Showing papers in "International Journal of Legal Medicine in 2016"

The pharmacology and toxicology of kratom: from traditional herb to drug of abuse

Abstract: Mitragyna speciosa (Rubiaceae), commonly known as kratom, is a tropical tree with a long history of traditional use in parts of Africa and Southeast Asia. In recent years, kratom has gained popularity for use as a recreational drug across the globe. Relatively new to the illicit market and used in a manner different from its traditional applications, preparations of kratom are touted by many as a safe and legal psychoactive product that improves mood, relieves pain, and may provide benefits in opiate addiction. Available literature was reviewed for M. speciosa via PubMed, Google Scholar, CINAHL, and EBSCO to summarize its traditional uses, phytochemical composition, pharmacology and toxicology of proposed active constituents, and potential for misuse and abuse. Research has demonstrated that both stimulant and sedative dose-dependent effects do exist, but a growing concern for the drug's effects and safety of use has resulted in national and international attention primarily due to an increase in hospital visits and deaths in several countries that are said to have been caused by extracts of the plant. The main active alkaloid substances in kratom, mitragynine and 7-hydroxymitragynine, present with a range of CNS stimulant and depressant effects mediated primarily through monoaminergic and opioid receptors. Recently, Palm Beach County, located in the southeastern corridor of Florida, has considered regulating kratom due to public safety concerns following the death of a young adult. At the local, state, and even federal levels, governments are now being confronted with the task of determining the safety and the possible regulation of kratom extracts. There are currently no standard analytical screening techniques for mitragynine and its metabolites following ingestion limiting its detection to more sophisticated techniques like liquid chromatography-mass spectrometry to determine kratom use. The growing concern of the abuse potential of kratom requires careful evaluation of its benefits and potential toxicities.

A standardized nomenclature for craniofacial and facial anthropometry.

Abstract: Standardized terms and methods have long been recognized as crucial to reduce measurement error and increase reliability in anthropometry. The successful prior use of craniometric landmarks makes extrapolation of these landmarks to the soft tissue context, as analogs, intuitive for forensic craniofacial analyses and facial photogrammetry. However, this extrapolation has not, so far, been systematic. Instead, varied nomenclature and definitions exist for facial landmarks, and photographic analyses are complicated by the generalization of 3D craniometric landmarks to the 2D face space where analogy is subsequently often lost, complicating anatomical assessments. For example, landmarks requiring palpation of the skull or the examination of the 3D surface typology are impossible to legitimately position; similar applies to median landmarks not visible in lateral photographs. To redress these issues without disposing of the craniometric framework that underpins many facial landmarks, we provide an updated and transparent nomenclature for facial description. This nomenclature maintains the original craniometric intent (and base abbreviations) but provides clear distinction of ill-defined (quasi) landmarks in photographic contexts, as produced when anatomical points are subjectively inferred from shape-from-shading information alone.

Death following ingestion of methylone

Abstract: Methylone is a synthetic derivative of cathinone It is sold principally on the Internet in powder form under the name «bath salts» Deaths following consumption are very rare This report details the first case of a death in France (a 21-year-old man), following ingestion of methylone during an evening with friends Anoxia was observed at the time of autopsy Toxicological analyses highlighted a consumption of methylone and cannabis However, biological analyses showed an absence of ethanol, cocaine, amphetamines, and opiate derivatives Likewise, no medications were found High concentrations of methylone were found in the peripheral blood (313 mg/L) and in the central blood (664 mg/L) Its presence in the gastric contents provides evidence that the substance was taken orally The dosage of δ9-tetrahydrocannabinol (THC) suggests a recent cannabis consumption (THC 129 μg/L, THC-COOH 293 μg/L, 11-OH-THC 49 μg/L) This case illustrates that the consumption of methylone, which has a reputation of being less «powerful» than ecstasy, is not without its dangers

Age estimation based on pulp cavity/chamber volume of 13 types of tooth from cone beam computed tomography images.

Abstract: The aims of this study are to identify which type of tooth has the strong relationship between age and pulp cavity/chamber volume among 13 types of tooth from the same dentition and to determine whether the inclusion of multiple types of tooth may improve the accuracy of age estimation. Cone beam computed tomography (CBCT) images from 115 females and 125 males aged between 16 and 63 years were analyzed. The DICOM data of all the images were imported into ITK-SNAP 2.4 for the calculation of pulp cavity/chamber volumes. Logarithmic regression analysis and multiple linear regression analysis were applied to establish the relationship between age and pulp cavity/chamber volumes. Among the 13 types of tooth, maxillary second molars have the largest R 2 (0.491, 0.642, and 0.498) and the smallest SEE (8.119, 6.754, and 8.022) in male, female, and pooled gender samples, respectively. The multiple linear regression analysis for the combination of multi-types of tooth indicated that a larger R 2 (0.627, 0.701, and 0.631) and smaller SEE (7.100, 6.258, and 6.970) than the counterpart calculated from the logarithmic regression analysis of a single type of tooth in male, female, and pooled gender samples, respectively. The pulp chamber volume of the maxillary second molars has the largest correlation coefficient with age. Using multiple types of tooth may improve the accuracy of age estimation compared with only one type of tooth used.

