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Contemporary criminal investigations are based on the statements made by the victim and the eyewitnesses. They also rely on the physical evidences found in the crime scene. These evidences, and more particularly biological ones, have a great judicial value in the courtroom. They are usually used to revoke the suspect's allegations and confirm or refute the statements made by the victim and the witnesses. Stains of body fluids are biological evidences highly sought by forensic investigators. In many criminal cases, the success of the investigation relies on the correct identification and classification of these stains. Therefore, the adoption of reliable and accurate forensic analytical methods seems to be of vital importance to attain this objective. Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) is a modern and universal analytical technique capable of fingerprint recognition of the analyte using minimal amount of the test sample. The current systematic review aims to through light on the fundamentals of this technique and to illustrate its wide range of applications in forensic investigations. ATR-FTIR is a nondestructive technique which has demonstrated an exceptional efficiency in detecting, identifying and discriminating between stains of various types of body fluids usually encountered in crime scenes. The ATR-FTIR spectral data generated from bloodstains can be used to deduce a wealth of information related to the donor species, age, gender, and race. These data can also be exploited to discriminate between stains of different types of bloods including menstrual and peripheral bloods. In addition, ATR-FTIR has a great utility in the postmortem investigations. More particularly, in estimating the postmortem interval and diagnosing death caused by extreme weather conditions. It is also useful in diagnosing some ambiguous death causes such as fatal anaphylactic shock and diabetic ketoacidosis.

In this paper, a conventional camera modified to capture multispectral images, has been used to locate latent forensic traces with a smart combination of wavelength filters, capturing angle, and illumination sources. There are commercial multispectral capture devices adapted to the specific tasks of the police, but due to their high cost and operation not well adapted to the field work in a crime scene, they are not currently used by forensic units. In our work, we have used a digital SLR camera modified to obtain a nominal sensitivity beyond the visible spectrum. The goal is to obtain forensic evidences from a crime scene using the multispectral camera by an expert in the field knowing which wavelength filters and correct illumination sources should be used, making visible latent evidences hidden from the human-eye. In this paper, we show a procedure to retrieve from latent forensic traces, showing the validity of the system in different real cases (blood stains, hidden/erased tattoos, unlocking patterns on mobile devices). This work opens the possibility of applying multispectral inspections in the forensic field specially for operational units for the location of latent through non-invasive optical procedures.

One of the first challenges that crime scene examiners have is determining if a substance is blood before performing analysis. Conventional methods of detecting blood involve the use of chemicals and different wavelengths of light in tandem with digital photography. However, these methods are destructive or provide false positives. Visible wavelength hyperspectral imaging (HSI) is a noncontact blood detection method that has been proven to provide accurate and reliable results. A novel application of this technique has been used for the detection and positive identification of bloodstained footwear marks, of different dilutions ranging from undiluted to 1:50 with distilled water, and on a range of substrates, and colors. Comparisons between HSI and conventional digital photography were made using a grading scale and analyzed using Mann-Whitney U-tests. The HSI technique was able to detect a statistically significant greater amount of tread detail on white tiles, laminate, carpet, and blue tiles compared with the digital photography technique, which was only superior on black tiles. Critically, the HSI technique was also able to determine that the footwear marks were made in blood. These results show that HSI will be useful in forensic investigations, where it is known that the perpetrator has walked through the victim's blood and left a trail of footwear marks at the crime scene. Even if the perpetrator had time to clean up afterward resulting in diluted stains, HSI would still be able to detect bloodstained footwear marks with a greater amount of detail compared with digital photography.

June of 1950 found the US forces poorly prepared to stop North Korean forces rolling into South Korea. First encounters scattered American soldiers and presented unique challenges for care of the casualties; battalion doctors and medics hustled the wounded along, sometimes themselves trapped, captured, or killed. Finally, within the Pusan perimeter American and South Korean resistance stiffened. It was in this defensive position the first Mobile Army Surgical Hospitals (MASH) were deployed far forward-heretofore simply a paper concept. MASH units performed magnificently, resuscitating and evacuating gravely wounded American and South Korean casualties, ushering in a new dimension to combat casualty care.

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 ellipses 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.

The most important task in a criminal investigation is to detect and identify the recovered biological stains beyond reasonable scientific doubt and preserve the sample for further DNA analysis. In the light of this fact, many presumptive and confirmatory tests are routinely employed in the forensic laboratories to determine the type of body fluid. However, the currently used techniques are specific to one type of body fluid and hence it cannot be utilized to differentiate multiple body fluids. Moreover, these tests consume the samples in due process, and thus it becomes a great limitation especially considering the fact that samples are recovered in minute quantity in forensic cases. Therefore, such limitations necessitate the use of non-destructive techniques that can be applied simultaneously to all types of bodily fluids and allow sample preservation for further analysis. In the current work, attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy has been used to circumvent the aforementioned limitations. The important factors which could influence the detection of blood such as the effect of substrates, washing/chemical treatment, ageing, and dilution limits on the analysis of blood have been analysed. In addition, blood discrimination from non-blood substance (biological and non-biological in nature) has also been studied. Chemometric technique that is PCA–LDA has been used to discriminate blood from other body fluids and it resulted in 100% accurate classification. Furthermore, blood and non-blood substances including fake blood have also been classified into separate clusters with a 100% accuracy, sensitivity, and specificity. All-inclusive, this preliminary study substantiates the potential application of ATR-FTIR spectroscopy for the non-destructive identification of blood traces in simulated forensic casework conditions with 0% rate of false classification.

Latent blood fingerprints (LBFPs) can provide critical information of foul play and help identify the suspects at violent crime scenes. The current methods for LBFP visualization are still not satisfactory because of the low sensitivity or complicated protocol. This study demonstrates a simple and effective LBFP visualization strategy by integrating a new amphiphilic fluorescent amino-functionalized conjugated polymer with the cotton-pad developing protocol. LBFPs on various substrates are visualized by simply covering them with the polymer solution-soaked cotton pads. The images display clear fingerprint patterns, ridge details, and sweat pores, even on very challenging substrates such as painted wood and multicolored can. The gray value analysis confirms semiquantitatively the enhancement of the contrast between ridges and furrows. Even LBFPs with various contaminations or aged for more than 600 days are effectively developed and visualized. The developed fingerprint images show superior stability over long storage time and against solvent washing. Moreover, the polymer causes no degradation of DNAs in the blood, suggesting the possibility of further DNA profiling and identification after development. The mechanistic investigation suggests that the formation of positive or inverted images can be attributed to the synergistic effects from the affinity between polymer and blood, and the affinity betwen polymer and substrate, as well as the slight quenching of polymer fluorescence by blood. Furthermore, the covalent bonding between the protonated primary amino group and proteins in blood endows the stability of the developed fingerprints. The result rationalizes the molecular design of the fluorescent polymer and sheds new light on the future strategies to effective LBFP visualization in practical applications.

