From rare events to systematic data collection: the RESCUED registry for sudden cardiac death in the young in Germany.

BACKGROUND: Approximately one-third of sudden cardiac deaths in the young (SCDY) occur due to a structural cardiac disease. Forty to fifty percent of SCDY cases remain unexplained after autopsy (including microscopic and forensic-toxicological analyses), suggesting arrhythmia syndromes as a possible cause of death. Due to the possible inheritability of these diseases, blood relatives of the deceased may equally be carriers of the causative genetic variations and therefore may have an increased cardiac risk profile. A better understanding of the forensic, clinical, and genetic data might help identify a subset of the general population that is at increased risk of sudden cardiac death. STUDY DESIGN: The German registry RESCUED (REgistry for Sudden Cardiac and UnExpected Death) comprises information about SCDY fatalities and clinical and genetic data of both the deceased and their biological relatives. The datasets collected in the RESCUED registry will allow for the identification of leading causes of SCDY in Germany and offer unique possibilities of scientific analyses with the aim of detecting unrecognized trends, risk factors, and clinical warning signs of SCDY. In a pilot phase of 24 months, approximately 180 SCDY cases (< 50 years of age) and 500 family members and clinical patients will be included. CONCLUSION: RESCUED is the first registry in Germany collecting comprehensive data of SCDY cases and clinical data of the biological relatives reviewed by cardiac experts. RESCUED aims to improve individual risk assessment and public health approaches by directing resources towards early diagnosis and evidence-based, personalized therapy and prevention in affected families. Trial registration number (TRN): DRKS00033543.

Detrimental effects of scene manipulations on temperature-based time since death estimation.

In forensic casework, time since death (TSD) estimations may play a crucial role to establish chains of events as well as for alibi assessment in homicide cases. Classical TSD estimation relies on reasonably stable ambient temperatures and a correct documentation of ambient and rectal temperatures. This constancy is in some cases disturbed by post-discovery alterations of the crime scene, e.g. opening a window. In order to develop a better understanding of this alteration-based detrimental impact on TSD estimation as well as to identify feasible recommendations for casework, the present pilot study examined ambient temperature effects of different window opening scenarios regarding various time intervals (5 to 360 min) in a furnished 10 m2 apartment during winter. In this context, in addition to the ambient temperature and thus the cooling rate of the room, re-approximation to initial room temperature, potential influences on a nomogram-based time since death estimation using a fictitious case, and the impact of the measurement height above the ground were investigated. Our data indicate a significant reduction of the mean temperature decrease rate after 15 min regardless of the remaining opening time and a correlation with the size of the respective opening surfaces. Re-approximation to initial room temperatures was observed with up to three times longer than the initial opening time. There was no evidence of a substantial advantage of temperature measurements above the level of the corpse (> 0.1 m). The limitations of the study and its applicability for forensic casework are critically reviewed.

Esophageal rupture through extreme sadomasochistic practice.

We report the case of a woman in her thirties who suffered an esophageal rupture while participating in extreme sadomasochistic practices. After herself seeking help in a hospital for complaints alleged to be from a fall, she was initially diagnosed with several broken ribs and a pneumothorax. The cause of the pneumothorax was later discovered to be an esophageal rupture. When confronted with this atypical injury for a fall, the woman admitted to have accidentally swallowed an inflatable gag, which her partner had afterwards inflated. In addition to the esophageal rupture, the patient also had numerous other externally visible injuries of various ages, reportedly also from sadomasochistic acts. Although an in-depth police investigation was conducted and a "slave contract" was found, the woman's consent to the extreme sexual practices performed by her life partner could not be substantiated conclusively. The man was convicted for intentional infliction of serious as well as dangerous bodily injury and sentenced to a long term in prison.

Establishment of a multiplex PCR-CE assay for the simultaneous and rapid analysis of age markers for Calliphora vicina pupae.

