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In Bloodstain Pattern Evidence, the concepts introduced in the author's first book, Blood Dynamics, are updated and applied to provide essential answers in the resolution of actual crimes. The book is accessible to all levels of investigators, regardless of academic background, and allows readers to develop a fundamental understanding of the underlying scientific principles behind bloodstain pattern evidence. Bloodstain Pattern Evidence builds on the fundamental ideas brought about by an understanding of Non-Newtonian dynamics, and illustrates through case work the practical forensic science applications of these principles to the analysis of bloodstain patterns. * Extensive case examples provide practical application of essential pattern analysis principles* Extensively illustrated with over 350 photos and line drawings * Takes a unique and scientific approach to bloodstain pattern analysis by exploring the fundamentals of fluid behavior

•Postmortem drug levels vary depending on sampling site and postmortem interval.•Morphine was quantified in cardiac and femoral blood in 103 postmortem cases.•The ratio between cardiac and femoral blood is a marker for postmortem redistribution.•13 variables potentially influencing this ratio were investigated.•Route of administration seems to have the highest impact on C/P ratio. In the field of forensic toxicology, many unexpected deaths are investigated as to whether toxicological substances may have caused or contributed to someone’s death. One of the factors that makes interpretation of the results of quantitative analysis in postmortem toxicology challenging, is that measured postmortem drugs levels may vary according to the sampling site and the interval between death and specimen collection. These site- and time-dependent variations are caused by ‘postmortem redistribution’ (PMR). Literature shows that there are several factors that determine the degree of PMR, such as cell and tissue changes after death, decomposition and the physicochemical characteristics of drugs. Blood from peripheral sites seems to be less affected by PMR than cardiac blood. Therefore, the ratio of cardiac blood concentration/peripheral blood concentration (C/P) of a drug is often used as a marker of the extent of postmortem redistribution. In this study, we investigated the relationship between different potentially important variables and the C/P ratio of morphine in humans in order to provide new insights that might assist in the interpretation of quantitative results in forensic casework. Toxicological results of all morphine positive postmortem cases investigated by the Netherlands Forensic Institute between January 1, 2010 and July 31, 2020 were reviewed. Morphine was quantified in both femoral and cardiac blood in a total of 103 cases. The C/P ratios were determined for all selected cases. To collect data for this study, all corresponding files were reviewed. C/P ratios were compared between subgroups by performing either a Mann-Whitney U test or a Kruskal-Wallis test, followed by a post-hoc Mann-Whitney U test. Bonferroni correction was performed to correct for the likelihood of a significant result by chance due to multiple testing. After Bonferroni correction, a p-value< 0.004 was considered statistically significant. The data suggests a relationship between grade of decomposition at autopsy, position of the corpse at discovery, route of administration, attempted resuscitation and the C/P ratio of morphine with p-values of 0.010, 0.026, 0.035 and 0.046, respectively. Grade of decomposition at autopsy, position of the corpse at discovery, route of administration and attempted resuscitation seem to be influencing the C/P ratio of morphine. Of these four variables, the route of administration seems to have the greatest impact.

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. •Using circRNAs as additional novel biomarkers for bloodstain age estimation for the first time.•The relative expression levels of circRNAs and mRNAs biomarkers significantly correlated with ageing points.•Different environments had a significant impact on the relative expression levels of certain blood biomarkers.•Sex differences did not affected the relationship between time and the relative expression levels of RNA.•The relative expression levels of circRNAs and mRNAs biomarkers can be used to estimate time since deposition.

