Search

Forensic DNA profiles are established by multiplex PCR amplification of a set of highly variable short tandem repeat (STR) loci followed by capillary electrophoresis (CE) as a means to assign alleles to PCR products of differential length. Recently, CE analysis of STR amplicons has been supplemented by high-throughput next generation sequencing (NGS) techniques that are able to detect isoalleles bearing sequence polymorphisms and allow for an improved analysis of degraded DNA. Several such assays have been commercialised and validated for forensic applications. However, these systems are cost-effective only when applied to high numbers of samples. We report here an alternative, cost-efficient shallow-sequence output NGS assay called maSTR assay that, in conjunction with a dedicated bioinformatics pipeline called SNiPSTR, can be implemented with standard NGS instrumentation. In a back-to-back comparison with a CE-based, commercial forensic STR kit, we find that for samples with low DNA content, with mixed DNA from different individuals, or containing PCR inhibitors, the maSTR assay performs equally well, and with degraded DNA is superior to CE-based analysis. Thus, the maSTR assay is a simple, robust and cost-efficient NGS-based STR typing method applicable for human identification in forensic and biomedical contexts.

The expression levels of intracellular heat shock proteins (Hsps), specialized chaperone proteins, increase in cases of cellular stress with protein misfolding and aggregation. In a previous study, we demonstrated that there is an extensive increase in intracellular Hsp27 and 70 expression levels in renal tissues in fire fatality cases. Hsp expression can be induced by not only heat, but also by tissue hypoxia. In cases of fatal hemorrhage, the individual suffers hypoxemia and consequently tissue hypoxia. Here, we examined 43 cases of fatal hemorrhage and a control group of 85 deaths not related to blood loss or temperature exposure. We evaluated Hsp27 and 70 protein expression levels in renal tissue using immunohistochemistry. The results revealed that no extensive Hsp27 or 70 expression is induced in the fatal hemorrhage cases. The renal Hsp levels were similar to those of the control group. Fatal blood loss does not cause relevant cell stress., Competing Interests: Declarations of interest None., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The interpretation of postmortem γ-hydroxybutyric acid (GHB) concentrations is challenging due to endogenous existence and postmortem GHB production in body tissues and fluids. As an additional complication, formation of GHB was also described in stored postmortem samples. We examined cardiac blood, femoral blood, vitreous humor, cerebrospinal fluid and urine of eight different corpses (male/female 5/3, aged 33-92 years, postmortem interval 1-6 days) where no intake of GHB or one of its precursors was assumed. All samples were collected during autopsy and divided into two aliquots. To one of the aliquots, sodium fluoride (NaF, 1% w/v) was added. Both aliquots were vortexed, further divided into seven aliquots and stored at -20°C. GHB concentrations were measured immediately and subsequently 1 day, 7 days, 2 weeks, 4 weeks, 3 months and 6 months, after sample collection using trimethylsilyl derivatization and gas chromatography, coupled to single quadrupole mass spectrometry. Similar progression curves of GHB concentrations were obtained for the different matrices in the individual corpses. Femoral and cardiac blood GHB concentrations were always found to be higher than in vitreous humor, cerebrospinal fluid, and urine irrespective of stabilization and storage time. None of the obtained GHB concentrations exceed the cutoff values for postmortem matrices commonly used for the identification of an exogenous GHB intake (urine, venous blood and cerebrospinal fluid: 30 mg/L, cardiac blood and vitreous humor 50 mg/L). No significant differences were found for the GHB concentrations measured immediately and 6 months after autopsy. However, we found a significant increase for the GHB concentrations 4 weeks as well as 3 months after sample collection, which was followed by a decrease nearly to initial values. There were no significant differences between samples with and without NaF addition. The data presented are useful for the interpretation of GHB concentrations in upcoming death cases, with special attention to storage conditions and different postmortem matrices., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Violence against the neck can result in a range of macromorphological and micromorphological findings. However, the forensic relevance of the carotid sinus in cases of violence against the neck remains controversial. In this follow-up study of 22 cases of suicidal and accidental strangulations, carotid bifurcations were examined histologically for morphological changes implying direct trauma, including haemorrhage and immunohistochemical expression of heat-shock proteins 27, 60, and 70 and aquaporin-3. These cases were compared with a control group (82 cases) without neck compression or head trauma and with variable causes of death. No relevant histopathological findings implying direct trauma of the carotid bifurcation were found. No cases showed positive aquaporin-3 staining and only five cases showed positive heat-shock protein-27 staining, all of which were hangings. Without massive trauma of the carotid bifurcation, histological alterations cannot be expected. Without signs of rapid death, findings of acute circulatory failure, macromorphological and micromorphological findings of neck compression, and reliable markers indicating relevant impact on the carotid bifurcation the diagnosis of a lethal reflex cannot be verified.Key pointsAmong 22 cases of strangulation causing death, there were 16 cases of hanging and 6 cases of ligature strangulationFew cases showed small haemorrhages located predominantly in the surrounding fat and connective tissues; however, the haemorrhages did not have any effects on the tissuesNeck compression had minimal effects on heat shock protein 27 expression in carotid artery tissueAquaporin-3 staining suggested it is not a useful marker for relevant neck pressure, or that there had not been any relevant neck impactOur findings suggested no direct evidence for reflex cardiac death resulting from a brief force against the neck., Competing Interests: The authors report there are no competing interests to declare., (© 2022 The Author(s). Published by Taylor & Francis Group on behalf of the Academy of Forensic Science.)