Insects, especially blow flies, are forensically relevant to determine the minimal postmortem interval (PMImin), based on the fact that they are usually the first colonisers of a body. By estimating the age of immature blow flies, interferences can be made about the time since death. Whilst morphological parameters are valuable for age estimation of blow fly larvae, gene expression profiling is more applicable for blow fly pupae. Here, the age-dependent changes in the gene expression levels during the development are analysed. 28 temperature-independent markers have already been described for the age estimation of pupae of the forensically important blow fly Calliphora vicina and are analysed by RT-qPCR. To allow simultaneous analysis of these age markers, a multiplex assay was developed in the present study. After reverse transcription, the markers are analysed simultaneously in an endpoint PCR and subsequently separated by capillary electrophoresis (CE). This method is highly attractive due to its quick and easy procedure and interpretation. The present age prediction tool was adapted and validated. The multiplex PCR assay reproduced the same expression profiles as the RT-qPCR assay based on the same markers. The statistical evaluation shows that the new assay has a lower precision but a better trueness for age determination compared to the RT-qPCR assay. Since the new assay is also qualified to estimate the age of C. vicina pupae and is practical, cost-effective and, even more importantly, time-saving, it is attractive for use in forensic casework.

Influence of microRNAs on iNOS expression in postmortem human infarction hearts.

MicroRNAs (miRNAs) are important post-transcriptional regulators in several diseases, including cancer, immunologic and cardiovascular diseases. A growing list of miRNAs are dysregulated in cardiac arrhythmias, contractility diseases, myocardial infarction (MI), sudden cardiac death (SCD), chronic heart failure and hypertrophy. However, the exact regulatory pathways, through which miRNAs exert their effects are often unclear. In this study, we measured the expression patterns of miR-21, miR-939 and miR-30e in postmortem human MI. The aim of the study was to examine the influence of these miRNAs on cardiac inducible nitric oxide synthase (iNOS) mRNA levels. We measured iNOS mRNA and miRNA expression patterns by means of qPCR. Further we used correlation analyses to determine causality between miRNA expression and cardiac iNOS levels. iNOS mRNA, miR-21, miR-939 and miR-30e were significantly upregulated in infarcted and non-infarcted regions of postmortem human MI hearts in comparison to healthy controls. While miR-21 and miR-939 showed their strongest expression in infarcted regions, miR-30e peaked in the non-infarcted myocardium. Further, we found a significant correlation between miR-939 and iNOS expression levels in controls and infarcted regions. The results indicate, that miR-939 is a regulator of cardiac iNOS expression. However, a massive iNOS activation might exceed the capability of miR-939 to keep its expression in balance. miR-21 and miR-30e do not seem to influence cardiac iNOS levels in MI. Further studies are needed to evaluate downstream targets of these miRNAs and their signaling pathways to clarify their role in human MI.

Forensic, legal, and clinical aspects of deaths associated with implanted cardiac devices.

As the population ages, the prevalence of heart failure and individuals wearing an implanted cardiac device is increasing. The combination of different underlying pathophysiologies and (the combination of) implanted cardiac devices can become a challenge with regard to the determination of cause and manner of death in such individuals. Additionally, heart disease is frequently associated with mental disease, ranging from anxiety and depression to suicidality and suicide (attempts). At the same time, the correct diagnosis of cause and manner of death is the basis for quality assurance, further therapeutic advances, legal safety, and suicide prevention. By that, an interdisciplinary field between legal medicine, clinicians, and law enforcement opens up. In this field, the different participants can simultaneously benefit from and need each other. For example, legal medicine experts need investigatory results and clinical expertise for the interpretation of readout data of implanted cardiac devices in order to correctly determine the cause of death. A correctly determined cause of death can assist law enforcement and help clinicians to further improve various therapeutic approaches based on correct mortality data collection. In addition, it is the basis for identification of suicides of device carriers, allowing psychological and psychiatric experts to better understand the burden of mental disease in this particular cohort. Against this interdisciplinary background, this manuscript summarizes information about psychiatric comorbidities and suicidality while being on a device. Thereby, basic information on complications and malfunctions of implanted cardiac devices, device-associated deaths with particular emphasis on device manipulation is displayed as basic information needed for correct determination of the cause of death. Also, legal and ethical issues in this field are outlined. The final result is a proposal of an interdisciplinary assessment workflow for a conjoint approach to improve the diagnosis of deaths associated with implanted cardiac devices. It will allow for a differentiation between an individual who died with or due to the device.

