Search

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 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.

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.

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.

Bloodstained fabrics found at crime scenes are likely to have had processing treatments, such as dyeing or printing, but the effect of the treatments on bloodstain morphology is not always considered. In order to study the effect of digital printing on bloodstain morphology, drip stains were created from five impact velocities (1.9–5.4 ms−1) on three different mass per unit areas (88–226 g/m²) of 100% cotton calico which had been digitally printed using reactive dye. Across all three printed fabrics, the bloodstains appeared visually similar, and no correlation was found between the dry bloodstain area and the impact velocity. When comparing the bloodstains on the printed fabric to those which had been created previously on the same fabric in a dyed and not-coloured state, the dry bloodstains on the printed fabric were statistically significantly larger (e.g. for the calico with the lightest mass per unit area, mean dry bloodstain area was 126.6, 64.4 and 44.3 mm² for the printed, dyed and not-coloured fabrics respectively). Examination of the larger bloodstains on the printed calico with the micro computed tomography scanner and scanning electron microscope, suggested that the printing process increased the wettability of the fabric, so the blood could spread more easily on the surface. This allowed the blood to coat the yarns, and wick into them before wicking along the intra-yarn spaces. The results presented in this paper showed that care must be taken when examining bloodstains at crime scenes. Depending on the fabric and the processing of the fabric the size of the blood stains may not increase with impact velocity as wicking may result in a larger bloodstain from a lower velocity. The bloodstain on the penetrated face of the fabric may be larger than on the impacted face and the same fabrics with different processing will produce different blood stain sizes and shapes.

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.

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.

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.

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.

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.

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.

Fingerprints and blood-groups constitute identifying characteristics of individuals. Correlation between dermatoglyphics and bloodgroups can be of use in victim identification. The aim of the present study was to determine accuracy of blood-group determination from saliva and to establish the association between distribution of fingerprint pattern and blood-groups in gender identification. 100 dental undergraduate students aged 18–22 years participated in the study. Fingerprint pattern was determined using Ink Method. ABO blood-grouping was done from saliva using the absorption-elution method. Present study showed that, most common fingerprint pattern was Loops (60%), followed by Whorls (26%) and Arches (14%). Most common blood-group was B, followed by A and O. Higher percentage of secretors in saliva was observed with females (86%) as compared to males (80%). In males, most common blood-group was AB while A was more common in females. In males, arches were most common finger print pattern (60%) whereas in females loops (55%) and whorls (52%) were more common. The study reports association between blood-group and dermatoglyphics which may help in gender identification in Forensic Medicine. It also suggests that saliva can used for ABO bloodgroup determination when blood stains are not available at crime-site.

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.

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.

In this study, the authors found that treating blood with 1 M HCl and 2% (w/v) 5-sulfosalicylic acid (SSA) in 1% (v/v) hydrogen peroxide mixture can produce photoluminescence of blood. SSA was added as a blood fixer. The photoluminescence was induced by irradiation of a forensic light source at 505 nm, which was detected using a 550 nm barrier filter. In this experiment, various level of acid and hydrogen peroxide were tested to find the optimal formulation of reagents, spot tests were conducted with diluted blood to test the sensitivity of this reagent, and impressions in blood left on porous/nonporous surfaces were enhanced. The sensitivity of this solution was slightly lower than Bluestar and was similar to leucocrystal violet or leucomalachite green on both porous/non-porous surfaces. The photoluminescence of blood treated with this reagent has been observed over 2 months. Using this reagent, it was possible to observe fingermarks or footwear impressions in blood on a black porous/non-porous surface. Through this, it was found that using this reagent could enhance bloodstains regardless of the porosity or color of the surface.

Necrophagous flies are presumed to feed on wet and dried blood at crime scenes, but no empirical information exists detailing fly interactions with dried bloodstains. In the present study, the foraging behavior of adult Calliphora vicina was characterized during interactions with dried bloodstains formed on a variety of porous, and non-porous materials that are commonly encountered in a household. Continuous digital recording and image analysis were used to monitor fly interactions with dried bloodstains and to determine mechanisms of stain modification. Flies displayed differential responses to bloodstains based on the porosity and topography of the surface material. For instance, blood that was not tightly adhered to the materials was flaked or dislodged by fly activity and was not consumed by the flies. On other non-porous surfaces, most stains were consumed following moistening by regurgitation. Feeding activity on such bloodstains frequently yielded partial, perimeter and skeletal stains. In contrast, adult flies rarely mechanically altered dried blood on porous fabrics and there was no evidence of modification due to feeding. Feeding avoidance behavior was observed due to tactile inhibition with blood dried on rough, uneven surfaces like cotton and denim.