Effect of body mass and clothing on carrion entomofauna

Abstract: Carcass mass largely affects pattern and rate of carrion decomposition. Supposedly, it is similarly important for carrion entomofauna; however, most of its likely effects have not been tested experimentally. Here, simultaneous effects of carcass mass and clothing are analyzed. A factorial block experiment with four levels of carcass mass (small carcasses 5–15 kg, medium carcasses 15.1–30 kg, medium/large carcasses 35–50 kg, large carcasses 55–70 kg) and two levels of carcass clothing (clothed and unclothed) was made in a grassland habitat of Western Poland. Pig carcasses (N = 24) were grouped into spring, early summer, and late summer blocks. Insects were sampled manually and with pitfall traps. Results demonstrate that insect assemblages are more complex, abundant, and long-lasting on larger carcasses, whereas clothing is of minor importance in this respect. Only large or medium/large carcasses were colonized by all guilds of carrion insects, while small or medium carcasses revealed high underrepresentation of late-colonizing insects (e.g., Cleridae or Nitidulidae). This finding indicates that carcasses weighing about 23 kg—a standard in forensic decomposition studies—give an incomplete picture of carrion entomofauna. Residencies of all forensically relevant insects were distinctly prolonged on larger carcasses, indicating that cadaver mass is a factor of great importance in this respect. The pre-appearance interval of most taxa was found to be unrelated to mass or clothing of a carcass. Moreover, current results suggest that rate of larval development is higher on smaller carcasses. In conclusion, this study demonstrates that carcass mass is a factor of crucial importance for carrion entomofauna, whereas the importance of clothing is small.

Postmortem muscle protein degradation in humans as a tool for PMI delimitation

Abstract: Forensic estimation of time since death relies on diverse approaches, including measurement and comparison of environmental and body core temperature and analysis of insect colonization on a dead body. However, most of the applied methods have practical limitations or provide insufficient results under certain circumstances. Thus, new methods that can easily be implemented into forensic routine work are required to deliver more and discrete information about the postmortem interval (PMI). Following a previous work on skeletal muscle degradation in the porcine model, we analyzed human postmortem skeletal muscle samples of 40 forensic cases by Western blotting and casein zymography. Our results demonstrate predictable protein degradation processes in human muscle that are distinctly associated with temperature and the PMI. We provide information on promising degradation markers for certain periods of time postmortem, which can be useful tools for time since death delimitation. In addition, we discuss external influencing factors such as age, body mass index, sex, and cause of death that need to be considered in future routine application of the method in humans.

Postmortem degradation of skeletal muscle proteins: a novel approach to determine the time since death.

Abstract: Estimating the time since death is a very important aspect in forensic sciences which is pursued by a variety of methods. The most precise method to determine the postmortem interval (PMI) is the temperature method which is based on the decrease of the body core temperature from 37 °C. However, this method is only useful in the early postmortem phase (~0-36 h). The aim of the present work is to develop an accurate method for PMI determination beyond this present limit. For this purpose, we used sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and casein zymography to analyze the time course of degradation of selected proteins and calpain activity in porcine biceps femoris muscle until 240 h postmortem (hpm). Our results demonstrate that titin, nebulin, desmin, cardiac troponin T, and SERCA1 degraded in a regular and predictable fashion in all samples investigated. Similarly, both the native calpain 1 and calpain 2 bands disintegrate into two bands subsequently. This degradation behavior identifies muscular proteins and enzymes as promising substrates for future molecular-based PMI determination technologies.

Genetic screening in sudden cardiac death in the young can save future lives

Abstract: Background Autopsy of sudden cardiac death (SCD) in the young shows a structurally and histologically normal heart in about one third of cases. Sudden death in these cases is believed to be attributed in a high percentage to inherited arrhythmogenic diseases. The purpose of this study was to investigate the value of performing post-mortem genetic analysis for autopsy-negative sudden unexplained death (SUD) in 1 to 35 year olds.

Fatal toxicity index of medicinal drugs based on a comprehensive toxicology database.

Abstract: The fatal toxicity index (FTI) is the absolute number of fatal poisonings caused by a particular drug divided by its consumption figure. Consequently, it is a useful measure in evaluating toxicity of the drug and its relevance in fatal poisonings. In this study, we assessed the FTI of medicinal drugs in 3 years (2005, 2009, and 2013) in Finland. As the measure of drug consumption, we used the number of defined daily doses (DDD) per population in each year. There were 70 medicinal drugs in Finland for which the mean FTI expressed as the number of deaths per million DDD over the three study years was higher or equal to 0.1. The Anatomical Therapeutic Chemical (ATC) classification system was used for the classification of the active ingredients of medicinal drugs according to the organ or system which they act on. Of these 70 drugs, 55 drugs (78.6 %) acted on the nervous system (denoted by ATC code N), 11 (15.7 %) on the cardiovascular system (C), three (4.3 %) on the alimentary tract and metabolism (A), and one (1.4 %) on the musculoskeletal system (M). The nervous system drugs consisted of 20 psycholeptics, (ATC code N05), 20 psychoanaleptics (N06), eight analgesics (N02), six antiepileptics (N03), and one other nervous system drug (N07). The highest individual FTIs were associated with the opioids methadone, dextropropoxyphene, oxycodone, tramadol, and morphine; the antipsychotics levomepromazine and chlorprothixene; and the antidepressants doxepin, amitriptyline, trimipramine, and bupropion. Buprenorphine was not included in the study, because most of the fatal buprenorphine poisonings were due to smuggled tablets. A clearly increasing trend in FTI was observed with pregabalin and possibly with bupropion, both drugs emerging as abused substances.