Suitable biomarkers for post-mortem differentiation of cardiac death causes: Quantitative analysis of miR-1, miR-133a and miR-26a in heart tissue and whole blood.

Cardiovascular diseases are the most common causes of death worldwide. Cardiac death can occur as reaction to myocardial infarction (MI). A diagnostic challenge arises for sudden unexpected death (SUD) cases with structural abnormalities (SA) or without any structural abnormalities (without SA). Therefore, the identification of reliable biomarkers to differentiate cardiac cases from each other is necessary. In the current study, the potential of different microRNAs (miRNAs) as biomarkers in tissue and blood samples of cardiac death cases was analyzed. Blood and tissue samples of 24 MI, 21 SUD and 5 control (C) cases were collected during autopsy. Testing for significance and receiver operating characteristic analysis (ROC) were performed. The results show that miR-1, miR-133a and miR-26a possess a high diagnostic power to discriminate between different cardiac death causes in whole blood and in tissue.

iNOS expressing macrophages co-localize with nitrotyrosine staining after myocardial infarction in humans.

Introduction: Inducible nitric oxide synthase (iNOS) produces micromolar amounts of nitric oxide (NO) upon the right stimuli, whose further reactions can lead to oxidative stress. In murine models of myocardial infarction (MI), iNOS is known to be expressed in infiltrating macrophages, which at early onset enter the infarcted zone and are associated with inflammation. In contrast cardiac tissue resident macrophages are thought to enhance regeneration of tissue injury and re-establish homeostasis. Both detrimental and beneficial effects of iNOS have been described, still the role of iNOS in MI is not fully understood. Our aim was to examine cell expression patterns of iNOS and nitrotyrosine (NT) production in human MI. Material and Methods: We examined in postmortem human MI hearts the iNOS mRNA expression by means of qPCR. Further we performed immunohistochemical stainings for cell type identification. Afterwards a distance analysis between iNOS and NT was carried out to determine causality between iNOS and NT production. Results: iNOS mRNA expression was significantly increased in infarcted regions of human MI hearts and iNOS protein expression was detected in resident macrophages in infarcted human hearts as well as in controls hearts, being higher in resident macrophages in MI hearts compared to control. Furthermore in MI and in healthy human hearts cells showing signs of NT production peaked within 10-15 µm proximity of iNOS+ cells. Discussion: These results indicate that, unexpectedly, resident macrophages are the main source of iNOS expression in postmortem human MI hearts. The peak of NT positive cells within 10-15 µm of iNOS+ cells suggest an iNOS dependent level of NT and therefore iNOS dependent oxidative stress. Our results contribute to understanding the role of iNOS in human MI.

Changes in the pattern of suicides and suicide attempt admissions in relation to the COVID-19 pandemic.

The consequences of the current COVID-19 pandemic for mental health remain unclear, especially regarding the effects on suicidal behaviors. To assess changes in the pattern of suicide attempt (SA) admissions and completed suicides (CS) in association with the COVID-19 pandemic. As part of a longitudinal study, SA admissions and CS are systematically documented and analyzed in all psychiatric hospitals in Frankfurt/Main (765.000 inhabitants). Number, sociodemographic factors, diagnoses and methods of SA and CS were compared between the periods of March-December 2019 and March-December 2020. The number of CS did not change, while the number of SA significantly decreased. Age, sex, occupational status, and psychiatric diagnoses did not change in SA, whereas the percentage of patients living alone while attempting suicide increased. The rate and number of intoxications as a SA method increased and more people attempted suicide in their own home, which was not observed in CS. Such a shift from public places to home is supported by the weekday of SA, as the rate of SA on weekends was significantly lower during the pandemic, likely because of lockdown measures. Only admissions to psychiatric hospitals were recorded, but not to other institutions. As it seems unlikely that the number of SA decreased while the number of CS remained unchanged, it is conceivable that the number of unreported SA cases increased during the pandemic. Our data suggest that a higher number of SA remained unnoticed during the pandemic because of their location and the use of methods associated with lower lethality.

