Fast microglial activation after severe traumatic brain injuries.

Traumatic brain injury is among the leading causes of death in individuals under 45 years of age. However, since trauma mechanisms and survival times differ enormously, the exact mechanisms leading to the primary and secondary injury and eventually to death after traumatic brain injury (TBI) remain unclear. Several studies showed the versatile functions of microglia, the innate macrophages of the brain, following a TBI. Earlier being characterized as rather neurotoxic, neuroprotective capacities were recently demonstrated, therefore, making microglia one of the key players following TBI. Especially in cases with only short survival times, immediate microglial reactions are of great forensic interest in questions of wound age estimation. Using standardized immunohistochemical methods, we examined 8 cases which died causatively of TBI with survival times between minutes and 7 days and 5 control cases with cardiovascular failure as the cause of death to determine acute changes in microglial morphology and antigen expression after TBI. In this pilot study, we detected highly localized changes in microglial morphology already early after traumatic damage, e.g., activated microglia and phagocyted erythrocytes in the contusion areas in cases with minute survival. Furthermore, an altered antigen expression was observed with increasing trauma wound age, showing similar effects like earlier transcriptomic studies. There is minute data on the direct impact of shear forces on microglial morphology. We were able to show localization-depending effects on microglial morphology causing localized dystrophy and adjacent activation. While rodent studies are widespread, they fail to mimic the exact mechanisms in human TBI response. Therefore, more studies focusing on cadaveric samples need to follow to thoroughly define the mechanisms leading to cell destruction and eventually evaluate their forensic value.

Post-mortem in situ stability of serum markers of cerebral damage and acute phase response.

The aim of the given study was to test the in situ stability of biochemical markers of cerebral damage and acute phase response in the early post-mortem interval to assess their usability for forensic pathology. A monocentric, prospective study investigated post-mortem femoral venous blood samples at four time points obtained within 48 h post-mortem starting at the death of 20 deceased, using commercial immunoassays for the ten parameters: S100 calcium-binding protein B (S100B), glial fibrillary acidic protein (GFAP), neuron-specific enolase (NSE), brain-derived neurotrophic factor (BDNF), interleukin-6 (IL-6), C-reactive protein (CRP), procalcitonin (PCT), ferritin, soluble tumor necrosis factor receptor type 1 (sTNFR1), and lactate dehydrogenase (LDH). Significant changes in serum levels were observed only later than 2 h after death for all markers. Inter-laboratory comparability was high, and intra-assay precision was sufficient for most markers. Most of the biomarker levels depended on the severity of hemolysis and lipemia but were robust against freeze-thaw cycles. Serum levels increased with longer post-mortem intervals for S100B, NSE, ferritin, sTNFR1, and LDH (for all p < 0.001) but decreased over this period for CRP (p = 0.089) and PCT (p < 0.001). Largely unchanged median values were found for GFAP (p = 0.139), BDNF (p = 0.106), and IL-6 (p = 0.094). Serum levels of CRP (p = 0.059) and LDH (p = 0.109) did not differ significantly between the final ante-mortem (resuscitation) and the first post-mortem sample (moment of death). Collecting the post-mortem blood sample as soon as possible will reduce the influence of post-mortem blood changes. Serum GFAP for detection of cerebral damage as well as serum IL-6 and CRP as proof of acute phase response seemed to be preferable due to their in situ stability in the first 2 days after death.

Frequency and intensity of pulmonary bone marrow and fat embolism due to manual or automated chest compressions during cardiopulmonary resuscitation.

Iatrogenic consequences of cardiopulmonary resuscitation (CPR) include sternal or rib fractures, pulmonary bone marrow embolisms (BME) and fat embolisms (FE). This report aimed to analyze the frequency and intensity of pulmonary BME and FE in fatal cases receiving final CPR efforts with the use of automated chest compression devices (ACCD) or manual chest compressions (mCC). The study cohort (all cardiac causes of death, no ante-mortem fractures) consisted of 15 cases for each group 'ACCD', 'mCC' and 'no CPR'. Lung tissue samples were retrieved and stained with hematoxylin eosin (n = 4 each) and Sudan III (n = 2 each). Evaluation was conducted microscopically for any existence of BME or FE, the frequency of BME-positive vessels, vessel size for BME and the graduation according to Falzi for FE. The data were compared statistically using non-parametric analyses. All groups were matched except for CPR duration (ACCD > mCC) but this time interval was linked to the existence of pulmonary BME (p = 0.031). Both entities occur in less than 25% of all cases following unsuccessful CPR. BME was only detectable in CPR cases, but was similar between ACCD and mCC cases for BME frequency (p = 0.666), BME intensity (p = 0.857) and the size of BME-affected pulmonary vessels (p = 0.075). If any, only mild pulmonary FE (grade I) was diagnosed without differences in the CPR method (p = 0.624). There was a significant correlation between existence of BME and FE (p = 0.043). Given the frequency, intensity and size of pulmonary BME and FE following CPR, these conditions may unlikely be considered as causative for death in case of initial survival but can be found in lower frequencies in autopsy histology.