Third molar maturity index (I3M) for assessing age of majority in a black African population in Botswana

Abstract: Assessment of legal age, also known as age of majority, is a controversial issue as there are few body biomarkers or evidence during late adolescence differentiating a subject from being a minor or adult. The third molar was recognized as a suitable site for age examination in late adolescence. We analyzed the development of the left mandibular third molar by the third molar maturity index (I3M) and a specific cut-off value of I3M = 0.08, established by Cameriere et al. in 2008 and used it for discriminating between minors and adult black Africans from Gaborone, Botswana. A final sample of panoramic radiographs (OPTs) of 1294 people (582 males and 712 females) aged between 13 and 23 years was evaluated. The real age decreased as I3M gradually increased. There was no statistically significant difference in the third molar development evaluated using I3M between males and females (p > 0.05) across different I3M classes. Results of 2 × 2 contingency tables for different cut-off values indicated that I3M = 0.08 was useful in discriminating between adults and minors. Precisely, for I3M = 0.08, the values of accuracy or overall fraction of correctly classified were 0.91 in males with a 95 % confidence interval (95 % CI) of 0.88 to 0.93 and 0.92 (95 % CI, 0.90 to 0.93) in females. Values of sensitivity of the test or the proportion of participants being 18 years and older were 0.88 (95 % CI, 0.87 to 0.90) in males and 0.88 (95 % CI, 0.90 to 0.93) in females, while values of specificity or proportion of individuals younger than 18 who have I3M <0.08 were 0.94 (95 % CI, 0.91 to 0.96) in males and 0.96 (95 % CI, 0.94 to 0.98) in females. Positive predictive values of the test, where the participants whose I3M <0.08 were adults, were 0.94 (95 % CI 0.91 to 0.96) in males and 0.97 (95 % CI, 0.94 to 0.98) in females, while negative predictive values of the test, where the participants whose I3M was ≥0.08 were minors, were 0.88 (95 % CI 0.85 to 0.90) in males and 0.97 (95 % CI, 0.94 to 0.98) in females. The likelihood ratios of the positive test (LR+) were 13.67 (95 % CI, 9.21 to 21.02) in males and 23.73 (95 % CI, 14.20 to 42.28) in females, while likelihood ratios of the negative test (LR-) were 0.12 (95 % CI 0.10 to 0.16) in males and 0.12 (95 % CI, 0.11 to 0.15) in females. Bayes post-test probabilities, p, were 0.94 (95 % CI 0.90 to 0.98) in males and 0.97 (95 %CI, 0.93 to 1.00) in females. These results indicate with high accuracy that I3M may be a useful alternative method in legal and forensic practice to discriminate individuals of black African origin who are around the legal adult age of 18 years in Botswana. Further studies should address the usefulness of this method and specific cut-off for different adolescent populations.

A SNP panel for identity and kinship testing using massive parallel sequencing.

Abstract: Within forensic genetics, there is still a need for supplementary DNA marker typing in order to increase the power to solve cases for both identity testing and complex kinship issues. One major disadvantage with current capillary electrophoresis (CE) methods is the limitation in DNA marker multiplex capability. By utilizing massive parallel sequencing (MPS) technology, this capability can, however, be increased. We have designed a customized GeneRead DNASeq SNP panel (Qiagen) of 140 previously published autosomal forensically relevant identity SNPs for analysis using MPS. One single amplification step was followed by library preparation using the GeneRead Library Prep workflow (Qiagen). The sequencing was performed on a MiSeq System (Illumina), and the bioinformatic analyses were done using the software Biomedical Genomics Workbench (CLC Bio, Qiagen). Forty-nine individuals from a Swedish population were genotyped in order to establish genotype frequencies and to evaluate the performance of the assay. The analyses showed to have a balanced coverage among the included loci, and the heterozygous balance showed to have less than 0.5 % outliers. Analyses of dilution series of the 2800M Control DNA gave reproducible results down to 0.2 ng DNA input. In addition, typing of FTA samples and bone samples was performed with promising results. Further studies and optimizations are, however, required for a more detailed evaluation of the performance of degraded and PCR-inhibited forensic samples. In summary, the assay offers a straightforward sample-to-genotype workflow and could be useful to gain information in forensic casework, for both identity testing and in order to solve complex kinship issues.