Gunshots through laminated glass: expelled compounded fragments as a function of bullet type.

In the frame of an experimental setting, the formation of round-shaped compounded glass fragments on the exit site after gunshots through a windshield was examined. For that purpose, a 9 × 19 mm pistol (HK P30) and two different cartridges containing (a) a full metal jacketed round-nosed projectile and (b) a deformation projectile were used. On the basis of 52 gunshots, the morphology, impact angles and terminal ballistics of occurring compounded glass fragments were examined. The results showed that the compounded glass fragments' morphology allowed for the differentiation of two used projectiles. Fragments were able to cause round-shaped defects in a single cotton layer (T-shirt) with subsequent penetration of up to 2.4 cm into ballistic gelatin (10%, 4 °C). As a function of the projectile type, the compounded glass fragments showed different reproducible impact angles that differed notably from the known conical pattern of expelled glass fragments after bullet penetration. These findings might help to explain the atypical morphology of gunshot wounds with laminated glass as an intermediate target and prevent possible misinterpretations when reconstructing the sequence of events.

Atypical wound trajectory after a tangential pistol shot.

Three intermediate-range shots from a Browning, model 1955, 7.65 mm caliber, pistol were fired from the driver's seat of a car at a woman in the passenger seat. She sustained three wounds: An, ultimately fatal, penetrating head wound, a graze wound across her forehead, and a tangential, perforating, wound, with bullet entry over the medial sternum and exit through the right flank. Neither postmortem CT nor forensic autopsy discovered bony thoracic injuries or perforations of the thoracoabdominal cavities. There was pulmonary contusion in the medial lobe of the right lung and hemorrhage in the adipose tissue around the right kidney. The tangential bullet had left an almost 40-cm-long wound channel through a pronounced layer of subcutaneous fat. Based on 3D reconstructed CT-data determinations, a straight bullet trajectory between entry and exit wounds would have traversed the abdominothoracic cavities, right lung, and liver. The actual trajectory, however, described a prominent curve, without signs of deflection by bone. Postulated explanations for this unusual bullet track are that the woman was twisting her body in a dynamic scene when the bullet struck; further, due to its shallow angle of incidence on the skin, the bullet was deflected to an intracutaneous path. Additionally, soft tissue resistance may have caused the bullet to yaw. Caution should, thus, be exercised when reconstructing bullet trajectories solely from entry and exist wounds, also for bullet wounds through basically homogenous soft tissues.

Particulate matter emissions during autopsies: a method to reduce exposure.

Particulate matter emitted during autopsies can serve as a vector for numerous viruses or bacteria and can lead to infections. Reducing the exposure of those particles in indoor working environments is, therefore, an important issue. To assess the health risk for employees in forensic medicine, we measure particulate matter in the ambient air during autopsies by using an aerosol spectrometer. The autopsies were performed with either an ordinary oscillating saw or an adapted saw with a suction unit. The particle emissions from both saws were compared to each other in order to evaluate whether a technical adaption leads to a particle reduction. Furthermore, the particle exposure reduction by wearing a face mask and variations in the background concentration in the room were analyzed. High particle concentrations were measured while using the ordinary saw. By using the adapted saw or wearing a face mask, the particle exposure could mostly be avoided. On the majority of the working days, an increase in the background concentration could be observed. Based on this knowledge, the use of a proper suction unit and wearing a face mask during autopsies is necessary. Besides, it is important to have sufficient ventilation in the room so that long-lasting high background concentrations can be prevented.

Variant interpretation in molecular autopsy: a useful dilemma.