Aconitum contains highly toxic alkaloids such as aconitine, hypaconitine, jesaconitine, and mesaconitine. Since Aconitum ingestion causes fatal intoxication, it is important to analyze aconitines and their metabolites in the blood. In forensic toxicology, postmortem drug redistribution is known as one factor that would hamper accurate evaluation of concentrations. Therefore, it is recommended to collect multiple blood samples from various sites and compare the results to avoid miss identification of causative compounds for intoxication. In this study, we evaluated aconitines and their metabolites in postmortem blood specimens from ten sites by QuEChERS extraction and liquid chromatography-tandem mass spectrometry (LC/MS/MS). The recovery rates and matrix effects of analytes were approximately 74–80% and 94–100%, respectively. The correlation coefficients were over 0.99. The validation studies revealed that accuracies and precisions were around 97–2% (intraday) and 100–4% (interday), respectively. Finally, the concentrations of aconitine and jesaconitine were from 2.72 to 7.20 ng/mL and from 14.9 to 26.3 ng/mL, respectively. The concentrations of mesaconitine were from 0.32 to 0.88 ng/mL in four samples and detected in two. The concentrations were highest in the right atrium and lowest in the femoral vein. Our results suggest that aconitine and jesaconitne are accumulated in right atrium blood after death, and that right atrium specimen is suitable for measuring aconitine compounds in fatal intoxication cases. •Postmortem redistribution of aconitines was estimated in an Aconitum ingestion case.•QuEChERS extraction was effective for cleaning contaminants from whole blood samples.•Aconitine and jesaconitine were accumulated in right atrium blood.•Our results showed that right atrium blood was suitable for quantifying aconitines.

Blood or bloodstains are encountered frequently in forensic investigations. Presumptive and more confirmatory tests for peripheral blood are well established, however, similar methods for menstrual blood identification are less so. D-dimer is a fibrin degradation product that occurs at high concentration in menstrual blood and therefore a potential target to screen for this body fluid. We evaluated three rapid tests to determine if they can discriminate menstrual blood from peripheral remote from a laboratory setting. Their sensitivity, specificity and robustness were also assessed. The assays were: a latex agglutination (Dade Dimertest Latex Assay), SERATEC PMB test and OneStep D-dimer RapidCard InstaTest, both of which are based on lateral flow immunochromatographic analysis. Of the three, greater sensitivity was observed using the OneStep D-dimer RapidCard InstaTest, regardless of whether liquid or a stain was used. This test also detected a result using the smallest volume of menstrual blood, 0.003125 μL. Specificity testing was based on six different body fluids (urine, saliva, peripheral blood, semen, sweats and vaginal fluid) resulting in all 30 samples testing negative for the D-dimer using the OneStep D-dimer RapidCard InstaTest. Mixtures at ratios 1:1, 1:3 and 1:9 (menstrual blood: the other biofluid or PBS) were tested and the results showed that D-dimer could be detected for all samples using either the Dade Dimertest Latex Assay or the OneStep D-dimer RapidCard InstaTest. The body fluids were exposed to environmental stresses such as various temperature (−20 °C, 4 °C, room temperature and 37 °C for 30, 90, 180 and 360 days) and fluctuations in humidity (42%, 76% and 100% humidity at room temperature for 1, 3, 5, 10 and 20 days): all samples were D-dimer positive using the OneStep D-dimer RapidCard InstaTest though the strength decreased relative to the increase of storage time and temperature or humidity. All 6 postmortem blood samples gave a positive result for D-dimer using the OneStep D-dimer RapidCard InstaTest and 2 samples gave a positive response using the Dade Dimertest Latex Assay and the SERATEC PMB test; peripheral blood postmortem samples can show an increase in D-dimer. Menstrual blood was recovered from the pads under the sample wells after testing using the two immunochromatographic assays from which STR alleles could be amplified successfully. The results presented here support the application of these commercial kits for effective identification of menstrual blood. [Display omitted] •Three commercial kits for D-dimer tests were evaluated for their applications on forensic menstrual blood identification.•All these three kits showed high sensitivity and specificity, and minimally influenced by the environmental stresses.•Peripheral blood postmortem samples showed an increase in D-dimer.•The menstrual blood recovered from the pads after completion of the immunochromatographic assays generated full STR profiles.