Bloodstains are among the most important biological types of forensic evidence collected from crime scenes. This study aimed to evaluate the effectiveness of age estimation of blood stains on cotton cloth using the Real-time PCR technique combined with a multidimensional prediction model. A tissue-specific mRNA (HBB) and 2 Housekeeping genes (GAPDH and B2M) were analyzed over a 150-day period. The combined correlation equation for estimating the age of a blood stain was identified as: Age (Day) = −1.205+4.794(2 −(ΔCT GAPDH − 2.14))+50.293(2 −(ΔCT HBB + 8.117))−5.778(2 −(ΔCT HBB + 8.117)) 2.

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.

An immunoassay was previously developed as a technique to improve methods for detection and analysis of fly artifacts found at crime scenes. The dot blot assay utilized a polyclonal antiserum (anti-md3) based on a unique digestive cathepsin D found in cyclorrhaphous Diptera. In this study, artifacts produced by adults of Calliphora vicina, Cynomya cadaverina, Sarcophaga bullata, and Protophormia terraenovae were examined using the immunoassay to determine if insect-derived stains could be distinguished from a range of human body fluid stains. A lift technique was developed which permitted transfer of fly artifacts from test materials to filter paper for dot blot analyses. All species readily deposited artifacts on all test household materials regardless of diet consumed. Despite differences in texture and porosity of the household materials, artifacts of all species transferred to the filter paper. With all fly species, anti-md3 serum bound to artifacts produced after feeding on semen, blood, feces, urine, and saliva. By contrast, anti-md3 serum did not react with any of the human fluids tested, nor with any of the lifts from household materials not exposed to flies. There was no evidence of false positives with any of the fly species tested, regardless of diet consumed. There was also no indication of false negatives with any of the dot blot assays. These observations suggest that immunoassays using anti-md3 serum performed on a simple lift of suspected fly artifacts can be used effectively as a confirmatory assay to distinguish fly regurgitate and fecal stains from human body fluids.

This paper discusses the effects of thickness, mass per unit area, sett, yarn linear density and twist of calico fabrics (100% cotton, plain woven) on the morphology of passive bloodstains. Horse blood was dropped vertically onto three calico fabrics with different mass per unit areas (85.1 g/m², 163.5 g/m² and 224.6 g/m²). Six different impact velocities were used (1.7 ms−1, 2.9 ms−1, 4.1 ms−1, 4.9 ms−1, 5.1 ms−1 and 5.4 ms−1). The dry bloodstains were largest on the calico with the lightest mass per unit area. The low yarn linear density and large inter-yarn spaces meant that the blood could wick into the yarns from all directions and along the intra-yarn spaces. The calico with the middle mass per unit area had the smallest mean dry bloodstain area for four out of the six velocities. The twist level for this calico was greater than for the calicos with a heavier or lighter mass per unit area. This reduced the amount of wicking which occurred along the yarns due to the tighter yarn structure. The calico with the heaviest mass per unit area had the highest yarn linear density resulting in a thicker fabric, so the blood could not as easily penetrate into the fabric. This resulted in a thicker wet blood layer remaining on the fabric surface, where it gradually wicked vertically into the yarns under gravity. Less wicking along the yarns occurred, resulting in a smaller bloodstain than on the fabric with the lightest mass per unit area. The correlation between impact velocity and mean dry bloodstain area was greater for the calicos with the medium and heaviest mass per unit area than for the calico with the lightest mass per unit area. For the calicos with the medium and heaviest mass per unit area, the distance the blood spread laterally at impact, which increased with the increase in impact velocity, had a greater influence on the dry bloodstain area than the amount of wicking.

This work characterizes fundamental features of static drip patterns simulated for forensic bloodstain pattern analysis. The purpose of this study was to determine if and how two independent variables, impact velocity and droplet number, influence the parent stain size, shape and satellite spatter distribution of drip patterns created with whole ovine blood. To do this, 500 drip patterns were created in a controlled environment at five varying impact velocities and ten different droplet numbers on a hard paper surface. Digital images of the dried patterns were processed and analyzed using Fiji (ImageJ). The data collected from each pattern support the hypotheses that drip patterns contain predictable and reproducible elements based on impact velocity and droplet number. Basic fluid dynamic principles demonstrate that the size of the parent stains, as well as the number and distribution of satellite stains increase with increasing Weber number. A decrease in the circularity of the parent stains was also noted. This study provides fundamental qualitative and quantitative data on observable elements of drip patterns that can be used practically by bloodstain pattern analysts for pattern identification and classification.