Macromorphological findings can be missing in cases of fatal hypothermia when the agonal period is very short because of a large difference between environmental and core body temperatures. Expression of heat shock proteins (Hsps) increases under endogenous and exogenous cellular stresses such as thermal stress. These stress proteins can be revealed by immunohistochemical staining. Forty-five cases of death due to hypothermia and a control group of 100 deaths without any antemortem thermal stress were examined for Hsp27, 60, and 70 expression in renal tissue because renal tissue is sensitive to cellular stress. The results revealed no significant difference between Hsp27, 60, and 70 expression in both groups (28.8% positive staining in the study group and 19.0% positive staining in the control group), which is contradictory to a previous study on expression of Hsp70 in renal tissue in cases of fatal hypothermia. Hence, it is currently unclear whether immunohistochemical staining of Hsps supports a morphological diagnosis of fatal hypothermia., Competing Interests: Declaration of Competing Interest None., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Forensic pathologists are routinely confronted with unclear causes of death or related findings. In some instances, difficulties arise in relation to questions posed by criminal investigators or prosecutors. Such scenarios may include questions about wound vitality or cause of death where typical or landmark findings are difficult to ascertain. In addition to the usual examinations required to clarify unclear causes of death or address specific questions, immunohistochemistry and genetic analyses have become increasingly important techniques in this area since their establishment last century. Since then, many studies have determined the usefulness and significance of immunohistochemical and genetic investigations on cellular structures and proteins. For example, these proteins include heat shock proteins (Hsp), which were first described in 1962 and are so called based on their molecular weight. They predominantly act as molecular chaperones with cytoprotective functions that support cell survival under (sub) lethal conditions. They are expressed in specific cellular compartments and have many divergent functions. Central family members include, Hsp 27, 60, and 70. This mini review investigates recent research on the Hsp family, their application range, respective forensic importance, and current limitations and provides an outlook on possible applications within forensic science., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Prangenberg, Doberentz, Mawick and Madea.)

Forensic pathologists are routinely confronted with unclear causes of death or findings. In some scenarios, it can be difficult to answer the specific questions posed by criminal investigators or prosecutors. Such scenarios may include questions about wound vitality or causes of death when typical or landmark findings are difficult to find. In addition to the usual subsequent examinations to clarify unclear causes of death or special questions, immunohistochemical analysis has become increasingly important since its establishment in the early 40s of the 20th century. Since then, numerous studies have been conducted to determine the usefulness and significance of immunohistochemical investigations on various structures and proteins. These proteins include, for example, aquaporins, which belong to the family of water channels. They enable the transport of water and of small molecules, such as glycerol, through biological channels and so far, 13 classes of aquaporins could have been identified in vertebrates. The classic aquaporin channels 1, 2, 4 and 5 are only permeable to water. The aquaporin channels 3, 7, 9, and 10 are also called aquaglycerolporins since they can also transport glycerol. This mini review discusses the immunohistochemical research on aquaporins, their range of applications, and respective forensic importance, their current limitations, and possible further implementations in the future., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Prangenberg, Doberentz and Madea.)

Decline in cognitive performance, an aspect of the normal aging process, is influenced by the endocannabinoid system (ECS). Cannabinoid receptor 1 (CB1) signaling diminishes with advancing age in specific brain regions that regulate learning and memory and abolishing CB1 receptor signaling accelerates cognitive aging in mice. We recently demonstrated that prolonged exposure to low dose (3 mg/kg/day) Δ 9 -tetrahydrocannabinol (THC) improved the cognitive performances in old mice on par with young untreated mice. Here we investigated the potential influence of cannabidiol (CBD) on this THC effect, because preclinical and clinical studies indicate that the combination of THC and CBD often exhibits an enhanced therapeutic effect compared to THC alone. We first tested the effectiveness of a lower dose (1 mg/kg/day) THC, and then the efficacy of the combination of THC and CBD in 1:1 ratio, same as in the clinically approved medicine Sativex ® . Our findings reveal that a 1 mg/kg/day THC dose still effectively improved spatial learning in aged mice. However, a 1:1 combination of THC and CBD failed to do so. The presence of CBD induced temporal changes in THC metabolism ensuing in a transient elevation of blood THC levels. However, as CBD metabolizes, the inhibitory effect on THC metabolism was alleviated, causing a rapid clearance of THC. Thus, the beneficial effects of THC seemed to wane off more swiftly in the presence of CBD, due to these metabolic effects. The findings indicate that THC-treatment alone is more efficient to improve spatial learning in aged mice than the 1:1 combination of THC and CBD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Nidadavolu, Bilkei-Gorzo, Krämer, Schürmann, Palmisano, Beins, Madea and Zimmer.)