Body fluid traces can provide highly valuable clues in forensic investigations. In particular, bloodstains are a common occurrence in criminal investigation, and the discrimination of menstrual and peripheral blood is a crucial step for casework involving rape and sexual assault. Most of the current protocols require the detection of characteristic menstrual blood components using sophisticated procedures that need to be performed in a laboratory. The present study uses attenuated total reflection Fourier transform-infrared (ATR FT-IR) spectroscopy as a nondestructive technique for discriminating menstrual and peripheral blood traces. This method incorporates statistical analysis and was evaluated by internal and external validation testing. A partial least squares discriminant analysis (PLSDA) classification model was created for differentiating the two types of blood in a binary manner. Excellent separation between menstrual and peripheral blood samples was achieved during internal validation. External validation resulted in 100% accuracy for predicting a sample as peripheral or menstrual blood. This study demonstrates that ATR FT-IR spectroscopy combined with chemometrics is a reliable approach for rapid and nondestructive discrimination of menstrual and peripheral bloodstains. It offers a significant advantage to forensic science due to the availability of portable instruments and the potential for bloodstain analysis at a crime scene. Graphical abstract.

The atomic force microscope (AFM) has found its way to the arsenal of tools available to the forensic practitioner for the analysis of samples at the nano and microscales. As a non-destructive probing tool that requires minimal sample preparation, the AFM is very attractive, particularly in the case of minimal or precious sample. To date, the use of the AFM has primarily been in the arena of imaging where it has been complementary to other microscopic examination tools. Forensic applications in the visual examination of evidence such as blood stains, questioned documents, and hair samples have been reported. While a number of reviews have focused on the use of AFM as an imaging tool for forensic analyses, here we not only discuss these works, but also point to a versatile enhancement in the capabilities of this nanoscale tool - namely its use for force spectroscopy. In this mode, the AFM can determine elastic moduli, adhesion forces, energy dissipation, and the interaction forces between cognate ligands, that can be spatially mapped to provide a unique spatial visualization of properties. Our goals in this review are to provide a context for this capability of the AFM, explain its workings, cover some exemplary works pertaining to forensic sciences, and present a critical analysis on the advantages and disadvantages of this modality. Equipped with this high-resolution tool, imaging and biophysical analysis by the AFM can provide a unique complement to other tools available to the researcher for the analysis and characterization of forensic evidence.

Two men were wrongfully convicted of murder in 2017 and sentenced to life imprisonment. After a physical altercation inside a flat, the victim (A) was found dead approximately 60 m away outside a residential address. He had sustained a number of injuries including a stab wound to the left side of his neck which was found to have divided the right carotid artery. The location where A was found was not regarded as a crime scene and not subjected to a specialist forensic examination by scientists as it was assumed that the fatal injury was sustained in the flat. The pathologist, who subsequently carried out the autopsy on A, was not asked to attend the scene. A review of the blood distribution at the scene in conjunction with the pathology findings indicated however that the fatal neck wound had been inflicted outside the flat, near to where the victim was found. An appeal against the convictions for murder was upheld in 2021 and a re-trial ordered. Following this second trial, both accused were acquitted of murder and released from custody. The new pathology and blood pattern evidence introduced at the second trial was a major part of the defense strategy which led to the acquittal of the accused. The case illustrates that a more inclusive and detailed crime scene strategy had been undertaken, including an assessment of the bloodstains present, in conjunction with discussion with the pathologist, then the likelihood is that the two men subsequently charged with murder would have been eliminated as suspects and a miscarriage of justice would have been avoided.

The physicochemical changes occurring in biomolecules in degrading bloodstains can be used to approximate the time since deposition (TSD) of bloodstains. This would provide forensic scientists with critical information regarding the timeline of the events involving bloodshed. Our study aims to quantify the timewise degradation trends and temperature dependence found in total RNA from bloodstains without the use of amplification, expanding the scope of the RNA TSD research which has traditionally targeted mRNA and miRNA. Bovine blood with ACD-A anticoagulant was deposited and stored in plastic microcentrifuge tubes at 21 or 4°C and tested over different timepoints spanning 1 week. Total RNA was extracted from each sample and analyzed using automated high sensitivity gel electrophoresis. Nine RNA metrics were visually assessed and quantified using linear and mixed models. The RNA Integrity Number equivalent (RINe) and DV200 were not influenced by the addition of anticoagulant and demonstrated strong negative trends over time. The RINe model fit was high (R

The chemical profiling of bloodstains is essential to link the suspect with the crime. The current study proposed a proof-of-concept methodology for the investigation of bloodstains by utilizing advanced ATR-FTIR spectroscopy coupled with new generation chemometric methods. Current study provides encouraging data to allow discrimination between human and animal blood though with small sample size. In this study, different models for the age estimation of human bloodstains are developed from the trained data sets of 1–175 days old bloodstains. The models such as curve estimation (CE), multiple linear regression (MLR), and partial least squares regressions (PLSR) are developed to determine the best prediction model for aged human bloodstains. The obtained results on the dating of bloodstains are very encouraging and also tested for unknown samples. The maximum dating errors are observed in the curve estimation models whereas, the other models MLR, PLSR show excellent age estimation of unknown bloodstains. These models represent an error of ~3 ± 1 days and ~4 ± 1 days in actual and estimated date, respectively, which is lowest ever reported so far. The present methodology is expected to provide a valuable insight into forensic society and hence, to the law enforcement community. The present methodology can further be explored for an ideal model by including all other external variables/factors and for more longer aging time.

Establishing the precise timeline of a crime can be challenging as current analytical techniques used suffer from many limitations and are destructive to the body fluids encountered at crime scenes. Raman spectroscopy has demonstrated excellent potential in forensic science as it provides direct information about the structural and molecular changes without the need for processing or extracting samples. However, its current applicability is limited to pure body fluids, as signals from the substrate underlying these fluids greatly influence the current models used for age estimation. In this study, we utilized Raman spectroscopy to identify selective spectral markers that delineate the bloodstain age in the presence of interfering signals from the substrate. The pure bloodstains and the bloodstains on the substrate were aged for two weeks at 21 ± 2 ℃ in the dark. Least absolute shrinkage and selection operator (LASSO) regression was employed to guide the feature selection in the presence of interference from substrates to accurately predict the bloodstain age. Substrate-specific regression models guided by an automated feature selection algorithm yielded low values of predictive root mean square error (0.207, 0.204, 0.222 h in logarithmic scale) and high R2 (0.924, 0.926, 0.913) on test data consisting of blood spectra on floor tile, facial tissue, and linoleum-polymer substrates, respectively. This framework for an automated feature selection algorithm relies entirely on pure bloodstain spectra to train substrate-specific models for estimating the age of composite (blood on substrate) spectra. The model can thus be easily applied to any new composite spectra and is highly scalable to new environments. This study demonstrates that Raman spectroscopy coupled with LASSO could serve as a reliable and nondestructive technique to determine the age of bloodstains on any surface while aiding forensic investigations in real-world scenarios.

A wet‐vacuum‐based collection method with the M‐Vac® was compared to a wet‐swabbing collection method by examining the recovery of diluted blood on 22 substrates of varying porosity. The wet‐vacuum method yielded more total nuclear DNA than wet‐swabbing on 18 porous substrates, recovering on average 12 times more DNA. However, both methods yielded comparable amounts of total DNA on two porous and two nonporous substrates. In no instance did wet‐swabbing significantly recover more DNA. The wet‐vacuum method also successfully collected additional DNA on previously swabbed substrates. Mitochondrial DNA yields were assessed, and outcomes were generally similar to the nuclear DNA outcomes described above. Results demonstrate that wet‐vacuuming may serve as an alternative collection method to swabbing on difficult porous substrates and could potentially recover additional DNA on previously swabbed substrates. However, swabbing remains the preferred collection method on substrates with visible stains and/or nonporous surfaces for reasons of convenience, simplicity, and lower cost relative to the wet‐vacuum method.