Next-generation sequencing of 100 candidate genes in young victims of suspected sudden cardiac death with structural abnormalities of the heart

Abstract: In sudden, unexpected, non-traumatic death in young individuals, structural abnormalities of the heart are frequently identified at autopsy. However, the findings may be unspecific and cause of death may remain unclear. A significant proportion of these cases are most likely caused by inherited cardiac diseases, and the cases are categorized as sudden cardiac death (SCD). The purpose of this study was to explore the added diagnostic value of genetic testing by next-generation sequencing (NGS) of a broad gene panel, as a supplement to the traditional forensic investigation in cases with non-diagnostic structural abnormalities of the heart. We screened 72 suspected SCD cases (<50 years) using the HaloPlex Target Enrichment System (Agilent) and NGS (Illumina MiSeq) for 100 genes previously associated with inherited cardiomyopathies and channelopathies. Fifty-two cases had non-diagnostic structural cardiac abnormalities and 20 cases, diagnosed with a cardiomyopathy post-mortem (ARVC = 14, HCM = 6), served as comparators. Fifteen (29 %) of the deceased individuals with non-diagnostic findings had variants with likely functional effects based on conservation, computational prediction, allele-frequency and supportive literature. The corresponding frequency in deceased individuals with cardiomyopathies was 35 % (p = 0.8). The broad genetic screening revealed variants with likely functional effects at similar high rates, i.e. in 29 and 35 % of the suspected SCD cases with non-diagnostic and diagnostic cardiac abnormalities, respectively. Although the interpretation of broad NGS screening is challenging, it can support the forensic investigation and help the cardiologist’s decision to offer counselling and clinical evaluation to relatives of young SCD victims.

Early markers for myocardial ischemia and sudden cardiac death

Abstract: The post-mortem diagnosis of acute myocardial ischemia remains a challenge for both clinical and forensic pathologists. We performed an experimental study (ligation of left anterior descending coronary artery in rats) in order to identify early markers of myocardial ischemia, to further apply to forensic and clinical pathology in cases of sudden cardiac death. Using immunohistochemistry, Western blots, and gene expression analyses, we investigated a number of markers, selected among those which are currently used in emergency departments to diagnose myocardial infarction and those which are under investigation in basic research and autopsy pathology studies on cardiovascular diseases. The study was performed on 44 adult male Lewis rats, assigned to three experimental groups: control, sham-operated, and operated. The durations of ischemia ranged between 5 min and 24 h. The investigated markers were troponins I and T, myoglobin, fibronectin, C5b-9, connexin 43 (dephosphorylated), JunB, cytochrome c, and TUNEL staining. The earliest expressions (≤30 min) were observed for connexin 43, JunB, and cytochrome c, followed by fibronectin (≤1 h), myoglobin (≤1 h), troponins I and T (≤1 h), TUNEL (≤1 h), and C5b-9 (≤2 h). By this investigation, we identified a panel of true early markers of myocardial ischemia and delineated their temporal evolution in expression by employing new technologies for gene expression analysis, in addition to traditional and routine methods (such as histology and immunohistochemistry). Moreover, for the first time in the autopsy pathology field, we identified, by immunohistochemistry, two very early markers of myocardial ischemia: dephosphorylated connexin 43 and JunB.

Genetic investigation of sudden unexpected death in epilepsy cohort by panel target resequencing

Abstract: Sudden unexpected death in epilepsy (SUDEP) is defined as the abrupt, no traumatic, witnessed or unwitnessed death, occurring in benign circumstances, in an individual with epilepsy, with or without evidence for a seizure and excluding documented status epilepticus (seizure duration ≥30 min or seizures without recovery), and in which postmortem examination does not reveal a cause of death. Although the physiopathological mechanisms that underlie SUDEP remain to be clarified, the genetic background has been described to play a role in this disorder. Pathogenic variants in genes associated with epilepsy and encoding cardiac ion channels could explain the SUDEP phenotype. To test this we use the next-generation sequencing technology to sequence a cohort of SUDEP cases and its translation into clinical and forensic fields. A panel target resequencing was used to study 14 SUDEP cases from both postmortem (2 cases) and from living patients (12 cases). Genes already associated with SUDEP and also candidate genes had been investigated. Overall, 24 rare genetic variants were identified in 13 SUDEP cases. Four cases showed rare variants with complete segregation in the SCN1A, FBN1, HCN1, SCN4A, and EFHC1 genes, and one case with a rare variant in KCNQ1 gene showed incomplete pattern of inheritance. In four cases, rare variants were detected in CACNA1A, SCN11A and SCN10A, and KCNQ1 genes, but familial segregation was not possible due to lack of DNA from relatives. Finally, in the four remaining cases, the rare variants did not segregate in the family. This study confirms the link between epilepsy, sudden death, and cardiac disease. In addition, we identified new potential candidate genes for SUDEP: FBN1, HCN1, SCN4A, EFHC1, CACNA1A, SCN11A, and SCN10A. Further confirmation in larger cohorts will be necessary especially if genetic screening for SUDEP is applied to forensic and clinical medicine. Nevertheless, this study supports the emerging concept of a genetically determined cardiocerebral channelopathy.