Identification of putrefied bodies is an important and common task in forensic routine. Usually, the identification of deceased is done by visual recognition, fingerprints, physical distinguishing marks (e.g. tattoos, scars and surgical implants) and/or dental examination. However, if morphologic characteristics are not recognizable, due to advanced putrefaction of the corpse, or recent medical records are not available, the DNA-based identification is favored. Thus, in order to find another reliable procedure for DNA extraction of putrefied samples regarding tissue selection, costs and time, two commercial forensic kits were compared: DNeasy® Blood & Tissue kit and SwabSolution™ kit. Both kits were used for DNA extraction from five soft tissues (brain, aorta, liver, kidney and psoas major muscle) and nails (finger- and toenail) obtained during the medicolegal autopsy of 20 putrefied corpses. DNeasy® Blood & Tissue kit was by quantitative comparison mostly superior to SwabSolution™ kit: it yielded more DNA of better quality (i.e. less degraded and inhibited). However, the qualitative results (DNA profiles) of both extraction procedures were similar. Among the analyzed tissue types, nails were proved as the most suitable for DNA-based identification of putrefied bodies., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Contact shots to the head often provoke a transfer of biological traces into firearm barrels, which are not visible at endoscopic inspection. STR-PCR can amplify these latent traces and assign them to the victim. Via RNA-DNA-co-extraction also miRNA can be detected, which allow a conclusion to be drawn about the body fluid or tissue. Molecular genetic analysis of experimental stains in firearm barrels requires the guarantee that the barrel is initially free of any nucleic acid. Twelve shots were fired to so-called "reference cubes" (10 % gelatine, 12 cm edge length, embedded paint-blood-pad) using three current handguns: from 20 and 30 cm distance, four at close range (1-2.5 cm) and six contact shots. After endoscopic examination and swabbing of the barrels, a previously described mechanical and chemical cleaning using DNAExitusPlus™ was performed. The inner surface of the barrel was thoroughly wiped off using moistened forensic swabs, which were submitted to RNA-DNA-co-extraction. The combined thorough mechanical cleaning with Ballistol® and the application of DNAExitusPlus™ eliminated any profilable DNA in all samples. However, in 10 of 12 samples RNA concentrations between 0.11 - 0.79 ng/μl were measured. Furthermore, in 9 of 12 samples blood-specific miRNA (miR-451a) was detected. Summarizing, none of the experimentally contaminated barrels was RNA-free despite the performed cleaning procedure. Further investigation showed, that even "professional" cleaning by a gunsmith did not remove RNA., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

γ-Hydroxybutyric acid (GHB) and its corresponding lactone γ-butyrolactone (GBL) are misused as knock out (k.o.) drugs. The short detection window and the major inter- and intra-individual variations of endogenous GHB concentrations in commonly used matrices such as blood and urine complicate the analytical proof of an exogenous GHB/GBL administration. We searched for an alternative way to prove an exogenous GHB/GBL administration via detection of methyl- and ethyl-4-hydroxybutyrate, which could arise in alcoholic solutions after spiking with GHB/GBL. A liquid chromatographic-triple quadrupole mass spectrometric method was developed and validated to quantitatively determine methyl- and ethyl-4-hydroxybutyrate in alcoholic beverages (limit of detection [LoD]: 5.8 and 3.4 ng/mL, respectively). A sample collective of alcoholic beverages (n = 47) revealed natural occurring amounts of ethyl-4-hydroxybutyrate (

The question whether an injury was sustained during life or not is one of the most important subjects in forensic medicine. Therefore, vital reactions have been a main research topic in forensic medicine for a long period and many renowned forensic pathologists have devoted important papers to this field. The research area ranges from macroscopically visible organ reactions, over tissue alterations (enzyme histochemistry, later on immunohistochemistry with a wide range of enzymes and other analytes, molecular pathology) to biochemical responses to injury. Especially in the field of immunohistochemistry and molecular pathology much progress has been achieved in the last years (e.g. heat-shock-proteins or positive aquaporine3-staining in mechanical skin trauma). Furthermore, 20 years after its implementation postmortem imaging also contributes to the detection and visualization of vital signs. The aim of the present review is to provide an update on forensically relevant vital signs/vital reactions. Systemic vital reactions especially of the circulatory and respiratory system as well as local vital reactions will be addressed. Vital reactions of different organ systems will be discussed in detail regarding pathogenesis and possible postmortem evolution. Current research on immunohistochemically detectable vital reactions (heat-shock-protein expression, aquaporine3-staining in mechanical trauma of the skin) will be addressed as well as biochemical vital reactions (agonochemical stress reaction, myoglobine in electrocution death, hypoxanthine as marker of hypoxia)., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Fentanyl is a potent synthetic opioid with a variety of possible applications. Transdermal fentanyl patches are regularly prescribed for patients with severe chronic or cancer-related pain. The potential for abuse is well-known and cases associated with illicit fentanyl intake are common. Fentanyl related fatalities due to unintentional misuse are relatively rare. This study focused on those instances and their identification in forensic examinations and adds new cases and consolidates the existing femoral blood concentrations in the event of fatal fentanyl patch misapplications. A total of 35 cases between 2010 and 2018 in which transdermal fentanyl patches were detected during forensic autopsies were identified and reviewed for the frequency of unspecific macroscopic signs of opioid intoxication. Furthermore, a detailed examination is presented for 11 cases in which toxicological results were available. The cause of death was eventually considered to be related to fentanyl patch misuse in 5 of these 11 cases. Co-administered drugs and signs of opioid intoxication, especially pulmonary edema, were frequently found. Lastly, it is advised to include norfentanyl and hair analysis in the interpretation of post-mortem fentanyl concentrations., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Gold standard for the estimation of the time since death in the early postmortem period is the temperature based nomogram method together with time of death dependent criteria of postmortem lividity, rigor mortis and supravital reactions. There is also a huge literature on chemical methods proposed for estimating the time since death which however play obviously no role in forensic practice. Especially the rise of vitreous potassium has been studied intensively. Also immunohistochemical methods have been proposed for estimating the time since death but obviously not yet applied in casework. We present a case where a woman was found murdered 8 days after having been seen last alive. Due to lack of putrefactive changes postmortem interval was thought to be not more than 2 days. However, immunohistochemical stainings and vitreous potassium concentration revealed that time since death was more than 6 days and the woman was obviously murdered immediately after she was seen lastly alive., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Mitochondrial DNA (mtDNA) heteroplasmy (intra-individual variation) varies among different human tissues and increases with age, suggesting that the majority of mtDNA heteroplasmies are acquired, rather than inherited. However, the extent to which heteroplasmic sites are shared across a tissue remains an open question. We therefore investigated heteroplasmy in two liver samples (one from each primary lobe) from 83 Europeans, sampled at autopsy. Minor allele frequencies (MAF) at heteroplasmic sites were significantly correlated between the two liver samples from an individual, with significantly more sharing of heteroplasmic sites in the control region than in the non-control region. We show that this increased sharing for the control region cannot be explained by recent mutations at just a few specific heteroplasmic sites or by the possible presence of 7S DNA. Moreover, we carried out simulations to show that there is significantly more sharing than would be predicted from random genetic drift from a common progenitor cell. We also observe a significant excess of non-synonymous vs. synonymous heteroplasmies in the protein-coding region, but significantly more sharing of synonymous heteroplasmies. These contrasting patterns for the control vs. the non-control region, and for non-synonymous vs. synonymous heteroplasmies, suggest that selection plays a role in heteroplasmy sharing.