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.

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.

In this study, author presented a color-crystal test, to confirm the presence of blood stain. In this test, blue colored micro-crystals were observed in case of positive blood sample. Since author included same reagents used for primary benzidine color test, hence in positive samples, used reagent impart blue color to the crystals and aid in proper visibility to the developed micro-crystals, observed under 20x and 40x magnification.

Bloodstain evidence can be very powerful evidence in assault related crimes. Determination of the distance that blood droplets may travel as a result of an impact into liquid blood may be of significance to corroborate or disprove a version of events, provide likely scenarios, or help determine the culpability of a person in determining their proximity to the blood shedding event. It was the aim of this research to determine the potential maximum distance blood droplets travel horizontally following a vertical impact into liquid blood. A custom apparatus was designed and constructed to replicate a vertical impact of a timber weapon, rotating on a fixed axis at one end, striking a pool of liquid blood. The device was positioned at three different levels of elevation to replicate an impact to the head of a person near ground level, a seated or kneeling height and standing height. Overall, the results indicated that the application of kinetic energy of between 1 and 5 J at a height of 1780 mm led to the blood droplets travelling a maximum horizontal distance of 5361 mm (and average maximum distance of 4981 mm). The horizontal distance blood droplets may travel upon impact does not appear to follow a linear trend with differing kinetic energy, but is affected by the applied force and release height in a curvilinear relationship. The results provide a valuable tool to bloodstain pattern analysts and investigators in determining search zones within a scene, as well as providing information about the proximity of an individual to an impact event.

Background Blood is one of the most common pieces of evidence encountered at the crime scene. Due to the viscous nature of blood, unique bloodstain patterns are formed which when studied can reveal what might have happened at the scene of the crime. Blood pattern analysis (BPA), i.e., the study of shape, size, and nature of bloodstain. The focus of this paper is to understand blood and BPA. An experimental finding to understand blood stain formation using Awlata dye was conducted within the university premises under laboratory conditions. Awlata (Alta), an Indian dye used for grooming of women, was used to create fake blood stains to understand the formation of bloodstains with respect to varying heights, and their relation with spines and satellite stains was determined. Results When the height of dropping fake blood increased, the distance of satellite stains emerging from the fake blood stains was also increasing. From the experimental finding, it was found that satellite stains were directly proportional to height of blood stain and spines were inversely proportional. Conclusion It can be concluded that blood is a vital source of information and when interpreted correctly it can be used as a source of information that can aid in investigations. Thus, a relation between formation of blood stains with relation to height was established. This finding using fake blood stains can help in carrying out future studies.

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.

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.

Bloodstain impact pattern Area of Origin (AO) estimation is an important but time-consuming process in criminal investigations. HemoVision is a software package that automates and accelerates this process. To date, however, no study has been published that evaluates HemoVision’s accuracy. Moreover, HemoVision relies on an automated variant of the tangent method to estimate a pattern’s AO, even though the use of front-view projections has been shown to provide biased AO estimates. Therefore, the goal of this paper is twofold. First, a novel AO estimation method is proposed, whereby AO estimation is formulated as a least-squares optimisation problem that operates in three dimensions directly, eliminating the need for front view projections. Second, ten impact patterns with known AO coordinates at both 50 cm and 100 cm with respect to the target wall are created and used to compare the proposed approach’s accuracy and robustness to the manual tangent method, HemoSpat, and HemoVision’s automated tangent method. Results show that for impacts at 100 cm or less to the target wall, the proposed approach achieves the lowest average error of 17.29 cm with the least uncertainty, and that it performs significantly better than the manual tangent and automated tangent methods at a 5% significance level. Moreover, it is shown to achieve errors of less than 30 cm at these distances with just nine stains, whereas the automated tangent method requires a minimum of 16 stains to achieve the same average error. ispartof: Forensic Science International vol:333 ispartof: location:Ireland status: Published online