The biology and ecology of Necrodes littoralis, a species of forensic interest in Europe

Abstract: Necrodes littoralis (Linnaeus, 1758) (Coleoptera: Silphidae), also known as the "shore sexton beetle," is a common silphid beetle that visits and breeds on large vertebrate cadavers. This study describes, for the first time, the involvement of N. littoralis on human corpses based on a large dataset of 154 French forensic cases. Various parameters regarding corpse location, decomposition stages, and entomofauna were extracted from each file. Compared to all of the forensic entomology cases analyzed between 1990 and 2013 (1028), N. littoralis was observed, on average, in one case out of eight; most of these cases occurred during spring and summer (73.5%). More than 90% of the cases were located outdoors, especially in woodlands, bushes, and fields. The decomposition stage of the corpse varied among cases, with more than 50% in the advanced decomposition stage, 36% in the early decomposition stage, and less than 10% in the fresh, mummified, or skeletonized stages. Regarding other necrophagous species sampled with N. littoralis, Calliphorid flies were found in 94% of the cases and Fanniidae/Muscidae in 65% of the cases. Chrysomya albiceps, a heliophilic species mostly located in the Mediterranean area, was present in 34% of the cases (only 20% in the whole dataset). The most common coleopteran species were Necrobia spp. (Coleoptera: Cleridae) and Creophilus maxillosus (Coleoptera: Staphylinidae); these beetles were observed in 27% of the cases. The over-representation of these species is likely due to similar requirements regarding the climate and decomposition stage. As N. littoralis is frequently observed and tends to become more common, we conclude that the developmental data for this species would be a precious tool for forensic entomologists in Europe.

Carcass mass has little influence on the structure of gravesoil microbial communities.

Abstract: Little is known about how variables, such as carcass mass, affect the succession pattern of microbes in soils during decomposition. To investigate the effects of carcass mass on the soil microbial community, soils associated with swine (Sus scrofa domesticus) carcasses of four different masses were sampled until the 15th day of decomposition during the month of June in a pasture near Lincoln, Nebraska. Soils underneath swine of 1, 20, 40, and 50 kg masses were investigated in triplicate, as well as control sites not associated with a carcass. Soil microbial communities were characterized by sequencing the archaeal, bacterial (16S), and eukaryotic (18S) rRNA genes in soil samples. We conclude that time of decomposition was a significant influence on the microbial community, but carcass mass was not. The gravesoil associated with 1 kg mass carcasses differs most compared to the gravesoil associated with other carcass masses. We also identify the 15 most abundant bacterial and eukaryotic taxa, and discuss changes in their abundance as carcass decomposition progressed. Finally, we show significant decreases in alpha diversity for carcasses of differing mass in pre-carcass rupture (days 0, 1, 2, 4, 5, and 6 postmortem) versus post-carcass rupture (days 9 and 15 postmortem) microbial communities.

A single-tube 27-plex SNP assay for estimating individual ancestry and admixture from three continents

Abstract: A single-tube multiplex assay of a small set of ancestry-informative markers (AIMs) for effectively estimating individual ancestry and admixture is an ideal forensic tool to trace the population origin of an unknown DNA sample. We present a newly developed 27-plex single nucleotide polymorphism (SNP) panel with highly robust and balanced differential power to perfectly assign individuals to African, European, and East Asian ancestries. Evaluating 968 previously described intercontinental AIMs from three HapMap population genotyping datasets (Yoruban in Ibadan, Nigeria (YRI); Utah residents with Northern and Western European ancestry from the Centre de'Etude du Polymorphism Humain (CEPH) collection (CEU); and Han Chinese in Beijing, China (CHB)), the best set of markers was selected on the basis of Hardy-Weinberg equilibrium (p > 0.00001), population-specific allele frequency (two of three δ values >0.5), according to linkage disequilibrium (r 2 < 0.2), and capable of being multiplexed in one tube and detected by capillary electrophoresis. The 27-SNP panel was first validated by assigning the ancestry of the 11 populations in the HapMap project. Then, we tested the 27-plex SNP assay with 1164 individuals from 17 additional populations. The results demonstrated that the SNP panel was successful for ancestry inference of individuals with African, European, and East Asian ancestry. Furthermore, the system performed well when inferring the admixture of Eurasians (EUR/EAS) after analyzing admixed populations from Xinjiang (Central Asian) as follows: Tajik (68:27), Uyghur (49:46), Kirgiz (40:57), and Kazak (36:60). For individual analyses, we interpreted each sample with a three-ancestry component percentage and a population match probability sequence. This multiplex assay is a convenient and cost-effective tool to assist in criminal investigations, as well as to correct for the effects of population stratification for case-control studies.

DNA and RNA profiling of excavated human remains with varying postmortem intervals.