DNA analysts in forensic laboratories are engaged in analysing and sampling bloodstains from bloodstained items. Detailed and precise descriptions of bloodstains on items of interest are very important for bloodstain pattern analysis (BPA). DNA and BPA reports were examined from forensic laboratories in Serbia ( N = 88). About 400 reports were observed from the past three years. First, we analysed descriptions of items (clothing and shoes) in DNA reports, and special attention was paid to descriptions of bloodstains. Subsequently, we estimated the value of descriptions of bloodstained items of interest in linking specific types of bloodstains to the obtained DNA profiles. Observed descriptions of bloodstained items in DNA reports are usually limited to phrases. A major problem exists in cases where several people were injured in the same bloodshed event. Connecting specific types of bloodstains to obtained DNA profiles is essential for the reconstruction of crime events. The complete analysis should therefore include detailed descriptions of all types of observed and sampled bloodstains. In DNA laboratories that are within a larger institute, it would be more appropriate and productive if BPA and DNA experts examined bloodstained items cooperatively. Moderately sized laboratories have a limited number of employees. So, in those DNA laboratories, it would be more appropriate to educate DNA analysts in the basic principles of BPA.

The use of alternative light sources (ALS) in bloodstain analysis has focused on dried (whole) blood, while information on detection of blood serum is lacking. Serum detection by ALS could provide valuable information at a crime scene, as serum may become separated from blood during clotting and cast off, especially in cases where the victim is moved. Additionally, a perpetrator may concentrate on the removal/scouring of dried blood with small amounts of serum going unnoticed, as it dries relatively clear on certain objects. In this report, the detection of human blood serum was evaluated using ultraviolet (UV) light at two different wavelengths. These results show that ultraviolet (UV) at 365 nm (UV365) was effective in the detection of even small amounts of blood plasma and serum, compared with UV at 395 nm, which was not. UV365 was also found to be useful in distinguishing blood imprints from clotting blood which had been transferred to material versus blood that had been added directly. Taken together, these results demonstrate that UV365 may be utilized as a simple, nondestructive method for blood serum detection.

The forensics discipline of bloodstain pattern analysis plays an important role in crime scene analysis and reconstruction. One reconstruction question is whether the blood has been spattered via gunshot or blunt impact such as beating or stabbing. This paper proposes an automated framework to classify bloodstain spatter patterns generated under controlled conditions into either gunshot or blunt impact classes. Classification is performed using machine learning. The study is performed with 94 blood spatter patterns which are available as public data sets, designs a set of features with possible relevance to classification, and uses the random forests method to rank the most useful features and perform classification. The study shows that classification accuracy decreases with the increasing distance between the target surface collecting the stains and the blood source. Based on the data set used in this study, the model achieves 99% accuracy in classifying spatter patterns at distances of 30 cm, 93% accuracy at distances of 60 cm, and 86% accuracy at distances of 120 cm. Results with 10 additional backspatter patterns also show that the presence of muzzle gases can reduce classification accuracy.

There is a recognized disconnect in priority and synergy between academic and practitioners in forensic science. In this work, we personally reflect on our experiences in conducting research studies that directly involve academic and practitioner stakeholders. We believe, amongst others, that this "gap" can be mitigated through regular and productive communication. We also emphasize the need to create stronger and national research strategies which identifies the current and pressing needs of enforcement officials in order to bring these needs directly to academia. As part of this, researchers should actively seek to make sure what they study will be relevant within the discipline. Our reflection is geared on direct feedback from an entomological study in large scale sampling of blowflies and workshops in bloodstain pattern analysis using a forensic blood substitute.

OBJECTIVES Our goal was to develop high throughput computer vision (CV) algorithms to detect blood stains in thoracoscopic surgery and to determine how the detected blood stains are associated with postoperative outcomes. METHODS Blood pixels in surgical videos were identified by CV algorithms trained with thousands of blood and non-blood pixels randomly selected and manually labelled. The proportion of blood pixels (PBP) was computed for key video frames to summarize the blood stain information during surgery. Statistical regression analyses were utilized to investigate the potential association between PBP and postoperative outcomes, including drainage volume, prolonged tube indwelling duration (≥5 days) and bleeding volume. RESULTS A total of 275 patients undergoing thoracoscopic lobectomy were enrolled. The sum of PBP after flushing (P < 0.022), age (P = 0.005), immediate postoperative air leakage (P < 0.001), surgical duration (P = 0.001) and intraoperative bleeding volume (P = 0.033) were significantly associated with drainage volume in multivariable linear regression analysis. After adjustment using binary logistic regression analysis, the sum of the PBP after flushing [P = 0.017, odds ratio 1.003, 95% confidence interval (CI) 1.000–1.005] and immediate postoperative air leakage (P < 0.001, odds ratio 4.616, 95% CI 1.964–10.847) were independent predictors of prolonged tube indwelling duration. In the multivariable linear regression analysis, surgical duration (P < 0.001) and the sum of the PBP of the surgery (P = 0.005) were significantly correlated with intraoperative bleeding volume. CONCLUSIONS This is the first study on the correlation between CV and postoperative outcomes in thoracoscopic surgery. CV algorithms can effectively detect from surgical videos information that has good prediction power for postoperative outcomes.

Bloodstains on fabrics may be washed or cleaned to eliminate incriminating evidence. These actions reduce the chances of obtaining an interpretable DNA profile. Previous studies have shown that conventional short-tandem repeat (STR) typing is affected by various factors associated with washing or laundering of stains. Here, we aim to increase the chances of obtaining interpretable STR profiles from laundered bloodstains using direct PCR. Preliminary investigations showed direct STR typing resulted in more alleles compared to conventional STR typing. We then further investigated the following factors with direct STR typing: fabric type (cotton, polyester, and denim), washing method (hand-washing and machine-washing), type of detergents (powder and liquid), washing temperature (cold to 90 °C), pretreatment agents (sodium hypochlorite and hydrogen peroxide), and the number of washes (one, three, and five). Direct PCR could be successfully used for STR typing from laundered bloodstains with very high success rates. Among the three fabric types, only denim negatively affected direct STR typing, while laundering of bloodstains on cotton and polyester had a negligible effect as mostly full profiles were obtained. Multiple washes resulted in a decrease in both the numbers of alleles and peak heights. Surprisingly, washing method, type of detergent, washing temperature, and pretreatment agents only had minimal to no effect on STR profile quality. Due to the robustness and sensitivity of direct STR typing from laundered bloodstains, the method could be beneficial for violent crime investigations in forensic DNA laboratories worldwide.