Hanging can be suicidal, accidental, or homicidal, and these backgrounds must be discriminated by police and forensic pathologists. We herein report a case involving a 33-year-old man who was found dead on the floor behind the entrance door of an apartment house. The man's brother declared that he had found him hanging in the gap between the stairs on the top floor. When his brother tried to cut him down, the victim fell three floors down through the gap between the stairs. Autopsy was performed to confirm suicidal hanging and a postmortem fall into the narrow gap. In this case, however, a homicide was suspected, and the version of events told by the victim's brother was initially doubted. Homicidal hanging may be uncommon, but intensive scene investigation and thorough autopsy are necessary in hanging cases to rule out homicide., (Copyright © 2019 Elsevier B.V. All rights reserved.)

5F-ADB is an indazole-based synthetic cannabinoid. In recent years, it has been detected in legal high products as well as in biological samples and is associated with serious adverse health, behavioral effects and even death. Due to the fast pace of the market of synthetic cannabinoids, data on such newly appearing substances are scarce. As pharmacological properties are often investigated in vitro or by using animal experiments, reports on synthetic cannabinoid findings in human samples along with corresponding case history descriptions are valuable for the interpretation of upcoming routine cases. Herein we report five cases with verified 5F-ADB consumption, including three fatalities, a case of driving under the influence of drugs as well as a case of grievous bodily harm. In four cases, 5F-ADB could be detected in blood or plasma. Concentrations were in the range of 0.11-0.57 μg/L. In one instance 5F-ADB consumption was verified by the detection of 5F-ADB metabolites in postmortem body fluids. The described cases illustrate various adverse effects including confusion (possibly even psychosis), collapse, loss of consciousness, unsafe driving style or changing moods that might be attributed to 5F-ADB., (Copyright © 2019 Elsevier B.V. All rights reserved.)