Cast-off spatter patterns exhibit linear trails of elliptical stains. These characteristic patterns occur by centrifugal forces that detach drops from a swinging object covered with blood or other liquid. This manuscript describes a method to reconstruct the motion, or swing, of the object. The method is based on stain inspection and Euclidean geometry. The reconstructed swing is represented as a three-dimensional region of statistical likelihood. The reconstruction uncertainty corresponds to the volume of the reconstructed region, which is specific to the uncertainties of the case at hand. Simple numerical examples show that the reconstruction method is able to reconstruct multiple swings that are either intersecting or adjacent to each other. The robustness, spatial convergence, computing time of the reconstruction method is characterized. For the purpose of this study, about 20 cast-off experiments are produced, with motion of the swinging object documented using video and/or accelerometers. The swings follow circular or arbitrary paths, and are either human- or machine-made. The reconstruction results are compared with the experimentally documented swings. Agreement between measured and reconstructed swings is very good, typically within less than 10 cm. The method used in this study is implemented as a numerical code written in an open source language, provided in an open access repository, for purposes of transparency and access.

In a bloodletting incident, the area of origin (AO) of an impact bloodstain pattern is crucial when establishing the sequence of events. The use of laser scanners and other three-dimensional (3D) technologies to document and analyse bloodstains have been the subject of previous papers, especially where AO analysis is concerned. FARO Zone 3D (FZ3D) is a relatively new software programme that can be used for bloodstain AO analysis. FZ3D requires a greater understanding of inter-observer errors associated with AO. This study looked at the inter-observer variation between 21 examiners when repeatedly calculating the AO six times for a single impact pattern on a plain white wall. An impact rig which consisted of a spring tension arm was positioned and fixed 45 cm from the X wall (right wall), and 45 cm from the Y wall (left wall). This experimental design resembles an impact blow for a bloodletting event. The AO was unknown to all examiners, making it a blind study. The collected results were documented in a Microsoft Excel datasheet and later analysed. From previous literature, a 30 cm acceptable allowance was utilised for AO analysis; however, there is currently no accepted standard error for this type of analysis. The overall total 3D mean error for all examiners was 5.62 cm. The maximum error for any one impact analysis was 24.27 cm. The variation of the data, which was collected by all examiners, was documented as X = 1.14 cm, Y = 1.24 cm, Z = 1.68 cm, and the total 3D error = 2.28 cm. The total 3D error for any one examiner and the variance between examiners did not exceed the 30 cm acceptable allowance utilised in previous literature.

Mammalian whole blood sources are often used for forensic research and training when human samples cannot be sourced. While porcine, ovine and equine blood have been shown to be viable alternatives to whole human blood for forensic purposes, procurement can still pose a problem, especially for smaller and remote institutions. This work explores the use of whole bovine blood for basic bloodstain simulation. Sample preparation through the addition of ACD-A anticoagulant was optimized and storability was explored. Viscosity, surface tension, density, and packed cell volume, four fluid properties relevant to bloodstain pattern analysis, were monitored over four days and in two temperature conditions. Linear mixed models accounting for variation in the donor demonstrated that these fluid properties of the bovine blood changed predictably over time and with temperature. Whole bovine blood with 12.5% v/v ACD-A was found to be viable for use in basic bloodstain simulation at ambient and physiological temperature.

The underlying physics in bloodstain formation on fabrics is not well understood, despite its importance in bloodstain pattern analysis (BPA). This paper presents a fundamental study of the formation of drip bloodstains on fabrics, by focusing on blood droplet impact and wicking dynamics. The bloodstains were created on plain woven fabric by the perpendicular impact of a single blood drop with seven different impact velocities. The whole droplet impact and wicking processes were captured by multiple cameras. Fabric properties were characterized in detail at different levels. The bloodstain formation process was classified into distinct stages, including the inertial impact, initial absorption, first wicking and second wicking stages. The subsequent wicking process greatly alters the impact-induced bloodstains, in terms of bloodstain area. The dimensionless impact-induced stain factor ( β i , e ) is strongly dependent on the impact velocity while the final stain factor ( β f , e ) after the second wicking stage is not. The contribution of the subsequent wicking in altering the stain factor (or stain area) is quantified and found to decrease with increasing impact velocity. The blood wicking dynamics on the fabric in the majority of the first wicking stage can be well described by a simple scaling: ( β e - β i , e ) / β i , e = C [ t - t i / t i ] 1 / 2 , where t i marks the end of the inertial impact stage. The wicking coefficient C , which represents the influence of droplet impact on the subsequent droplet wicking, is found to scale as C ∼ W e - 0.34 . In the end, brief comments are provided regarding (1) the influence of the evaporation on the blood drop post-impact wicking dynamics and (2) the shape of bloodstains formed on fabrics, with a few suggested research directions for future work.