One of the most important types of evidence in certain criminal investigations is traces of human blood. For a detailed investigation, blood samples must be identified and collected at the crime scene. The present study aimed to evaluate the potential of the identification of human blood in stains deposited on different types of floor tiles (five types of ceramics and four types of porcelain tiles) using a portable NIR instrument. Hierarchical models were developed by combining multivariate analysis techniques capable of identifying traces of human blood (HB), animal blood (AB) and common false positives (CFP). The spectra of the dried stains were obtained using a portable MicroNIR spectrometer (Viavi). The hierarchical models used two decision rules, the first to separate CFP and the second to discriminate HB from AB. The first decision rule, used to separate the CFP, was based on the Q-Residual criterion considering a PCA model. For the second rule, used to discriminate HB and AB, the Q-Residual criterion were tested as obtained from a PCA model, a One-Class SIMCA model, and a PLS-DA model. The best results of sensitivity and specificity, both equal to 100%, were obtained when a PLS-DA model was employed as the second decision rule. The hierarchical classification models built for these same training sets using a PCA or SIMCA model also obtained excellent sensitivity results for HB classification, with values above 94% and 78% of specificity. No CFP samples were misclassified. Hierarchical models represent a significant advance as a methodology for the identification of human blood stains at crime scenes.

In forensic crime scene investigations, biological fluids such as blood are commonly found in soil. However, the analysis of blood-stained soil can be challenging due to the presence of inhibitors which limit the effective extraction and amplification of the deoxyribonucleic acid (DNA) required to produce a reportable DNA profile. There are some extraction methods that have been applied to blood-stained soil in forensic science, but these have produced sporadic results. This research has taken a number of different extraction methods from the fields of ancient DNA and environmental DNA and broken them down into the individual steps of pre-treatment, incubation, separation and purification. These steps were assessed independently then combined into various extraction methods to determine the best technique that can effectively and reliably profile human DNA from blood-stained soil. Testing involved assessment of three extraction buffers, (cetyltrimethylammonium bromide, guanidine thiocyanate, and proteinase K), four pre-treatment methods, (polyvinylpyrrolidone, ethylenediaminetetraacetic acid, hydrochloric acid, and sodium hydroxide), three separation steps, (centrifugation, phenol chloroform, and chloroform) and four purification steps, (size exclusion chromatography, bind elute columns, isopropanol precipitation and silica magnetic beads). The most effective procedure was found to be a polyvinylpyrrolidone pre-treatment with a proteinase K extraction buffer followed by magnetic silica bead purification with or without centrifugation. However, centrifugation separation was found to be equally effective after the pre-treatment step as after the incubation step. Our results shows that most of the current forensic procedures would benefit from the addition of a pre-treatment step prior to processing through the automated DNA profiling pipeline.

Insects found at a crime scene can produce traces referred to as fly artifacts (FA) due to their movement over the corpse and the manner in which they feed upon it. These can be detrimental for carrying out criminal investigations. Confusing a FA with a genuine bloodspot can lead to misinterpretations, also taking into consideration that FA may contain a human DNA profile. The aim of the present study was to employ scanning electron microscopy (SEM) for the analysis of FA produced by Calliphora vomitoria on hard surfaces and fabrics that are commonly present at crime scenes. FA and control bloodstains were produced under experimental conditions on metal, glass, plaster, cotton, and polyester. After macroscopic analysis, FA were examined at standard low (20–40 ×), medium low (300–600 ×), and high ultrastructural (1200 ×) magnification through a SEM Stereoscan 360, Leica, Cambridge. SEM analysis enabled the identification of distinctive features of FA on hard surfaces, namely, amorphous crystals, micro-crystals with a morphology similar to those of uric or micro-crystals with a comparable morphology to cholesterol, absent in controls. Moreover, red blood cells (RBC) were absent in FA but were always present in controls. On cotton, for both FA and controls, the drop was almost completely absorbed and thus indistinguishable from the underlying fabric texture. On polyester, FA showed amorphous/crystal-like deposits and no RBC, as observed on hard surfaces, except for those showing a completely flat surface. SEM analysis appeared to be suitable for differential diagnosis between FA and genuine bloodstains on hard surfaces, although the results may be inconclusive on tested fabrics.

Thrombus formation and infections caused by bacterial adhesion are the most common causes of failure in blood-contacting medical devices. Reducing the interaction of pathogens using repellent surfaces has proven to be a successful strategy in preventing device failure. However, designing scale-up methodologies to create large-scale repellent surfaces remains challenging. To address this need, we have created an all-polymeric lubricant-infused system using an industrially viable swelling-coagulation solvent (S-C) method. This induces hierarchically structured micro/nano features onto the surface, enabling improved lubricant infusion. Poly(3,3,3-trifluoropropylmethylsiloxane) (PTFS) was used as the lubricant of choice, a previously unexplored omniphobic nonvolatile silicone oil. This resulted in all-polymeric liquid-infused surfaces that are transparent and flexible with long-term stability. Repellent properties have been demonstrated using human whole blood and methicillin-resistant Staphylococcus aureus (MRSA) bacteria matrices, with lubricated surfaces showing 93% reduction in blood stains and 96.7% reduction in bacterial adherence. The developed material has the potential to prevent blood and pathogenic contamination for a range biomedical devices within healthcare settings., E.A. thanks Spanish Ministry of Science and Innovation for the FPI Grant (BES-2017-080057). T.F.D. acknowledges support from NSERC Discovery Grant, Ontario Early Researcher Award and McMaster Start-up funds to T.F.D. T.F.D and Z.H.D are Tier II Canada Research Chairs.

Bloodstains found at crime scenes represent a crucial source of information for investigative purposes. However, in forensic practice, no technique is currently used to estimate the time from deposition of bloodstains. This preliminary study focuses on the age estimation of bloodstains by exploiting the color variations over time due to the oxidation of the blood. For this purpose, we used a colorimetric methodology in order to easily obtain objective, univocal and reproducible results. We developed two bloodstain age prediction algorithms: a short-term and a long-term useful model for the first 24h and 60 days, respectively. Both models showed high levels of classification accuracy, particularly for the long-term model. Although a small-scale study, these results improve the potential application of colorimetric analysis in the time-line reconstruction of violent criminal events.

Blood is the primary material for quantitative toxicological analysis and interpretation of the results. However, in some cases like hit-and-run, it may be collected after a long time since the incident, resulting in the complete elimination or a significant decrease in the concentrations of potentially present drugs. If the suspect was injured and bloodstains were revealed, they can become the best or sometimes, the only material to be used to prove driving under the influence of drugs. In this context, the aim of this work was to develop a simple procedure that would allow for the estimation of drug concentrations in forensic bloodstains of unknown volume. In this work endogenous amino acids: valine and leucine were used to determine the unknown volume of blood from which the stain was formed. In order to isolate the analytes from bloodstains on different surfaces (plastic and cotton) the elution mixture consisted of an acetonitrile: water (60:40) and sodium chloride (0.9%) was used. The developed protocol was tested on 32 authentic forensic samples (mostly cases of people driving under the influence of amphetamine). The results of bloodstains analyses were compared with the results of whole blood analyses. The accuracy was in the range from -87.7 to +471.0%. However, for most of the cases, more accurate results were obtained. The differences between amphetamine values estimated from bloodstains and determined from whole blood were analysed using the Bland-Altman difference plots that revealed significant agreement. The variables concentrations estimated from bloodstains and concentrations calculated from whole blood were found to be strongly correlated. The Pearson correlation coefficients were: for plastic r(29) = 0.85 and for cotton r(25) = 0.91. This is the first work in which quantitative analyses of drugs in bloodstains have been attempted. A special protocol was developed for this purpose. The conducted research indicates that estimation of the concentration of drugs in bloodstains from the blood of an unknown volume is possible. The developed protocol has been extensively tested on authentic forensic samples and the obtained results were successfully compared with drug concentrations determined in whole blood. Nevertheless, the proposed method of estimating drug concentrations in bloodstains is characterized by many disadvantages, and the determined values should be treated with great caution, at best as estimates.