Sudden cardiac death (SCD) in adolescents and young adults may be the first manifestation of an inherited arrhythmic syndrome. Thus identification of a genetic origin in sudden death cases deemed inconclusive after a comprehensive autopsy and may help to reduce the risk of lethal episodes in the remaining family. Using next-generation sequencing (NGS), a large number of variants of unknown significance (VUS) are detected. In the majority of cases, there is insufficient evidence of pathogenicity, representing a huge dilemma in current genetic investigations. Misinterpretation of such variants may lead to inaccurate genetic diagnoses and/or the adoption of unnecessary and/or inappropriate therapeutic approaches. In our study, we applied current (ACMG) recommendations for variant classification in post-mortem genetic screening of a cohort of 56 SCD victims. We identified a total 53 rare protein-altering variants (MAF < 0.2%) classified as VUS or worse. Twelve percent of the cases exhibited a clinically actionable variant (pathogenic, likely pathogenic or VUS - potentially pathogenic) that would warrant cascade genetic screening in relatives. Most of the variants detected by means of the post-mortem genetic investigations were VUS. Thus, genetic testing by itself might be fairly meaningless without supporting background data. This data reinforces the need for an experienced multidisciplinary team for obtaining reliable and accountable interpretations of variant significance for elucidating potential causes for SCDs in the young. This enables the early identification of relatives at risk or excludes family members as genetic carriers. Also, development of adequate forensic guidelines to enable appropriate interpretation of rare genetic variants is fundamental.

Assessment of renal glomerulosclerosis and thickness of the carotid intima-media complex as a means of age estimation in Western European bodies.

INTRODUCTION: The estimation of age-at-death of unidentified cadavers is a central aspect of the identification process. With increasing age, the incidence of glomerulosclerosis and the thickness of the carotid wall have been observed to also increase. This correlation has been demonstrated in various international histological studies. The aim of our study was to assess whether these correlations also apply to a Western European population. METHODOLOGY: In this retrospective observational study, kidney and common carotid artery samples from 216 cases autopsied at the Institute of Legal Medicine at the Justus-Liebig University in Giessen, Germany, were examined. Only cases with available tissue samples from both body sides were included. Exclusion criteria were poor sample quality and an age younger than 21 years. After histological processing, the tissue samples were assessed and digitally evaluated. Regression and classification analyses were used to investigate the correlation between age-at-death and intima-media thickness and age-at-death and the incidence of renal glomerular sclerosis. RESULTS: Of the 216 autopsy cases, 183 were included for evaluation. Analysis of the carotid artery segments showed a strong correlation (Pearson correlation coefficient r = 0.887) between the intima-media-complex thickness and chronological age. Classification of the glomerulosclerotic incidence showed a correlation of 37.7-43.1% with the predicted age group. DISCUSSION: Both the intima-media thickness and the proportion of sclerotic glomeruli can be used to estimate age in Western European cadavers. On the basis of these results, both methods are suited to supplement other already established methods for age-at-death estimation in the identification of an unknown cadaver.

Postmortem fluorescence angiography of the explanted human heart.

Within the scope of this technical report, the feasibility of indocyanine green (ICG) as a fluorescent agent for postmortem angiography of the heart is tested. The study included 4 deceased persons with no respective medical history of heart diseases. The basic patterns of findings in ICG fluorescence angiography associated with healthy hearts are presented. The method can easily be integrated into a workflow without restricting the macroscopic or histologic diagnostics. This paper represents the fundamental technical and analytical basis for upcoming studies concerning the possibilities and limitations of fluorescence angiography in the diagnosis of heart pathology.

Retrograde dye perfusion of the proximal aorta - A postmortem technical study.