Blood-containing mixtures often appear in murder and robbery cases, and their identification plays a significant role in solving crimes. In recent years, the co-detection of DNA methylation markers (CpG) and single nucleotide polymorphism (SNP) markers has been shown to be a promising tool for the identification of semen and its donor. However, similar research on blood stains that are frequently found at crime scenes has not yet been reported. In this study, we employed blood-specific CpG-linked SNP markers (CpG-SNP) for blood-specific genotyping and the linking of blood and its donor. The tissue-specific CpG markers were screened from the literature and further verified by combining bisulfite conversion with amplification-refractory mutation system (ARMS) technology. Meanwhile, adjacent SNP markers with a minor allele frequency (MAF) greater than 0.1 were selected within 400 bp upstream and downstream of the CpG markers. SNP genotyping was performed using SNaPshot technology on a capillary electrophoresis (CE) platform. Finally, a multiplex panel, including 19 blood-specific CpG linked to 23 SNP markers, as well as 1 semen-specific CpG, 1 vaginal secretion-specific CpG, and 1 saliva-specific CpG marker, was constructed successfully. The panel showed good tissue specificity and blood stains stored at room temperature for up to nine months and moderately degraded (4 < DI < 10) could be effectively identified. Moreover, it could also be detected when blood content in the mixed stains was as low as 1%. In addition, 15 ng of DNA used for bisulfite conversion was required for obtaining a complete profile. The cumulative discrimination power of the panel among the Han population of northern China could reach 0.999983. This is the first investigation conducted for the simultaneous identification of blood and its donor regardless of other body fluids included in mixed stains. The successful construction of the panel will play a vital role in the comprehensive analysis of blood-containing mixtures in forensic practice.

Analyzing all biological evidence at a crime scene presents serious time, budget, and labor constraints. Therefore, selecting valid evidence is crucial for efficient screening. The ABO blood group is a marker that can serve as valid evidence for identifying investigative leads in criminal case. Conventional identification of ABO blood groups using serological methods has only been for blood and is difficult to apply to other body fluids. ABO genotyping was conducted by analyzing single nucleotide polymorphisms (SNP) representative of each blood group. However, this method is time-consuming, expensive, and requires sophisticated instruments. In this study, we developed rapid ABO genotyping method using loop-mediated isothermal amplification (LAMP) and multiplex real-time polymerase chain reaction (PCR). Three SNP sites in the ABO gene (nt 261, 526, and 803) were selected to classify the ABO genotypes. For the specificity test, we performed sequencing of 60 saliva samples to confirm that the genotyping. We conducted experiments to apply ABO genotyping using two amplification methods to mock forensic sample using cotton swab and filter paper. As a result, using LAMP, we successfully identified six ABO genotypes within 30 min at a constant temperature (65 ℃). Moreover, by using multiple real-time PCR, it was possible to detect not only the major group but also the subgroup of the ABO genotype (ex. cis-AB). The amplification results using the new methods were in concordance with the sequencing results. Therefore, these ABO genotyping methods are expected to select valid evidence successfully and efficiently at the crime scene.

Monozygotic (MZ) twins cannot be distinguished using conventional forensic STR typing because they present identical STR genotypings. However, MZ twins do not always live in the same environment and often have different dietary and other lifestyle habits. Metabolic profiles are deyermined by individual characteristics and are also influenced by the environment in which they live. Therefore, they are potential markers capable of identifying MZ twins. Moreover, the production of proteins varies from organism to organism and is influenced by both the physiological state of the body and the external environment. Hence, we used metabolomics and proteomics to identify metabolites and proteins in peripheral blood to discriminate MZ twins. We identified 1749 known metabolites and 622 proteins in proteomic analysis. The metabolic profiles of four pairs of MZ twins revealed minor differences in intra-MZ twins and major differences in inter-MZ twins. Each pair of MZ twins exhibited distinct characteristics, and four metabolites—methyl picolinate, acesulfame, paraxanthine, and phenylbenzimidazole sulfonic acid—were observed in all four MZ twin pairs. These four differential exogenous metabolites conincidently show that the different external environments and life styles can be well distinguished by metabolites, considering that twins do not all have the same eating habits and living environments. Moreover, MZ twins showed different protein profiles in serum but not in whole blood. Thus, our results indicate that differential metabolites provide potential biomarkers for the personal identification of MZ twins in forensic medicine.