The estimation of bloodstain formation time is still an unsolved problem in forensic science and lacks accurate quantitative methods. Whether DNA can be adopted to estimate bloodstain formation time is still controversial, and there is no study to confirm the potential of mtDNA markers. To address these issues, a triple quantification method based on the ratio of mtDNA fragments of different lengths of COⅠ (mitochondrially encoded cytochrome c oxidase Ⅰ) for estimating bloodstain formation time was established. A total of 152 samples (140 old samples, 12 fresh samples) were collected and tested, and the absolute copies of different-sized fragments of COⅠ (304 bp, 120 bp, 41 bp) in all samples were quantified by SYBR Green real-time qPCR. The natural logarithms of two copy number ratios (304 bp/41 bp, 120 bp/41 bp) of COⅠ in old samples were calculated, which were used as degradation indexes to evaluate the degradation degree of mtDNA. The 140 old human blood samples from 1 to 14 years of storage were accumulated from casework of forensic practice to establish the method of estimating bloodstain formation time and used to analyze the impact of gender factors on the two degradation indexes, and 10 animal samples and 2 fresh human samples were collected to verify the human specificity of the method. There was a high correlation between degradation indexes and bloodstain formation time (the absolute values of correlation coefficients of these two degradation indexes were 0.901 and 0.758 respectively). A method with triple quantification and dual indexes estimating bloodstain formation timewas successfully established, which was highly human-specific. There was no statistically significant difference in degradation indexes between different gender samples (P 0.05). This study confirmed that mtDNA can be utilized to estimate bloodstain formation time, which provides a new solution to the forensic problem of estimating the time of bloodstain formation.

Research informed by practice is more likely to have a greater impact on society. However, forensic cases are confidential and thus, real-life data regarding the details of violent crime is usually withheld from the public and academia. Through a partnership between the authors institution and a UK police service, casefiles from 78 criminal investigations from 2012 to 2020 involving Bloodstain Pattern Analysis (BPA) were examined and quantified. The most common methods of assault and weapons used were identified as well as the frequency of different bloodstain pattern classifications. The results of this study will help inform researchers and supply forensic training providers with data derived from forensic practice. Despite a significant body of literature exploring impact patterns and software for calculating the Area of Origin (AO), impact pattern was classified at only 22% of scenes, with sharp-force trauma being the most prevalent form of assault. This paper recommends a review of the BPA terminology, to include additional commonly encountered patterns that are not defined by the current standard.

Objective Although the disease has been eliminated in Turkey malaria continues to be a threat due to increase in the number of people coming from or going to countries where the disease is endemic. In this study, we aimed to evaluate blood smears sent to the National Malaria Reference Laboratory within the malaria surveillance system. Methods From March 2016 to July 2018 a retrospective study was conducted to compare the results of Malaria Reference Laboratory and Public Health Laboratories. A total of 16.827 blood stains were sent to our laboratory for approval. Results In Public Health Laboratories, 315 (1.88%) of the smears were positive, 16.510 (98.12%) were negative, and in the National Malaria Reference Laboratory 252 (1.50%) were positive, 16.466 were negative. In the Public Health Laboratories, one of the two samples considered to be malaria suspected was positive in the National Malaria Reference Laboratory and one was negative. In Public Health Laboratories 35.88% of smears were P. falciparum, 27.30% were Plasmodium spp., 20.96% were P. vivax, 14.92% were mixed infection, 0.63% were P. malariae, 0.31% were P. ovale, and in the Reference Laboratory 49.60% were Plasmodium spp., 29.37% were P. falciparum, 16.27% were P. vivax, 4.36% were mixed infection, 0.40% were P. malariae. Conclusion In order to malaria surveillance system to be maintained in a healthy manner, preparation, staining, coding, packaging, transportation of blood slides is very important. Also if necessary, continuing training of laboratory staff working in malaria diagnosis is crucial.

Fabricating simple, accurate and user-friendly diagnostic device for “point of care testing” (POCT) applications is one of the most challenging objectives in the analytical field. Hemin detection is important for drugs monitoring, diagnosis, and forensic latent bloodstain imaging. Herein is developed, luminol chemiluminescence biosensor for hemin detection using artesunate as coreactant. A possible mechanism to account for the chemiluminescence reaction is discussed. Hemin was detected using both photomultiplier tube (PMT) and smartphone as detector. The detection limit for hemin using smartphone as detector is 20 nM, enabling the visual detection of hemin in blood sample with a dilution factor of blood up to 120,000. While PMT detector is used, the system is able to detect hemin down to 0.22 nM. In addition to high sensitivity, this sensing system exhibit high selectivity. It can successfully distinguish bloodstain from other stains while applying the system for point of care testing using smart phone as detector. Moreover, the system can detect artesunate with a linear range from 0.1 nM to 1.0 μM with a limit of detection of 0.078 nM.

Bloodstain pattern analysis (BPA) is an integral part of crime scene investigation. For violent crimes involving gunshots, standard practice in police departments worldwide have some physical limitations. For instance, the effect of gravity and air drag on trajectories of blood droplets are neglected using current reconstruction methods, which results in a well-known overestimation of the height of the source of blood. As a consequence, more sophisticated models for blood spatter trajectory reconstruction are being developed, two of which are highlighted in the present work. They allow the prediction of bloodstain patterns produced from backward spattered blood droplets from blunt and sharp bullets. Our recent models attribute the splashing of blood to the Rayleigh-Taylor instability which arises when blood is accelerated towards lighter air. This physically-based description comes with the powerful predictive capability to correlate features of bloodstain patterns with the specific bullet and gun that produced them, as well as with the body position. The results of the numerical models were compared with four experiments simulating blood spatter deposition on a vertical wall through the number of stains produced, average stain area, and average impact angle at the surface, and the agreement found is fairly good. Moreover, further insight is obtained by probing and explaining the influence of observable parameters on the resulting spatter pattern, with the goal of aiding BPA experts evaluating a crime scene.

