a crime scene is human may not be realised until after costly and sample-consuming tests are performed. To address the question of blood origin, the novel application of visible-near infrared hyperspectral imaging (HSI) is used for the detection and discrimination of human and animal bloodstains. The HSI system used is a portable, non-contact, non-destructive method for the determination of blood origin. A support vector machine (SVM) binary classifier was trained for the discrimination of bloodstains of human (n = 20) and five animal species: pig (n = 20), mouse (n = 16), rat (n = 5), rabbit (n = 5), and cow (n = 20). On an independent test set, the SVM model achieved accuracy, precision, sensitivity, and specificity values of 96, 97, 95, and 96%, respectively. Segmented images of bloodstains aged over a period of two months were produced, allowing for the clear visualisation of the discrimination of human and animal bloodstains. The inclusion of such a system in a forensic investigation workflow not only removes ambiguity surrounding blood origin, but can potentially be used in tandem with HSI bloodstain age determination methods for rapid on-scene forensic analysis., (© 2023. The Author(s).)

In the recent past, hyper-spectral imaging has found widespread application in forensic science, performing both geometric characterization of biological traces and trace classification by exploiting their spectral emission. Methods proposed in the literature for blood stain analysis have been shown to be effectively limited to collaborative surfaces. This proves to be restrictive in real-case scenarios. The problem of the substrate material and color is then still an open issue for blood stain analysis. This paper presents a novel method for blood spectra correction when contaminated by the influence of the substrate, exploiting a neural network-based approach. Blood stains hyper-spectral images deposited on 12 different substrates for 12 days at regular intervals were acquired via a hyper-spectral camera. The data collected were used to train and test the developed neural network model. Starting from the spectra of a blood stain deposited in a generic substrate, the algorithm at first recognizes whether it is blood or not, then allows to obtain the spectra that the same blood stain, at the same time, would have on a reference white substrate with a mean absolute percentage error of 1.11%. Uncertainty analysis has also been performed by comparing the ground truth reflectance spectra with the predicted ones by the neural model.

Background: Among the physical evidence, bloodstain is one of the most common biological pieces of evidence at the crime scene, especially in violent crimes. Bloodstains are frequently seen at the crime scene and can be easily detected due to their color and structure. Because blood and bloodstains can potentially be evidence of a crime, offenders often tend to clean or wash them to get rid of relevant evidence. Some researchers think that washed bloodstains cannot obtain enough quality DNA for DNA profiling. However, some studies have shown that bloodstains on a piece of fabric can be used for DNA profiling even after washing. This study, it is aimed to determine whether a sufficient amount of DNA can be obtained for DNA profiling used for forensic purposes from blood-stained nylon and cotton fabrics washed at different temperatures such as 40 °C, 60 °C and 90 °C. Results: In this study, bloodstains were created on cotton and nylon fabrics in a representative crime scene. After washing with laundry detergent at 40 °C, 60 °C and 90 °C was performed, studies were conducted in order to make bloodstains visible and obtain DNA for genetic profiling. The result is that more DNA is lost due to easy exposure to external factors in the non-absorbent nylon fabric type compared to the absorbent cotton fabric. Moreover, the rates of obtaining DNA from bloodstains on different types of fabrics washed at several temperatures are shown in this study. Conclusions: It was determined that while a sufficient amount of DNA for the forensic genetic profiling can be obtained from cotton and nylon fabrics washed at 40 and 60 °C, a sufficient amount could not be obtained from the ones washed at 90 °C. And that shows even if bloodstained fabrics are washed at 60 °C, the fabrics still can be considered as physical evidence of the crime and can be associated with the scene, perpetrator and victim triangle. [ABSTRACT FROM AUTHOR]

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.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

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., (© 2024 The Authors. Journal of Forensic Sciences published by Wiley Periodicals LLC on behalf of American Academy of Forensic Sciences.)

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., (© 2024 The Authors. Journal of Forensic Sciences published by Wiley Periodicals LLC on behalf of American Academy of Forensic Sciences.)

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 R 2 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

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., (© 2023 Wiley-VCH GmbH.)

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., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Objective To explore the effect of benzidine test and related reagents on DNA analysis of bloodstain. Methods A total of 970 bloodstain filter paper samples with 1 μL venous blood were collected, and 10 of them acted as control samples. After benzidine test and related reagent processing, DNA of 960 samples was extracted by Chelex-100 and silica bead methods and then multiplex amplified by AmpF?詛STRTM IdentifilerTM Plus PCR kits. The results of STR typing were compared between different groups. Results DNA were extracted immediately after benzidine test. Totally STR loci （3.80±1.34） were detected by silica bead method, while no STR loci were obtained by Chelex-100 method. Thirteen samples （21.7%） with whole STR typing results were obtained by drying after benzidine test, and the STR locus number （12.90±1.49） which obtained by silica bead method was much higher than by Chelex-100 method （4.70±1.96） （P

• Blood is a magical life force & its journey has been nothing short of a fascinating voyage, to say the least. • The author alleges the blood stains on the Shroud of Turin to be the possible blood of God's son. Further, literature proves that the said blood stains were shown to belong to an AB and MNS-positive individual, with halos surrounding the blood stains compatible with serum containing trace amounts of albumin and immunoglobulins. • Conceivably enough, the ongoing debate is fierce between the religious sect on one hand and the scientists on the other. [ABSTRACT FROM AUTHOR]

Objective To develop a device of trace bloodstains imaging and age analysis, so as to provide a non-destructive, simple and objective method for age estimation of bloodstains at the crime scene. Methods Based on the principle of digital imaging and color pattern analysis, the mobile terminal of the device was used to collect images of bloodstains of different ages. The time-dependent pattern of 6 parameters （R, G, B, C, Y, M） reflecting the changes of color of images of different ages was obtained by computer image analysis. A multiparameter comprehensive inference equation of bloodstains age was established and embedded into the device software to realize the intelligent inference of the bloodstains age. Then the capability and reliability of the device was verified. Results This integrated device of bloodstains imaging and age analysis could quickly collect bloodstains at the crime scene and automatically analyze and infer the age of bloodstains combined with related intelligence software. In the blind test, the detection accuracy of this device was 95% in both natural light airtight group and dark airtight group, and 80% in the natural light ventilation group. Conclusion The integrated device of trace bloodstains imaging and age analysis can be used in a simple manner, which provides a new objective method for bloodstains age estimation.

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.

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., Competing Interests: Declaration of Competing Interest The authors report no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)