We report a fatal γ-hydroxybutyric acid (GHB) intoxication of a forty-year old man. According to an acquaintances' statement, the deceased had drunk a beverage containing GHB approximately five hours before he was found. Postmortem GHB concentrations were determined using gas chromatography coupled to single quadrupole mass spectrometry after simple protein precipitation with methanol and derivatization with BSTFA (1% TMCS). Concentrations in body fluids and tissues of the deceased were as follows: cardiac blood 384 mg/L, femoral blood 358 mg/L, urine 864 mg/L, brain tissue 211 mg/kg, liver tissue 201 mg/kg, kidney tissue 492 mg/kg, bile 334 mg/L and gastric content 2025 mg/L. In an exhibit (liquid in a plastic bottle found next to the decedent) analyzed 29 days after the intake 27.6 g/L GHB were found with an increasing content during storage depending on the pH of the liquid (17 months after the intake: 70.0 g/L GHB and 121.2 g/L after adjusting the exhibit to a pH of 10 before extraction). GHB concentrations in head hair of the deceased (overall length approx. 4 cm, measured in segments of 0.5 cm) were measured using liquid chromatography coupled to triple quadrupole mass spectrometry. Concentrations in unwashed and washed hair samples were 91.9-174 ng/mg and 49.2-134 ng/mg, respectively. All cut-off values for postmortem matrices generally used for the identification of an exogenous GHB intake, which are further discussed within this publication, were exceeded. A lethal GHB intoxication can be assumed by a combination of toxicological findings, police investigations and exclusion of other causes of death., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In the last decades, more and more new psychoactive substances (NPS) were introduced on the drug market which were sold as "legal" alternatives for classic drugs and misused medications. Due to an increased number of available substances and a growing utilization by users of common drugs but also by inexperienced users because of the supposed "legal" status, also undesired adverse effects of these NPS, at worst leading to death, became apparent. This review summarizes fatalities previously described in scientific literature which were attributed to the use of NPS or such cases, in which intake of NPS was proven or even assumed to contribute to death. This summary includes an overview of substances involved (particularly synthetic cannabinoids ("spice"), novel opioids and synthetic cathinones ("bath salts")) as well as of postmortem concentrations determined in various biological matrices. The compiled data assist forensic toxicologists with the interpretation of death cases involving NPS., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Providing evidence of asphyxia death is a challenging issue in forensic pathology. Besides helpful macroscopical signs (e.g. strangulation mark, lung edema), recent data from literature indicate that the time of protracted asphyxia suffices to trigger an increase of giant cells and a migration of inflammatory cells from the bone marrow to the lung, thus offering a help in diagnosis of asphyxia death. In search of new valid asphyxia markers, the present study examined this hypothesis and investigated the leading role of pre-existing lung tissue cells and their functional state in reaction patterns to asphyxia. In specimens of suffocated human lungs following a short (n = 13) and a long asphyxia terminal episode (n = 15), and controls (sudden cardiovascular (n = 11) and traumatic deaths (n = 7)), the count of alveolar phagocytes, megakaryocytes, giant and mast cells, using H&E and toluidine blue staining, was performed. To show macrophage activation, immunohistochemical stainings for CD68, late (25F9) and early (MRP-8/-14) stage inflammatory markers were used. Measuring concentration of tryptase in femoral blood acted as a parameter for mast cell degranulation and consequently their activation. Results showed the lack of specificity of macroscopical parameters despite an association of suffocation with heavy lung edema. No significant differences in the numbers of inflammatory cells in the lungs of different case groups were detected. The doubling of MRP-8- and a five-fold elevation of MRP-14-positive cells compared to cardiovascular controls, proved an early activation state of pre-exiting monocytes in protracted asphyxia. These activated monocytes induced the degranulation of mast cells, resulting in slightly elevated tryptase levels in suffocation compared to cardiovascular controls. In summary, the duration of asphyxia (max. 20 min in cases investigated) only suffices to cause changes on molecular level, being not detectable in any specific macroscopical or histological form in the lung. Despite a potential utility of this molecular insight in individual cases, these results point to the classic doctrine of the evaluation of a rounded overall picture, accentuating on the proof of the ligature tool and the marks of suffocation process., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Heat-shock proteins (HSPs) are expressed during cellular stress, especially thermal stress, to support protein homeostasis. Extensive investigations have revealed that HSP27 is rapidly and intensively expressed in the pulmonary and renal tissues of most cases of death due to fire, contrary to cases without premortem thermal influences, which are negative for HSP expression. As such, HSP27 can be used as a marker of vitality in the investigation of fire-related deaths. Without positive HSP27 expression or only slight expression, one can conclude that the deceased was not alive at the time of heat stress. This is presented in two case reports of men who were shot and later burned. HSP espression could not be found in these cases. In the very early supravital period, particularly the latency and supravital periods, metabolic processes can continue after irreversible cardiac arrest. Extensive cellular stress can lead to an immunohistochemically detectable expression of HSP in this portmortem period. This is shown in the case report of a 49-year-old man who immediately died and burned following the massive detonation of an air mine. Immunohistochemical studies of recovered renal tissues have revealed HSP27 expression in the vessel walls as well as renal tubules, as presented in a third case report. This HSP expression can be considered as a phenomenon of supravitality. Heat stress induces massive cellular stress and the expression of HSP under global ischemic conditions. When vitality is to be determined in cases of suspected fire deaths, the possibility of supravital expression has to be considered., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Fatty acid conjugates of hydroxy-metabolites of tetrahydrocannabinol (THC) or cannabinol have already been reported as metabolites in rats. In the herein presented investigation, palmitic acid esters of THC and its primary metabolite 11-hydroxy-delta9-tetrahydrocannabinol (11-OH-THC) were synthesized using esterification with palmitic acid chloride. Structural elucidation of the products was conducted using nuclear magnetic resonance spectroscopy (NMR) and liquid chromatography coupled to quadrupole time of flight mass spectrometry (LC-QToF-MS). For the confirmation of a previous cannabis use, body fluids (femoral blood, heart blood, urine, bile) of 27 death cases (all with known cannabis use), including adipose tissue homogenates of six of these cases as well as eleven plasma samples (probably all with regular cannabis use, confirmed by a high 11-nor-9-carboxy-delta9-tetrahydrocannabinol (THC-COOH) concentration (except one sample, >200ng/mL), were tested for THC and its main metabolites 11-OH-THC and THC-COOH using gas chromatography coupled to mass spectrometry (GC-MS). These samples as well as further tissue homogenates of autopsy cases (liver, kidney, brain) were additionally tested for the presence of THC palmitic acid ester or 11-OH-THC palmitic acid diester by means of a liquid chromatographic triple quadrupole mass spectrometric (LC-QQQ-MS) method, in order to evaluate a possible presence of these conjugates in humans. In none of the analyzed samples (in total 196 specimens; plasma (N=11), femoral blood (N=23), heart blood (N=25), urine (N=23), bile (N=27), liver (N=27), kidney (N=27), brain (N=27), adipose tissue (N=6)), palmitic acid esters of THC or 11-OH-THC could be proven. Even if the existence of these esters in human samples cannot be ruled out definitely, suitability as cannabis consumption markers does not seem likely based on our findings., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Since there are no characteristic morphological findings post mortem diagnosis of diabetes mellitus and identification of diabetic coma need to be confirmed by suitable biomarkers. The postmortem identification of preexisting hyperglycemia or diabetic coma can be difficult if the matrices for the determination of the established biomarkers are not available or the obtained results are close to the established cut-off values. 