Physicochemical property changes observed in a degrading bloodstain can be used to estimate its time since deposition (TSD) and provide a timestamp to the sample's age. Many of the time-dependent processes that occur as a bloodstain degrades, such as DNA fragmentation and changes in hemoglobin structure, also exhibit temperature-dependent behaviours. Previous studies have demonstrated that pairing high-resolution automated gel electrophoresis and visible absorbance spectroscopy could be used to quantify the rate of degradation of a bloodstain in relation to time and storage substrate. Our study investigates such trends with an added factor, extreme temperatures. Passive drip stains were stored in either microcentrifuge tubes or on FTA cards at either -20°C, 21°C or 40°C and tested over 11 time points spanning 15 days. For both storage substrates, the wavelength at maximum absorbance for the Soret band and the maximum absorbance of the Alpha band showed a negative trend over time suggesting that spectral shifts are informative for TSD estimates. The ratio of the maximum peak height for DNA fragments lengths of 500-1000 base pairs to 1000-5000 base pairs was the most informative DNA variable in relation to time for both substrates. Cross-validation suggested the appropriate fit of the models with the data and reasonable predictive ability. We integrated both DNA concentration and hemoglobin visible absorbance metrics using principal component analysis (PCA) into a single model. Adding the random effect of the donor to the PCA model accounted for a large portion of the variation as did storage method and temperature. Additionally, canonical correspondence showed that temperature corresponded differently to the response variables for FTA card and microcentrifuge tube samples, suggesting a substrate specific effect. This study confirms that pairing DNA concentration and hemoglobin's visible absorbance can provide insight on the effect of different environmental and storage conditions on bloodstain degradation. While the level of uncertainty surrounding TSD estimates still precludes its use in the field, this study provides a valuable framework that improves our understanding of variation surrounding TSD estimates, which will be critical to any eventual application.

Evidence of dried blood is very valuable in forensic science. Since the discovery of luminescence with Luminol and dried blood spots (DBSs) in 1928, interest and research on blood have continued to date. One of the most important factor that DBSs have is genes. However, the current use of distilled water (DDW) to collect and extract blood samples has disadvantages related to DNA stability. Therefore, this study aimed to develop an extraction reagent that is most suitable for gene extraction from DBSs. Blood was collected from 45 healthy adult men and women in vacuum blood containers without coagulants or anticoagulants. The collected blood was dried in various settings to check the performance of the extraction reagent. Extraction with Tris-EDTA (TE) and phosphate-buffered saline (PBS) was found more suitable in terms of gene interference effects compared with DDW; their performance was also compared with those of the newly developed extraction reagents. Upon comparing the results of polymerase chain reaction for human genomic DNA samples using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene as the target, the performance of the newly developed extraction reagents, modified TE and PBS, was found to be relatively good. To determine the optimal composition of the developed extraction reagents, 12 new extraction reagents were developed with different pH and sodium concentrations. Among them, the best results were found when the DNA was extracted using extraction reagent No. 3 with pH 8.0 and containing 1 M NaCl. Next, the four extraction reagents, DDW, TE, PBS, and No. 3 were compared under nine different temperature and humidity conditions. Similarly, under various environmental conditions, extraction reagent No. 3 performed better than other reagents. It is proposed that modified TE and PBS mixed extraction reagents are the most suitable for collecting and preserving crime site samples. The proposed composition for a DNA extraction reagent can contribute greatly to crime scene reconstruction.