Abstract: When postmortem intervals (PMIs) increase such as with longer burial times, human remains suffer increasingly from the taphonomic effects of decomposition processes such as autolysis and putrefaction. In this study, various DNA analysis techniques and a messenger RNA (mRNA) profiling method were applied to examine for trends in nucleic acid degradation and the postmortem interval. The DNA analysis techniques include highly sensitive DNA quantitation (with and without degradation index), standard and low template STR profiling, insertion and null alleles (INNUL) of retrotransposable elements typing and mitochondrial DNA profiling. The used mRNA profiling system targets genes with tissue specific expression for seven human organs as reported by Lindenbergh et al. (Int J Legal Med 127:891-900, 27) and has been applied to forensic evidentiary traces but not to excavated tissues. The techniques were applied to a total of 81 brain, lung, liver, skeletal muscle, heart, kidney and skin samples obtained from 19 excavated graves with burial times ranging from 4 to 42 years. Results show that brain and heart are the organs in which both DNA and RNA remain remarkably stable, notwithstanding long PMIs. The other organ tissues either show poor overall profiling results or vary for DNA and RNA profiling success, with sometimes DNA and other times RNA profiling being more successful. No straightforward relations were observed between nucleic acid profiling results and the PMI. This study shows that not only DNA but also RNA molecules can be remarkably stable and used for profiling of long-buried human remains, which corroborate forensic applications. The insight that the brain and heart tissues tend to provide the best profiling results may change sampling policies in identification cases of degrading cadavers.

A 17-month time course study of human RNA and DNA degradation in body fluids under dry and humid environmental conditions

Abstract: Blood, saliva, and semen are some of the forensically most relevant biological stains commonly found at crime scenes, which can often be of small size or challenging due to advanced decay In this context, it is of great importance to possess reliable knowledge about the effects of degradation under different environmental conditions and to use appropriate methods for retrieving maximal information from limited sample amount In the last decade, RNA analysis has been demonstrated to be a reliable approach identifying the cell or tissue type of an evidentiary body fluid trace Hence, messenger RNA (mRNA) profiling is going to be implemented into forensic casework to supplement the routinely performed short tandem repeat (STR) analysis, and therefore, the ability to co-isolate RNA and DNA from the same sample is a prerequisite The objective of this work was to monitor and compare the degradation process of both nucleic acids for human blood, saliva, and semen stains at three different concentrations, exposed to dry and humid conditions during a 17-month time period This study also addressed the question whether there are relevant differences in the efficiency of automated, magnetic bead-based single DNA or RNA extraction methods compared to a manually performed co-extraction method using silica columns Our data show that mRNA, especially from blood and semen, can be recovered over the entire time period surveyed without compromising the success of DNA profiling; mRNA analysis indicates to be a robust and reliable technique to identify the biological source of aged stain material The co-extraction method appears to provide mRNA and DNA of sufficient quantity and quality for all different forensic investigation procedures Humidity and accompanied mold formation are detrimental to both nucleic acids

DNA transfer-a never ending story. A study on scenarios involving a second person as carrier.

Abstract: The transfer of DNA directly from one item to another has been shown in many studies with elaborate discussions on the nature of the DNA donor as well as material and surface of the items or surrounding features. Every DNA transfer scenario one can imagine seems to be possible. This evokes more and more intricate scenarios proposed by lawyers or attorneys searching for an explanation of the DNA of a certain person on a distinct item with impact on a crime. At court, the forensic genetic scientist has to comment on the probability of these scenarios thus calling for extensive studies on such settings. Here, the possibility of an involvement of a second person as a carrier of the donor's DNA in a variety of different scenarios including three pairs of people and two kinds of items (textiles and plastic bags) was investigated. All transfer settings were executed with and without gloves on the carrier's hands. DNA left on the items was isolated and analyzed using the Powerplex® ESX17 kit. In 21 out of 180 samples, all alleles of the donor DNA could be obtained on the second item (12%), on eight samples, the donor's DNA was dominant compared to all other alleles (38% of samples with complete donor profile). Additionally, 51 samples displayed at least more than half of the donor's alleles (28%). The complete DNA profile of the carrier was found in 47 out of 180 samples (42 partial profiles). In summary, it could be shown that a transfer of donor DNA from epithelial cells through a carrier to a second item is possible, even if the carrier does not wear gloves.

Sex-specific age association with primary DNA transfer

Abstract: Practicing forensic scientists who are called to provide expert witness testimony are often asked to explain both the presence and the absence of DNA on objects that have been handled by perpetrators with bare hands. Unwashed hands, depending on what they have come in contact with previously, may become the vehicle of both primary and secondary transfer of DNA. In this study, we investigated the propensity of primary and secondary transfer of DNA from unwashed bare hands of 128 individuals onto plastic tubes. Our experiments, carried out in triplicate, have shown that DNA was not detected on all the touched tubes, secondary transfer of DNA, through unwashed hands, was small, and in the majority of cases primary DNA transfer could be distinguished from secondary DNA transfer. A statistically significant association was demonstrated between percent DNA profile deposited on plastic tubes, through unwashed hands, and the age of male individuals.

Human cranial vault thickness in a contemporary sample of 1097 autopsy cases: relation to body weight, stature, age, sex and ancestry.