Introduction: Multiple cardiovascular conditions can lead to unexpected fatality, which is defined as sudden cardiac death. One of these potentially underlying conditions is aortic regurgitation, which can be caused by discrete changes of the geometry of the proximal aorta. To analyze aortic valve competency and furthermore to elucidate underlying pathological alterations of the coronary arteries and the vasa vasorum a perfusion method to simulate a diastolic state was designed. Material and methods: A postmortem approach with retrograde perfusion of the ascending aorta with methylene blue was applied to three bodies. The procedure comprised cannulation of the brachiocephalic trunk, clamping of the aortic arch between brachiocephalic trunk and left carotid artery, infusion of 250 ml of methylene blue, and optical clearing of the superficial tissue layers after perfusion. Organs were examined directly following perfusion and after optical clearing. Results: Assessment and visualization of aortic valve competency and the vasa vasorum were possible in all three instances. Visualization of the coronary perfusion was impaired by postmortem thrombus formation. Optical clearing did not provide additional information. Discussion: The method presented here is a time- and cost-efficient way of visualizing aortic valve competency and the vasa vasorum. The visualization of the vasa vasorum highlights the potential of this method in basic research on diseases of the great arteries and coronaries. However, for a time-efficient functional analysis of the coronaries, other methods must be applied.

It is all about the insects: a retrospective on 20 years of forensic entomology highlights the importance of insects in legal investigations.

This study highlights the importance of insect evidence by evaluating 949 insect-associated cases, including 139 entomological reports, from 2001 to 2019 at the Institute of Legal Medicine Frankfurt/Germany. With a high number of cases in the summer months and a low number in the colder season, 78.5% of the bodies were found indoors, regardless of year or month. In more than 80% of the cases, where PMI information was available (n = 704), the presumed PMI ranged from 1 to 21 days, a period during which entomological evidence can provide a day-specific estimate of PMImin. In cases where insects have been identified to species level (n = 279), most bodies were infested by one or two species with a maximum of 10 different species. Overall, a total of 55 insect species were found. Information on biology, activity and distribution of the most abundant taxa is given and applied for 5 case histories estimating different PMImins of up to over 6 months. Despite proved importance and scientific development of forensic entomology, insects are still rarely considered as a tool in forensic case work. The main reasons are a lack of awareness and (too) late involvement of a forensic entomologist. Our work shows that forensic entomology is an independent discipline that requires specialist expertise.

Genetic analysis of sudden unexpected death cases: Evaluation of library preparation methods to handle heterogeneous sample material.

Over the past years, next-generation sequencing (NGS) technologies revolutionized the possibilities in a broad range of application areas. Also in the field of forensic genetics, NGS continuously gained in importance and attentiveness. A significant number of sudden cardiac deaths (SCD) in the young is due to heritable arrhythmia syndromes emphasizing the need of examining the genetic basis in these cases also with regard to the identification of relatives and/or patients being at risk. As a result, high-throughput methods became of increasing value in molecular autopsy investigations enabling the analysis of a broad spectrum of genes. Most standard protocols are optimized for high-quality samples and frequently not directly applicable to challenging forensic sample material. In the present study, we intended to examine a comprehensive gene panel associated with SCD and inherited arrhythmogenic disorders. We compared three different hybridization-based library preparation technologies in order to implement a suitable NGS workflow for heterogeneous, forensic as well as diagnostic sample material. The results obtained indicated, that the Illumina technologies Nextera DNA Flex and TruSeq were compatible with samples exhibiting varying levels of degradation. In comparison, the TruSight method also resulted in good sequencing data, but seemed to be more dependent on DNA integrity. The preparation protocols evaluated in our study are not restricted to molecular autopsy investigations and might be helpful for and transferrable to further forensic research applications.

To Be There or Not to Be There, That Is the Question-On the Problem of Delayed Sampling of Entomological Evidence.

The aim of the current study was to analyze two major pitfalls in forensic entomological casework: delayed evidence sampling and the effect of low-temperature storage of the body. For this purpose, temperature profiles of heavily infested corpses during cooling and cases in which insect evidence was collected both at the scene and during autopsy were evaluated with regard to species composition and development stages found. The results show that the temperature in the body bags remained at higher average temperatures up to 10 °C relative to the mortuary cooler, therefore, sufficient for larval development, with significant differences in temperature between larval aggregations on one and the same body. In addition, we found large differences both in species number, species composition, and the developmental stages found at the scene and during the autopsy. These data and observations underscore the importance of sampling evidence at the scene and recording temperatures throughout the cooling period of a body.