Rapid DNA profiling of casework samples is a powerful tool that can support law enforcement agencies in the quick apprehension of perpetrators before they re-offend or escape the jurisdiction. This present study evaluated the feasibility of direct PCR amplification, using the microFLOQ™ Direct swab, for generating DNA profiles (from bloodstains) within 3 h. The swab tip is coated with nylon fibers pre-treated with cell lysing agent, which allows for the direct PCR amplification of collected samples without DNA extraction and quantification, thereby shortening the time required to obtain a DNA profile. Samples collected were directly amplified using GlobalFiler™ PCR Amplification Kit with and without the presence of a PCR additive. Addition of the PCR additive enhanced the peak heights of DNA profiles by approximately 2 fold. Hence, an additive could improve results obtained in the absence of a DNA purification step, especially since casework samples may contain PCR inhibitors. Subsequently, these swabs, amplified using the GlobalFiler™ PCR Amplification Kit with PCR additive, were evaluated on common substrates encountered in routine casework samples submitted with bloodstains, such as denim jeans, knife blade, tissue paper, leather belt, shirt, and blood swabs. The minimum peak heights observed were generally above the analytical and stochastic thresholds established by the laboratory. Finally, the microFLOQ™ Direct swab workflow was compared to the laboratory’s standard workflow of DNA profiling comprising of conventional processing steps such as extraction using the DNA-IQ™ chemistry on Maxwell® 16, followed by quantification, amplification and capillary electrophoresis. The average peak heights of the DNA profiles generated by direct PCR amplification were similar or exceeded those generated using the standard workflow. This study clearly demonstrates that direct PCR amplification using microFLOQ™ Direct swab can be used in a rapid workflow to obtain DNA profiles from casework samples.

Cocaine (COC) is one of the most widely abused drugs in the world and its sensitive and its reliable measurement in blood is of great importance in the field of forensic and clinical toxicology. Additionally, the determination of COC metabolites such as benzoylecgonine (BZE), cocaethylene (CE), ecgonine methyl ester (EME), and norcocaine (NCOC) are also of complementary diagnostic value. The quantification of COC and metabolites in dried blood spots (DBS) may be an alternative to conventional collection methods with several advantages, including easier, on-site, collection, transportation and storage. In this study, we present a simple and comprehensively validated UPLC–MS/MS assay to measured COC, BZE, EME, NCOC and CE in DBS. The evaluated assay was linear from 5–500 ng mL−1. Precision assays presented CV% of 1.27–6.82, and accuracy in the range of 97–113.78%. Low haematocrit values had a negative impact in the assay accuracy. COC, BE, NCOC and CE measurements can be made reliably in DBS stored for 14 days at room temperature, as well as at −20 °C and 45 °C. All evaluated compounds can be measured in DBS maintained at −20 °C for 14 days. DBS sampling can be used for the clinical evaluation of the exposure to COC, being an alternative for collection, short-term storage and transportation of blood at room and high temperatures.

In cases of crimes involving blood, the perpetrators often attempt to remove the traces they have left behind. Setting fire to the crime scene, aside from cleaning measures, seems to achieve this goal and presents a major challenge for crime scene investigators. There is only very little published information available on the effect of fire and extreme heat on blood and the detection thereof. After exposure to high temperatures of or exceeding 1.000 °C, blood is deemed to be undetectable. This study exposed 11 different potentially crime-relevant objects using a standardized and controlled procedure to temperatures of 300 °C, 700 °C, and 1.000 °C documenting the influence of heat on bloodstains and the detection of blood. The results of the forensic collection of blood traces with and without liquid latex confirmed the advantage of using the latex method. Almost all objects showed a clear luminescence-caused visualization of traces of blood after removing the soot with a latex lift. There were also fewer false positive results than in tests not using latex.

The estimation of bloodstain age is an important factor in forensic analysis. Previously, we have reported a smartphone-based colorimetric system for age estimation of bloodstain, in which Whole blood and EDTA whole blood were dropped on 4 different materials (700 μL) and captured using a smartphone for 72 h. In order to enhance sensitivity and accuracy of the previous system, the current work is dedicated towards the application of pattern recognition and classification of bloodstain images based on a smartphone. Three detection methods (blood pool, crack ratio, and colorimetric analysis) in terms of 6 steps of drying process of the bloodstain (coagulation, gelation, edge desiccation, center desiccation, crack propagation, and final desiccation) were applied to estimate age of the bloodstain accurately. Three parameters from the bloodstain images were then classified as comparing to those of stored reference images with similar trends in database. The bloodstain age was successfully determined by 9 h, 18 h, and 48 h with respect to the three detection methods mentioned above, respectively. The differences in bloodstain images were clearly distinguished every hour by using smartphone-based pattern recognition analysis. Therefore, our system is expected to shed a light on the field of forensic science by estimating bloodstain age in real time.

Foraging behavior of necrophagous flies commonly leads to distortion of human bloodstains and production of artifacts that confound reconstruction efforts at crime scenes. Currently there is no reliable method for detection of fly-derived stains or distinction of the artifacts from human bloodstains. To overcome these deficiencies, a confirmatory test was developed based on immunological detection of cathepsin D found in digestive fluids of Musca domestica and Protophormia terraenovae. Anti-serum (anti-md3 serum) was generated toward a 17-amino acid synthetic peptide based upon predicted antigenic amino acid sequences for the propeptide and mature enzyme of cathepsin D proteinase from larvae of M. domestica. The serum was used to test the hypothesis that digestive artifacts produced by an array of necrophagous flies associated with human decomposition could be detected with the immunoassay. Anti-md3 serum was able to bind artifacts from 27 species of flies representing 9 families. The antiserum reacted with both regurgitate and defecatory stains, but not transfer patterns. Stains from 4 fly species displayed no reactivity with anti-serum in dot blot assays. Anti-md3 serum did not bind to either human or bovine blood stains on filter paper. However, when both types of blood were spiked with synthetic md3 peptide the antiserum was able to bind. Dot blot assays displayed positive reactions with stains produced from larvae and teneral adults of Sarcophaga bullata, and with artifacts as old as 7-years after deposition. These observations indicate that the immunoassay permits distinction of artifacts from a wide range of species from human bloodstains, from multiple development stages, and from artifacts that remain at crime scenes for many months to years after deposition. Further work is needed to determine whether the detection of fly artifacts using the antiserum is suitable for non-laboratory conditions., Highlights • Antiserum was generated that detects digestive cathepsin D in necrophagous Diptera. • Anti-md3 serum specifically reacted with regurgitate and defecatory stains produced by flies after consuming human blood. • The antiserum did not bind human blood or blood from other mammals. • Anti-md3 shows promise for use in a confirmatory test that reliably distinguishes fly artifacts from human bloodstains.

Determining the time since deposition (TsD) of bloodstains can provide forensic investigators with additional clues, as it can corroborate eyewitness accounts, limit the number of suspects, and help confirm alibis. Bloodstains are the most common bodily fluid stains at crime scenes. In this study, we examined the relative expression levels (REs) of circRNAs and mRNAs data in bloodstains over ten time points by Real-time quantitative polymerase chain reaction (qPCR), to determine the utility of the relative expression levels of RNA markers for TsD estimation. Forensic samples more than just appear in indoor settings, we also evaluated the use of RNA degradation rate to indicate the age of bloodstains in different environments including indoor and outdoor conditions. The expression levels of six blood-specific mRNA markers (GYPA, CD93, ALAS2, SPTB, HBB, HBA), three highly expressed circRNAs in human peripheral blood (hsa_circ_0001445, hsa_circ_0000972, hsa_circ_0000095) and three reference genes (18 S, ACTB and U6) were analyzed across numerous ageing time points. Analysis of the degradation rates of individual RNAs under indoor and outdoor conditions showed that they exhibited a unique degradation profile during the four-month storage interval, with both circRNAs and mRNAs linearly showing continuous degradation, while U6 is more stable than other reference gene markers. In the current study, we firstly used circRNAs as additional novel biomarkers for bloodstain age estimation, and at the same time proved that different environments had a significant impact on the REs of certain blood biomarkers, and sex differences did not affect the age estimation of bloodstains. The REs of the selected RNA molecules in this study showed a non-linear relationship with bloodstain age and the mathematical formula for estimating the bloodstain age based on the relative expression levels of hsa_circ_0001445, ALAS2 and HBB can be used to estimate the TsD of bloodstains from the REs of bloodstains of unknown age, which represent a potentially effective approach to looking for time-dependent changes and TsD estimation.