Abstract: The relation between human cranial vault thickness (CVT) and various elements of the physical anthropological biological profile is subject of ongoing discussion. Some results seem to indicate no correlation between CVT and the biological profile of the individual, whereas other results suggest that CVT measurements might be useful for identification purposes. This study assesses the correlation between CVT and body weight, stature, age, sex, and ancestry by reviewing data of 1097 forensic autopsies performed at the Netherlands Forensic Institute (NFI). In subadults (younger than 19 years of age at the time of death), all frontal, temporal, and occipital CVT measurements correlated moderately to strongly with indicators of growth (body weight, stature, and age). Neither sex nor ancestry correlated significantly with cranial thickness. In adults, body weight correlated with all CVT measurements. No meaningful correlation was found between CVT and stature or age. Females showed to have thicker frontal bones, and the occipital region was thicker in the Negroid subsample. All correlation in the adult group was weak, with the distribution of cranial thickness overlapping for a great deal between the groups. Based on these results, it was concluded that CVT generally cannot be used as an indicator for any part of the biological profile.

Phosphatidylethanol (PEth) in blood samples from "driving under the influence" cases as indicator for prolonged excessive alcohol consumption.

Abstract: Phosphatidylethanol (PEth) is considered as specific biomarker of alcohol consumption. Due to accumulation after repeated drinking, PEth is suitable to monitor long-term drinking behavior. To examine the applicability of PEth in "driving under the influence of alcohol" cases, 142 blood samples with blood alcohol concentrations (BAC) ranging from 0.0-3.12‰ were analyzed for the presence of PEth homologues 16:0/18:1 (889 ± 878 ng/mL; range

Obtaining appropriate interval estimates for age when multiple indicators are used: evaluation of an ad-hoc procedure.

Abstract: When an estimate of age is needed, typically multiple indicators are present as found in skeletal or dental information. There exists a vast literature on approaches to estimate age from such multivariate data. Application of Bayes' rule has been proposed to overcome drawbacks of classical regression models but becomes less trivial as soon as the number of indicators increases. Each of the age indicators can lead to a different point estimate ("the most plausible value for age") and a prediction interval ("the range of possible values"). The major challenge in the combination of multiple indicators is not the calculation of a combined point estimate for age but the construction of an appropriate prediction interval. Ignoring the correlation between the age indicators results in intervals being too small. Boldsen et al. (2002) presented an ad-hoc procedure to construct an approximate confidence interval without the need to model the multivariate correlation structure between the indicators. The aim of the present paper is to bring under attention this pragmatic approach and to evaluate its performance in a practical setting. This is all the more needed since recent publications ignore the need for interval estimation. To illustrate and evaluate the method, Kohler et al. (1995) third molar scores are used to estimate the age in a dataset of 3200 male subjects in the juvenile age range.

Performance of post-mortem CT compared to autopsy in children

Abstract: Radiological techniques such as non-enhanced post-mortem computed tomography (PMCT) play an increasingly important role in death investigations, especially in cases of non-medicolegal context of death, where the consent of the next of kin is required to perform autopsy Such consent is often difficult to obtain for deceased children, and radiological methods may be an acceptable alternative The aim of our study was to evaluate the performance of PMCT explorations compared to medicolegal conventional autopsies in children and its potential usefulness in non-medicolegal situations We retrospectively reviewed a group of 26 children aged 0–12 years who died of different causes, which were investigated by both conventional autopsy and PMCT We compared the findings extracted from radiological and autopsy reports All findings were grouped according to their importance with respect to cause of death and to the anatomical structure they covered: organs, vascular system, soft tissue, and skeletal system A significantly larger number of findings were detected by autopsy compared to PMCT Autopsy proved to be superior to PMCT, notably at detecting organ, soft tissue, and vascular findings, while PMCT was superior at detecting bone findings However, no statistically significant differences were found between the methods concerning the essential findings used to define the cause of death In children, PMCT was less sensitive than conventional autopsy for detecting general findings However, most essential findings were detected by both methods PMCT was superior to autopsy for the detection of bone lesions in children Up to today, very rare literature exists concerning PMCT in children, especially in a forensic setting This article investigates the advantages and limitations of PMCT compared to autopsy in a unique study group and discusses possibilities for future developments

DNA barcoding and NMR spectroscopy-based assessment of species adulteration in the raw herbal trade of Saraca asoca (Roxb.) Willd, an important medicinal plant.