1,5-Anhydroglucitol (1,5-AG), the 1-deoxy form of glucose, competes with glucose for renal reabsorption. Therefore low serum concentrations of 1,5-AG, reflect hyperglycemic excursions over the prior 1-2 weeks in diabetic patients. To evaluate postmortem 1,5-AG concentrations in vitreous humor (VH) and cerebrospinal fluid (CSF), a liquid chromatographic mass spectrometric method for the quantification of 1,5-AG in VH and CSF was developed and validated according to international guidelines. In order to establish a cut-off for the identification of an ante mortem existing diabetes and the diagnosis of a diabetic coma in deceased the relationships between 1,5-AG concentrations in VH and CSF to other diabetes associated biochemical parameters of 47 non-diabetic, 86 diabetic and 9 cases of diabetic coma were examined. In 83 of these cases, both matrices could be obtained and analyzed. Comparisons of the respective HbA1c, Glucose in VH or Sum-formula of Traub to 1,5-AG concentrations in VH and CSF resulted in correlation coefficients R 2 ≤0.2. For the application of 1,5-AG concentrations in VH against CSF, a linear regression gave a correlation coefficient of R 2 =0.955. Comparable linear correlations of 1,5-AG concentrations could be observed between VH and femoral venous blood (FVB) (R 2 =0.839) as well as between CSF and FVB (R 2 =0.756). Due to overlapping concentration ranges, the determination of a reliable cut-off for the differentiation of diabetic disease to diabetic coma cases was not possible. However, the 1,5-AG concentrations in VH and CSF in cases of deceased diabetics were significantly lower (p<0.05) than in non-diabetic deceased and therefore indicate a pre-existing diabetes or even a diabetic coma as the cause of death., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Because of the lack of characteristic morphological findings post mortem diagnosis of diabetes mellitus and identification of diabetic coma can be complicated. 1,5-Anhydroglucitol (1,5-AG), the 1-deoxy form of glucose, competes with glucose for renal reabsorption. Therefore low serum concentrations of 1,5-AG, reflect hyperglycemic excursions over the prior 1-2 weeks in diabetic patients. Next to clinical applications determination of 1,5-AG can also be used in forensic analysis. To investigate the elimination of 1,5-AG, a liquid chromatographic-mass spectrometric method for the determination of 1,5-AG and creatinine in urine was developed and validated according to international guidelines. To evaluate ante mortem concentrations of 1,5-AG spot urine samples of 30 healthy subjects, 46 type 1 and 46 type 2 diabetic patients were analyzed. 1,5-AG urine concentrations of diabetic patients were significantly (p<0.001) lower (mean: 1.54μg/ml, n=92) compared to concentrations of healthy subjects (mean: 4.76μg/ml, n=30) which led to the idea that 1,5-AG urine concentrations post mortem might help in the interpretation of a diabetic coma post mortem. Urine of 47 deceased non-diabetics, 37 deceased diabetic and 9 cases of diabetic coma were measured. Comparison of blood and urine 1,5-AG concentrations in clinic samples (linear, R 2 =0.13) and forensic samples (linear, R 2 =0.02) showed no correlation. Urinary levels of 1,5-AG in deceased diabetic (mean 6.9μg/ml) and in non-diabetic patients (mean 6.3μg/ml) did not show a significant difference (p=0.752). However, urinary 1,5-AG concentrations in deceased due to diabetic coma (mean: 1.7μg/ml) were significantly lower than in non-diabetic (mean: 6.3μg/ml, p=0.039) and lower than in diabetic cases (mean: 4.7μg/ml, p=0.058). The determination of a reliable cut-off for the differentiation of diabetic to diabetic coma cases was not possible. Normalization of urinary 1,5-AG concentrations with the respective creatinine concentrations did not show any gain of information. In clinical (serum) and forensic blood samples a significant difference between all groups could be detected (p<0.05). Comparison of blood and urine 1,5-AG concentrations in clinical samples (linear, R 2 =0.13) and forensic samples (linear, R 2 =0.02) showed no correlation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Due to a lack of reference values for blood concentration of metformin in the literature, the forensic evaluation of metformin findings in blood samples is difficult. Interpretations with regard to the assessment of blood concentrations as well as an estimation of the ingested metformin amounts are often vague. Furthermore, post mortem evaluation of death due to lactic acidosis because of metformin is difficult since renal performance or lactate concentrations can not always reliably be determined after death. To describe a concentration range in clinical samples after chronic use of metformin, metformin serum concentrations were determined in serum samples of 95 diabetic patients receiving daily doses of 500mg-3000mg of metformin. The analyses of metformin was carried out using a validated high performance liquid chromatograph coupled to triple quadrupole mass spectrometry (LC-QQQ-MS). On average, metformin concentrations were 1846ng/mL (2 =0.707). Results of the herein presented study are useful for the interpretation of analytical metformin findings in forensic toxicology. The utility of the described concentration range is demonstrated by discussing a death case involving a fatal lactate acidosis after metformin intake and renal failure. Usefulness of the parameters metformin blood concentration, lactate concentration and glomerular filtration rate in post mortem cases of lactic acidosis due to metformin intoxication are discussed., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Recently, phase-II-metabolites of γ-hydroxybutyric acid (GHB), namely GHB-β-O-glucuronide and GHB-4-sulfate, were implemented in the scope of drug testing methods The clearance of GHB from the circulation is extremely fast due to its incorporation into the metabolic pathway of the citrate cycle. The elimination half-life of GHB from blood was reported to be dose dependent between 30 and 50min resulting in narrow detection windows of less than 12h after illicit administration or cases of drug facilitated sexual assault regardless of the biological matrix used. As sulfated metabolites tend to show prolonged half-lives and slower elimination kinetics compared to unmodified or glucuronidated drugs, the potential of GHB-4-sulfate in prolonging the detection of GHB administration was assessed. Its urinary concentrations were determined in n=100 samples from athletes and n=50 samples from sport students, and the resulting data were used to calculate a preliminary reference population-based threshold for urinary GHB-sulfate concentration. The threshold was then compared to concentrations found in post-administration urine samples collected from 3 volunteers who administered GHB within the setting of a clinical trial. Due to the large inter-individual variability of concentrations found in the reference population, GHB-4-sulfate itself was not suitable to prolong the detection times for GHB applications, even when specific gravity-corrected values were used. Therefore, a metabolomics-based approach was applied to the reference population samples and evaluated regarding other urinary metabolites that potentially correlate with the urinary excretion of GHB-4-sulfate and GHB-β-O-glucuronide in order to find a suitable marker to normalize urinary concentrations. The most promising candidate was found at a molecular mass of 321.0696 and was preliminarily identified as β-citryl-glutamic acid., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Gamma-hydroxybutyric acid (GHB) can be used as a knock-out drug in drug facilitated crime (DFC). Due to its rapid metabolism and resulting narrow detection window, uncovering GHB use in DFC still constitutes a problem. In this experiment we determined the GHB and GHB-β-O-glucuronide (GHB-Gluc) concentrations in hair samples after single and chronic GHB exposures. Hair samples of three narcoleptic patients therapeutically taking sodium oxybate (GHB-sodium-salt) were collected. Patients 1 (P1) and 2 (P2) took the medication for nine and six years, respectively. P1 took daily the pharmaceutical Xyrem ® in a total dose of 5.78g GHB at bed time (2.89g) and four hours (2.89g) later. P2 took a dose of 3.10g GHB at bed time and an additional dose of 2.68g GHB four hours later. Patient 3 (P3) was newly diagnosed with narcolepsy and started his therapy with oral dose of 6g (divided in three portions of 2g GHB) within 24h. The hair samples were extracted both with and without forerunning washing steps. GHB and GHB-Gluc were determined by a published ultra-high performance liquid chromatography-tandem mass spectrometry method using GHB-d 6 and GHB-Gluc-d 4 as internal standards. GHB and GHB-Gluc concentrations in unwashed hair samples of P1 and P2 were determined in a range of 0.56-1.30ng/mg and <0.48-0.85ng/mg, respectively. In washed hair samples of P1 and P2 the concentrations were in a range of <0.32-0.68ng/mg and <0.48-1.20ng/mg for GHB and GHB-Gluc, respectively. The determined concentrations were within the published endogenous range. The confirmed results showed that the washing procedure before extraction causes a minor decrease of GHB concentrations in hair (difference: <1ng/mg). The investigations showed that a single GHB exposure might not be determined by hair analysis of GHB and GHB-Gluc. The chronical intake of therapeutic sodium oxybate with doses up to 7g per night was also not confirmed by hair analysis maybe due to hair treatments. Therefore, GHB hair analysis should be assessed critically and determined negative results could not exclude GHB exposures., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In cases of death caused by hypothermia, histological analysis can be used to determine the cause of death. Certain histological alterations of the pituitary glands in hypothermia have been reported in the literature, including haemorrhage, hyperaemia and cellular vacuolization of cells in the anterior lobe. In the present study, the validity of these morphological alterations as markers for fatal accidental hypothermia was investigated in autopsy material. A total of 34 pituitary glands in cases of verified fatal accidental hypothermia were examined histologically (haematoxylin and eosin, ferric, azan) and immunohistochemically (LCA, ACTH, C5b-9). The findings were compared with 61 cases in a control group. Hyperaemia was found in 50.0% of the study group cases and 59.0% of the control group cases. Cellular vacuolization was observed in one case (2.9%) in the study group and one case (1.6%) in the control group. Acute or recent haemorrhage in the glandular tissue was never detected. In our study, the histopathological characteristics described in the literature as pathognomonic for hypothermia could not be confirmed. Furthermore, histological differences in the pituitary glands between fatal hypothermia cases and control group cases were not observed.