Dried bloodstains at crime scenes provide abundant information for analyzing criminal identity of victims or suspects, morphological characteristics, and biological and chemical compounds. Therefore, they are considered important evidence by investigators at crime scenes. Moreover, the age of bloodstains can be used to determine the timeline of incidents at crime scenes; Inappropriately handled bloodstains may cause degradation of blood components. In this study, we identified a novel marker, hemoglobin subunit beta protein, as an internal standard to determine the age of bloodstains at crime scenes. We found that the target spot between 20 and 30 kDa in two-dimensional electrophoresis gradually increased in size. The hemoglobin subunit beta protein was identified from this spot using liquid chromatography-tandem mass spectrometry and verified using western blotting. Sample bloodstains were exposed to various environmental conditions (humidity: 30%, 60%, 90% at room temperature [RT]). Furthermore, the hemoglobin subunit protein extracted from the sample bloodstains at various time points (0 h to 30 d) was dissolved in our newly developed buffer solution and in deionized or distilled water. We also analyzed the expression levels of the protein in the sample bloodstains, dried at RT and under various humidity over time, using western blotting. In addition, we evaluated the protein extraction capacity of deionized or distilled water and the newly developed buffer from the sample bloodstains over time. At RT and 60% humidity, using the newly developed buffer, the hemoglobin subunit beta protein levels showed a gradually increasing pattern. Finally, we quantitated human hemoglobin subunit beta protein using western blotting and enzyme-linked immunosorbent assay, which revealed significant differences among the samples. In particular, the time points from 36 h to 30 days were considered for analysis. Thus, the hemoglobin subunit beta protein dried at RT and 60% humidity and further dissolved in the newly developed buffer solution can be used to determine the age of bloodstains at crime scenes.

A 2-year-old boy presented with history of generalised itchy eczematous rashes for one year and loose stool containing blood stains for 10 days. There was a history of skin abscess (left cervical region) at 15 months of age requiring incision and drainage and pneumonia at 18 months of age requiring

Blood-drop trajectory analysis can provide investigators with retrospective information regarding the spatial positioning of an injured person. To assist with bloodstain pattern analysis, various commercially available software have been developed and deployed. A systematic review was conducted to understand the extent of experimental validation and applications of blood-drop trajectory analysis software to case work. Ninety-two sources between 1987 and 2020 were identified including peer-reviewed studies and commercial websites. Thirty-four of these were validation studies, of which, only two involved impact patterns generated from greater than 1 m from the main target surface. Fifteen software were identified during this review with six documented to have been applied in casework. The reviewed software do not appear to fully satisfy relevant forensic validation criteria, based on publicly available literature. In some cases, software underwent limited experimental validation prior to real-world application with subsequent references to this in later literature. This review provides forensic investigators and bloodstain pattern analysts with a comprehensive overview of all available software options, knowledge of the extent of research into validating these techniques and highlights documented applications of these software in criminal cases.

Both latent sebaceous and blood fingerprints may provide valuable information for forensic investigation. To detect both types of fingerprints with no need to predistinguish them, a new adaptive developing strategy was proposed. A cationic conjugated polymer with poly[p-(phenylene ethylene)-alt-(thienylene ethynylene)] backbone (PPETE-NMe3+) was synthesized, which was dissolved in N,N-dimethylformamide (DMF) to form the developing solution. Fingerprints were developed by a simple dropping and incubating process without any pre-/post-treatments. Fluorescent photographs of the developed fingerprints on various substrates demonstrated that this developing strategy was effective for both types of fingerprints on nonporous substrates. Gray value analysis further confirmed the enhancement of the legibility of the fingerprint images. The preliminary mechanism exploration suggested that certain weak interactions, such as hydrophobic interaction and electrostatic interaction, may synergistically contribute to the interaction between the polymer and fingerprint components. The molecular design of the polymer combined with an appropriate solvent endowed the developing system the adaptiveness toward different types of fingerprints. This adaptive developing strategy made the fingerprint-developing process more efficient and may be further extended to more practical application scenes.

The historical analysis and the results of research activities of the Department of Forensic Medicine of the Sechenov University on the study of blood stains as important material evidence in forensic medicine and forensic science are presented. The personal contribution to the solution of this problem of outstanding forensic doctors whose life and work was associated with the department was noted.Представлены исторический анализ и результаты научно-исследовательской деятельности кафедры судебной медицины Сеченовского университета по изучению следов крови как важных вещественных доказательств в судебной медицине и криминалистике. Отмечен личный вклад в решение данной проблемы выдающихся судебных медиков, жизнь и деятельность которых была связана с кафедрой.

Copper is a metal that is commonly found in many evidence types, such as weapons, and household items, that are often retrieved from crime scenes. We used copper and brass sheets, cartridge cases, and metal decor items as substrates for deposition of blood to study inhibition induced by metals. Another objective was to compare the efficiency of STR profiling following extraction, quantification, amplification, and capillary electrophoresis with a direct amplification method which only requires amplification and capillary electrophoresis. Samples that were designated for extraction, were collected, extracted, and purified using the Qiagen EZ1 DNA Investigator® Kit. Direct amplification was performed using Copan microFLOQ® Direct Swabs to collect minute amount of blood. Complete STR profiles were obtained from 54% of the extracted samples and from 88% of directly amplified samples. Substrate type and amplification method (two-way ANOVA, F(5, 131) = 3.59, p = 4.50 × 10−3) impacted profile completeness but collection time did not (one-way ANOVA, F(1, 131) = 0.017, p = 0.90). Composition of the substrates was determined with scanning electron microscopy (SEM), and inhibition caused by copper was evaluated for each substrate. When substrate composition was analyzed in combination with amplification method, a substrate’s copper percent was not associated with increased allelic dropout (one-way ANOVA, F(1, 176) = 0.70, p = 0.40) but amplification alone was statistically significant (one-way ANOVA, F(1, 176) = 40.64, p = 1.56 × 10−9). Using the Copan microFLOQ® Direct Swabs, inhibition induced by metal observed was not as pronounced. In addition, less variability in profile completeness was detected, profiles were generated within a very short period, and only minute amount of bloodstain was needed, allowing for additional analysis of the same samples. Our results directly quantify the effect of copper composition on DNA profiling and suggest that while copper percentage is an observational contributor to allele dropout it is not wholly responsible for these events.