Abstract: Saraca asoca (Roxb.) Willd, commonly known as “Asoka” or “Ashoka,” is one of the most important medicinal plants used in raw herbal trade in India. The bark extracts of the tree are used in the treatment of leucorrhea and other uterine disorders besides also having anti-inflammatory, anti-bacterial, anti-pyretic, anti-helminthic, and analgesic activity. The indiscriminate and rampant extraction of the wood to meet the ever-increasing market demand has led to a sharp decline in naturally occurring populations of the species in the country. Consequently, the species has recently been classified as “vulnerable” by the International Union for Conservation of Nature (IUCN). Increasing deforestation and increasing demand for this medicinal plant have resulted in a limited supply and suspected widespread adulteration of the species in the raw herbal trade market. Adulteration is a serious concern due to: (i) reduction in the efficacy of this traditional medicine, (ii) considerable health risk to consumers, and (iii) fraudulent product substitution that impacts the economy for the Natural Health Product (NHP) Industry and consumers. In this paper, we provide the first attempt to assess the extent of adulteration in the raw herbal trade of S. asoca using DNA barcoding validated by NMR spectroscopic techniques. Analyzing market samples drawn from 25 shops, mostly from peninsular India, we show that more than 80 % of the samples were spurious, representing plant material from at least 7 different families. This is the first comprehensive and large-scale study to demonstrate the widespread adulteration of market samples of S. asoca in India. These results pose grave implications for the use of raw herbal drugs, such as that of S. asoca, on consumer health and safety. Based on these findings, we argue for a strong and robust regulatory framework to be put in place, which would ensure the quality of raw herbal trade products and reassure consumer confidence in indigenous medicinal systems.

Systematic review of the toxicological and radiological features of body packing.

Abstract: Body packing is the term used for the intracorporeal concealment of illicit drugs, mainly cocaine, heroin, methamphetamine, and cannabinoids. These drugs are produced in the form of packages and are swallowed or placed in various anatomical cavities and body orifices. Basing on these two ways of transportation a distinction between body stuffers and body pushers can be made, with the former described as drug users or street dealers who usually carry small amounts of drugs and the latter as professional drug couriers who carry greater amounts of drugs. A review of the literature regarding body packing is presented, with the aim to highlight the toxicological and radiological features related to this illegal practice. Raising awareness about the encountered mean body levels of the drugs and the typical imaging signs of the incorporated packages could be useful for clinicians and forensic pathologists to (a) identify possible unrecognized cases of body packing and (b) prevent the serious health consequences and deaths frequently occurring after the packages' leakage or rupture or the packages' mass obstructing the gastrointestinal lumen.

Whiplash-Associated Disorders : Clinical and medico-legal guidelines on the methods of ascertainment.

Abstract: The manuscript presents the International Guidelines developed by the Working Group on Personal Injury and Damage under the patronage of the International Academy of Legal Medicine (IALM) regarding the Methods of Ascertainment of any suspected Whiplash-Associated Disorders (WAD).The document includes a detailed description of the logical and methodological steps of the ascertainment process as well as a synoptic diagram in the form of Flow Chart.

Response of forest soil euglyphid testate amoebae (Rhizaria: Cercozoa) to pig cadavers assessed by high-throughput sequencing

Abstract: Decomposing cadavers modify the soil environment, but the effect on soil organisms and especially on soil protists is still poorly documented. We conducted a 35-month experiment in a deciduous forest where soil samples were taken under pig cadavers, control plots and fake pigs (bags of similar volume as the pigs). We extracted total soil DNA, amplified the SSU ribosomal RNA (rRNA) gene V9 region and sequenced it by Illumina technology and analysed the data for euglyphid testate amoebae (Rhizaria: Euglyphida), a common group of protozoa known to respond to micro-environmental changes. We found 51 euglyphid operational taxonomic units (OTUs), 45 of which did not match any known sequence. Most OTUs decreased in abundance underneath cadavers between days 0 and 309, but some responded positively after a time lag. We sequenced the full-length SSU rRNA gene of two common OTUs that responded positively to cadavers; a phylogenetic analysis showed that they did not belong to any known euglyphid family. This study confirmed the existence of an unknown diversity of euglyphids and that they react to cadavers. Results suggest that metabarcoding of soil euglyphids could be used as a forensic tool to estimate the post-mortem interval (PMI) particularly for long-term (>2 months) PMI, for which no reliable tool exists.

Whole mitochondrial genome genetic diversity in an Estonian population sample.

Abstract: Mitochondrial DNA is a useful marker for population studies, human identification, and forensic analysis. Commonly used hypervariable regions I and II (HVI/HVII) were reported to contain as little as 25 % of mitochondrial DNA variants and therefore the majority of power of discrimination of mitochondrial DNA resides in the coding region. Massively parallel sequencing technology enables entire mitochondrial genome sequencing. In this study, buccal swabs were collected from 114 unrelated Estonians and whole mitochondrial genome sequences were generated using the Illumina MiSeq system. The results are concordant with previous mtDNA control region reports of high haplogroup HV and U frequencies (47.4 and 23.7 % in this study, respectively) in the Estonian population. One sample with the Northern Asian haplogroup D was detected. The genetic diversity of the Estonian population sample was estimated to be 99.67 and 95.85 %, for mtGenome and HVI/HVII data, respectively. The random match probability for mtGenome data was 1.20 versus 4.99 % for HVI/HVII. The nucleotide mean pairwise difference was 27 ± 11 for mtGenome and 7 ± 3 for HVI/HVII data. These data describe the genetic diversity of the Estonian population sample and emphasize the power of discrimination of the entire mitochondrial genome over the hypervariable regions.