After contact shots to the head biological traces inside firearm barrels can be found. This study was conducted to simulate and to evaluate such staining. Five current handguns of four inch barrel length in the calibre .22 long rifle, 7.65mm Browning, 9mm Luger and .38 special were used to perform 24 contact shots on silicone coated, gelatine filled box models using the triple contrast method. The staining was documented by endoscopy and swabs gathered from both ends of the barrel were analysed by quantitative PCR. With the exception of the .22 revolver, all firearms showed distinct staining which decreased from the muzzle to the rear end of the barrel. The pattern was varied, showing droplets, elongated forms or stripes. In 14 of 24 shots, staining reached the chamber. The staining results were comparable to real suicide cases., (Copyright © 2017 Elsevier B.V. All rights reserved.)

For the estimation of time since death in the early postmortem interval, a large repertoire of valid methods is available. With increasing time since death the estimation of the postmortem interval becomes more imprecise and unreliable because validated methods are missing. Wehner et al. developed a method to estimate the time since death using immunohistochemical staining. Proteins undergo degradation after death and this reduces their stainability. Previous studies have explored the development of a method to estimate the time since death using immunohistochemical staining. A systematic analysis (Wehner et al., 1999-2002) demonstrated that calcitonin can still be stained 4 days, thyreoglobulin 5 days, glucagon 6 days, and insulin 12 days after death. After 12 days, calcitonin and thyroglobulin can no longer be stained, glucagon after 14 days, and insulin after 29 days. The aim of the present study was to test this original data on independent case material. Included in this control study were 105 cases with known time since death (between several hours and 22 days). Pancreatic tissue was stained for insulin and glucagon, and the thyroid gland for thyreoglobulin and calcitonin. The original findings could be generally confirmed, however for calcitonin and thyreoglobulin we observed earlier negative stainings. Altogether immunohistochemistry may still be useful as an additional method for estimating time since death in forensic cases., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A male driver was checked during a traffic stop. A blood sample was collected 35min later and contained 7.3ng/mL THC, 3.5ng/mL 11-hydroxy-THC and 44.6ng/mL 11-nor-9-carboxy-THC. The subject claimed to have used two commercially produced products topically that contained 1.7ng and 102ng THC per mg, respectively. In an experiment, three volunteers (25, 26 and 34 years) applied both types of salves over a period of 3days every 2-4h. The application was extensive (50-100cm 2 ). Each volunteer applied the products to different parts of the body (neck, arm/leg and trunk, respectively). After the first application blood and urine samples of the participants were taken every 2-4h until 15h after the last application (overall n=10 urine and n=10 blood samples, respectively, for each participant). All of these blood and urine samples were tested negative for THC, 11-hydroxy-THC and 11-nor-9-carboxy-THC by a GC-MS method (LoD (THC)=0.40ng/mL; LoD (11-hydroxy-THC)=0.28ng/mL; LoD (THC-COOH)=1.6ng/mL;. LoD (THC-COOH in urine)=1.2ng/mL). According to our studies and further literature research on in vitro testing of transdermal uptake of THC, the exclusive application of (these two) topically applied products did not produce cannabinoid findings in blood or urine., (Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.)