Blood is one of the most common body fluids discovered at crime scenes involving violent actions. It is one of the most important types of forensic evidence since it allows for the identification of the individual providing that there is a match with a known DNA profile. Determining the time since deposition (TSD) can assist investigators in establishing when the crime occurred or if a bloodstain present is actually related to the investigated event. To develop a forensically sound method for determining the TSD of a bloodstain, it is necessary to understand the underlying biochemical mechanisms occurring during aging. As biochemical processes occurring in blood are necessary for the continued survival of living organisms, they are important subjects of human biology and biomedicine and are well understood. However, the biochemistry of bloodstain aging ex vivo is primarily of interest to forensic scientists and has not yet been thoroughly researched. This preliminary study utilizes steady-state fluorescence spectroscopy to probe the changes in fluorescence properties of peripheral and menstrual blood up to 24-h post deposition. Peripheral and menstrual blood exhibited similar kinetic changes over time, assigned to the presence of the fluorophores: tryptophan, nicotinamide adenine dinucleotide (NADH), and flavins in both biological fluids. The biochemical mechanism of blood aging ex vivo is discussed.

Fly artifacts resulting from insect activity could act as confounding factors on a crime scene and interfere with bloodstain pattern analysis interpretation. Several techniques have been proposed to distinguish fly artifacts from human bloodstains based on morphological approach and immunological assay, but a DNA-based method has not been developed so far. Even if in forensic genetic investigations the detection of human DNA is generally the primary goal, fly artifacts can provide useful information on the dynamics of crime events. The present study provides a molecular method to detect fly DNA from artifacts deposited by Calliphora vomitoria after feeding on human blood through the analysis of the mitochondrial cytochrome oxidase gene subunit I (COI). Fly artifacts originated from digestive process and of different morphology spanning from red and brownish/light brown, circular and elliptical stains to artifacts with sperm-like tail or a tear-shaped body were collected. The COI amplification was successfully obtained in 94% of fly artifact samples. The method showed high sensitivity and reproducibility, and no human DNA contamination was observed, offering specificity for use in confirmatory test. This molecular approach permits the distinction of fly artifacts from genuine bloodstains and the identification of fly’s species through the COI region sequencing by protocols usually applied in forensic genetic laboratories. Supplementary Information The online version contains supplementary material available at 10.1007/s00414-021-02643-7.

Bloodstains are frequently encountered at crime scenes and they provide important evidence about the incident, such as information about the victim or suspect and the time of death or other events. Efforts have been made to identify the age of the bloodstain's donor through genomic approaches, but there are some limitations, such as the availability of databases and the quality dependence of DNA. There is a need for the development of a tool that can obtain information at once from a small blood sample. The aim of this study is to identify bloodstain metabolite candidates that can be used to determine donor age. We prepared bloodstain samples and analyzed metabolites using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). Eighteen molecular features (MFs) were selected as candidates using volcano plots and multivariate analysis. Based on the MS/MS spectrum of the MFs, the following nine metabolites were identified from the METaboliteLINk database: Δ2-cis eicosenoic acid, ergothioneine, adenosine 5'-monophosphate, benzaldehyde, phenacylamine, myristic acid ethyl ester, p-coumaric acid, niacinamide, and N-arachidonoyl-L-alanine. These nine age markers at high or low abundances could be used to estimate the age of a bloodstain's donor. This study was the first to develop metabolite age markers that can be used to analyze crime scene bloodstains.

Although the analysis of bloodstain pattern evidence left at crime scenes relies on the expert opinions of bloodstain pattern analysts, the accuracy and reproducibility of these conclusions have never been rigorously evaluated at a large scale. We investigated conclusions made by 75 practicing bloodstain pattern analysts on 192 bloodstain patterns selected to be broadly representative of operational casework, resulting in 33,005 responses to prompts and 1760 short text responses. Our results show that conclusions were often erroneous and often contradicted other analysts. On samples with known causes, 11.2% of responses were erroneous. The results show limited reproducibility of conclusions: 7.8% of responses contradicted other analysts. The disagreements with respect to the meaning and usage of BPA terminology and classifications suggest a need for improved standards. Both semantic differences and contradictory interpretations contributed to errors and disagreements, which could have serious implications if they occurred in casework.

In recent years, growing interest in deep learning neural networks has raised a question on how they can be used for effective processing of high-dimensional datasets produced by hyperspectral imaging (HSI). HSI, traditionally viewed as being within the scope of remote sensing, is used in non-invasive substance classification. One of the areas of potential application is forensic science, where substance classification on the scenes is important. An example problem from that area&mdash, blood stain classification&mdash, is a case study for the evaluation of methods that process hyperspectral data. To investigate the deep learning classification performance for this problem we have performed experiments on a dataset which has not been previously tested using this kind of model. This dataset consists of several images with blood and blood-like substances like ketchup, tomato concentrate, artificial blood, etc. To test both the classic approach to hyperspectral classification and a more realistic application-oriented scenario, we have prepared two different sets of experiments. In the first one, Hyperspectral Transductive Classification (HTC), both a training and a test set come from the same image. In the second one, Hyperspectral Inductive Classification (HIC), a test set is derived from a different image, which is more challenging for classifiers but more useful from the point of view of forensic investigators. We conducted the study using several architectures like 1D, 2D and 3D convolutional neural networks (CNN), a recurrent neural network (RNN) and a multilayer perceptron (MLP). The performance of the models was compared with baseline results of Support Vector Machine (SVM). We have also presented a model evaluation method based on t-SNE and confusion matrix analysis that allows us to detect and eliminate some cases of model undertraining. Our results show that in the transductive case, all models, including the MLP and the SVM, have comparative performance, with no clear advantage of deep learning models. The Overall Accuracy range across all models is 98&ndash, 100% for the easier image set, and 74&ndash, 94% for the more difficult one. However, in a more challenging inductive case, selected deep learning architectures offer a significant advantage, their best Overall Accuracy is in the range of 57&ndash, 71%, improving the baseline set by the non-deep models by up to 9 percentage points. We have presented a detailed analysis of results and a discussion, including a summary of conclusions for each tested architecture. An analysis of per-class errors shows that the score for each class is highly model-dependent. Considering this and the fact that the best performing models come from two different architecture families (3D CNN and RNN), our results suggest that tailoring the deep neural network architecture to hyperspectral data is still an open problem.

The age estimation of blood traces provides important leads for the chronological assessment of criminal events and their reconstruction. To determine bloodstain age, experimental comparative data from a laboratory environment are used. Under these conditions the utilization of anticoagulants such as EDTA helps to suppress the blood clotting mechanism to allow the examination over a longer time period. This unnatural prevention of blood coagulation is highly questionable when estimating bloodstain age, since the blood’s physical and chemical properties are altered. For this reason, the authors determined actual influence of EDTA on blood spectra over time in order to formulate a statement as to whether this effect can be measured. Human and porcine blood samples were aged under controlled conditions. The resulting UV/VIS spectra were separated into their individual components using signal separation techniques, allowing the changes in the ratios of the individual hemoglobin derivatives to be observed over time. The results show a significant influence of EDTA on the conversion of oxyhemoglobin to methemoglobin and a minor influence on the conversion of methemoglobin to hemichrome within the relevant time range of 5–100 h. The use of EDTA thus slows down the aging process of blood spots. To illustrate the great influence of EDTA, spectra of untreated pig blood samples were included as comparison data. These show that the difference between EDTA-treated and untreated blood samples is as great as the difference between human blood and pig blood. As a consequence of our findings experimental comparative data for the age estimation of bloodstains should never result from EDTA-treated blood.