Fingerprint enhancement often includes either physical or chemical approaches, such as fingerprint powder or cyanoacrylate fuming, to improve the quality of a fingerprint for visualization and analysis. However, these methods become more complex when fingerprints are partial bloody, and these procedures may interfere with downstream DNA analysis. Columnar thin film (CTF) deposition is a type of nanotechnology that utilizes an evaporant material to enhance a fingerprint under low-pressure conditions. Short tandem repeat (STR) analysis is the traditional method employed in crime laboratories. When DNA is of poor quality and quantity, like that often obtained from fingerprints, little to no genetic information may be obtained. Single nucleotide polymorphisms (SNPs) may be used to glean additional information when STR analysis fails. In this pilot study, 100 partial bloody fingerprints were collected from two donors and deposited on five different crime scene substrates, in which half were enhanced with CTFs and were graded for quality by an IAI-certified latent fingerprint examiner. CTF-developed fingerprints, on average, had higher grades compared to non-developed partial bloody fingerprints. STR analysis using Fusion 6C was performed to assess inhibition from the evaporant materials, in which no inhibition was observed. Sequencing of SNPs using the Precision ID Identity Panel was also employed, in which genetic information that could not be obtained from STRs was acquired with SNPs. Various sample types (i.e. pristine, low quality, and contaminated) utilized in this project demonstrated the acceptable performance of the Precision ID Identity Panel.

Bloodstain pattern analysis, one of the areas of forensic science, is performed to analyze the physical characteristics of bloodstains, including their size, shape, and distribution, to reconstruct a crime scene. A bloodstain pattern analyst should obtain through experiments and education the capabilities to both understand the generation mechanisms of bloodstains and identify the characteristics of the bloodstains. Experiments and education about bloodstain pattern analysis are carried out by using human blood taken from subjects, animal blood (porcine or bovine) supplied from butcheries, and blood substitute products developed in other countries. However, these kinds of blood have many limitations in their application due to various problems. The blood substitute developed in the present study is more similar to human blood than other blood substitute products developed in other countries with regard to the physical properties, including viscosity, viscoelasticity, and surface tension, as well as the drip bloodstain patterns depending on the surface and coordinate characteristics of drip stains impact angle. The blood substitute developed in the present study is more practical, because the materials that are used in its preparation are readily available in the market and do not include chemicals that are harmful to the human body, and the blood substitute has luminol reaction functionality and pattern transfer bloodstain (bloodstain fingerprint, bloodstain footprint, etc.) dyeing functionality.

Age estimation is one of the most important components in the practice of forensic science, especially for body fluids or stains at crime scenes. Recent studies have focused on the application of DNA methylation for chronological age determination in the field of forensic genetics. However, the amount of DNA and the complex bisulfite conversion process make applying this method in trace or degraded samples difficult. MicroRNAs (miRNAs), a group of small noncoding RNAs, have great potential in forensic science due to their antidegradation property and tissue specificity. Certain miRNAs are highly age-related and may have potential utility in age prediction. In this study, the expression profile of miRNAs from blood samples was explored using massive parallel sequencing; age-related miRNAs were subsequently selected for age prediction. We then established age prediction models for bloodstains based on six age-related miRNAs using seven machine learning models. Results revealed that the mean absolute error (MAE) was 5.52 and 7.46 years in male and female bloodstain samples, respectively, using the AdaBoost algorithm. This pilot study demonstrates the possibility of performing forensic age prediction using miRNAs and may provide useful information in future case investigations.

The interpretation of sharp force fatality dynamics may be difficult in some cases, but a contribution to analysis of the phenomenon may be provided by case studies. Therefore, the purpose of our study is focused on identifying, in observed sharp force fatalities, reliable parameters that can differentiate a homicidal and suicidal manner of death, with particular reference to criminological parameters. Data derived from sharp force fatality cases in Padua and Venice from 1997 to 2019, anonymized and collected in Excel, included personal, circumstantial, clinical, and psychopathological-criminological data, as well as crime scene investigation, necroscopic, and toxicological data. Statistical analyses were performed using chi-square and Wilcoxon rank-sum tests. Possible predictors of homicide were analyzed by logistic regression. Six parameters (bloodstains distant from the body, clothing lacerations, hesitation/defense wounds, number of injuries, and potential motives) were significantly different in the two groups (p < 0.05). An independent statistical association between potential motives explaining the crime (p < 0.001; OR 27.533) and homicide on multiple logistic regression analysis was highlighted. The absence of clothing lacerations was inversely related to homicide (p = 0.002, OR 0.092). To the best of our knowledge, this is one of very few Italian studies concerning the differential diagnosis between homicidal and suicidal sharp force fatalities. The dynamics of the event is established in most cases by the integrated evaluation of data from crime scene investigation and the autopsy. Nevertheless, in an atypical scenario, a psychopathological-criminological analysis may provide essential elements, and particular attention should be given to the identification of potential explanatory motives.