Analysis of the anesthetic agent propofol in biological samples by LC-MS/MS is a great challenge due to weak fragmentation and poor ionization efficacy of propofol resulting in weak signal intensities. Improvements of the ionization and fragmentation efficacy can be achieved by conversion of propofol to its dimethylimidazolesulfonyl (DMIS) derivative by a derivatization reaction using 1,2-dimethylimidazole-4-sulfonyl chloride (DMISC). This DMIS derivative produced intense [M + H] + ions in positive-ion LC-ESI-MS/MS with the dimethylimidazole moieties representing the most abundant product ions. Derivatization of serum samples is achieved by direct conversion of the acetonitrile supernatant of a protein precipitation with DMISC followed by a double liquid-liquid extraction using n-hexane. Reliability of the method was confirmed under consideration of the validation parameters selectivity, linearity, accuracy and precision, analytical limits, and processed sample stability. Linearity was demonstrated over the whole calibration range from 5 to 1000 ng/ml with the use of a 1/x 2 weighting. Stability of the processed samples was verified for a time period of up to 25 h. Due to its high sensitivity, appropriate quantification and detection limits (LLoQ = 5 ng/ml, LoD = 0.95 ng/ml) for toxicological propofol analyses could be achieved. Applicability of the method to biological samples could be verified by analysis of a human serum sample collected after propofol-induced sedation. Graphical abstract A novel derivatization strategy using 1,2-dimethylimidazole-4-sulfonyl chloride (DMISC) was developed to improve the ionization and fragmentation efficacy of propofol for LC-ESI-MS/MS analysis.

The precision of death time estimation by vitreous potassium using 5 different formulas is compared on a random sample of 600 cases. Mean differences, standard deviations and 95% limits of confidence between real and extrapolated time since death were calculated. The best results were obtained using equations with "mean" slopes of about 0,17 or 0,19mmol/l per hour. Equations with a steeper or flatter slope reveal already great mean deviations between real and extrapolated time since death and do not allow a reliable death time estimation., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

This study evaluated the normal number of inflammatory cells in the heart in the first year of life using two methods to compare their ability to quantitate physiological myocardial infiltration. Eight endomyocardial samples from both ventricles were obtained at autopsy from 56 structurally normal hearts during the first year of life. In each sample the numbers of leucocytes, T-lymphocytes and macrophages were counted once in 20 randomly chosen high-power fields (400×) as well as in a 10mm(2) area of randomly chosen myocardial tissue (100×) by two independent investigators. Compared to the literature a greater representative proportion of myocardial tissue was analyzed. The results of the enumeration in mm(2) were converted into high-power-fields to compare both methods. The mean numbers and standard deviations for leucocytes, T-lymphocytes and macrophages were calculated. Both counting methods showed similar results with low inflammatory cell counts per single heart and staining. A greater understanding of the physiological myocardial infiltration by leucocytes, T-lymphocytes and macrophages is important for postmortem forensic cases, and for the interpretation of endomyocardial biopsies in infants., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)