Proposals for best-quality immunohistochemical staining of paraffin-embedded brain tissue slides in forensics.

Immunohistochemistry (IHC) has become an integral part in forensic histopathology over the last decades. However, the underlying methods for IHC vary greatly depending on the institution, creating a lack of comparability. The aim of this study was to assess the optimal approach for different technical aspects of IHC, in order to improve and standardize this procedure. Therefore, qualitative results from manual and automatic IHC staining of brain samples were compared, as well as potential differences in suitability of common IHC glass slides. Further, possibilities of image digitalization and connected issues were investigated. In our study, automatic staining showed more consistent staining results, compared to manual staining procedures. Digitalization and digital post-processing facilitated direct analysis and analysis for reproducibility considerably. No differences were found for different commercially available microscopic glass slides regarding suitability of IHC brain researches, but a certain rate of tissue loss should be expected during the staining process.

Acute phase response after fatal traumatic brain injury.

An inflammatory response occurring after fatal traumatic brain injury (TBI) initiates time-dependent cascades of acute phase response. This may offer the potential to monitor postmortem biomarker levels of several pro-inflammatory cytokines to gain information about the cause of death and the trauma survival time. Cerebrospinal fluid (CSF) and serum samples were collected from forensic autopsies of 95 adult cadavers after postmortem intervals up to 6 days. The cases were divided according to their cause of death into fatal TBI (n = 46) with different survival times and age- and gender-matching non-TBI fatalities as controls (n = 49). Quantitative marker levels of interleukin-6 (IL-6), ferritin, soluble tumor necrosis factor receptor type 1, C-reactive protein, and lactate dehydrogenase were analyzed using immunoassays. Standardized statistical tests were performed to differentiate causes of death and survival time of TBI cases. The CSF IL-6, ferritin, and LDH levels after TBI were significantly higher than those in the controls (p < 0.001). Only serum IL-6 values showed comparable differences (p < 0.05). Both CSF and serum ferritin levels were discriminative between early and delayed death after TBI (p < 0.05). There were partly distinctive correlations between marker levels in both fluids with rising values after longer survival. There were up to moderate correlation between the marker levels and the postmortem interval due to postmortem hemolysis. However, neither CSF nor serum level ranges were affected by the age or gender of the subjects. This study is the first to measure all five proteins systematically in postmortem trauma cases. Ferritin and IL-6 proved themselves to be interesting postmortem biomarkers to provide specific information on the injury pattern and the survival time of traumatic fatalities. Such forensic investigations could serve as inexpensive and fast laboratory tests.

Chest compression-associated injuries in cardiac arrest patients treated with manual chest compressions versus automated chest compression devices (LUCAS II) - a forensic autopsy-based comparison.

The aim of this autopsy study was to investigate chest-compression associated injuries to the trunk in out-of-hospital and in-hospital non-traumatic cardiac arrest patients treated with automated external chest compression devices (ACCD; all with LUCAS II devices) versus exclusive manual chest compressions (mCC). In this retrospective single-center study, all forensic autopsies between 2011 and 2017 were included. Injuries following cardiopulmonary resuscitation (CPR) in patients treated with mCC or ACCD were investigated and statistically compared using a bivariate logistic regression. In the seven-year period with 4433 autopsies, 614 were analyzed following CPR (mCC vs. ACCD: n = 501 vs. n = 113). The presence of any type of trunk injury was correlated with longer resuscitation intervals (30 ± 15 vs. 44 ± 25 min, p < 0.05). In comparison with mCC, treatment with ACCD led to more frequent skin emphysema (5 vs 0%, p = 0.012), pneumothorax (6 vs. 1%, p = 0.008), lung lesions (19 vs. 4%, p = 0.008), hemopericardium (3 vs 1%, p = 0.025) and liver lesions (10 vs. 1%, p = 0.001), all irrespective of confounding aspects. Higher age and longer CPR durations statistically influenced frequency of sternal and rib fractures (p < 0.001). The mean number of fractured ribs did not vary significantly between the groups (6 ± 3 vs. 7 ± 2, p = 0.09). In this cohort with unsuccessful CPR, chest compression-related injuries were more frequent following ACCD application than in the mCC group, but with only minutely increased odds ratios. The severity of injuries did not differ between the groups, and no iatrogenic injury was declared by the forensic pathologist as being fatal. In the clinical routine after successful return of spontaneous circulation a computed tomography scan for CPR-associated injuries is recommended as soon as possible.

X-chromosomal 21-indel marker panel in German and Baltic populations.

In order to verify specific biallelic X-indels and their characteristic properties in distinct populations, one German and three Baltic population groups (Estonia, Latvia, and Lithuania) have been analyzed by a short amplicon method, which also enables detection of degraded DNA samples. To combine 21 indels in a single multiplex PCR, all products were arranged according to their expected amplicon length (~40-160 bp) on the basis of three different fluorochromes. Separation of PCR products was carried out in a single capillary electrophoresis. Data evaluating was performed including five further indel markers which have already been tested in identical samples, resulting in altogether 26 markers. The majority of the genetic material showed combinations of insertion elements (L-fragments). Combinations of deletion elements (S-fragments) in contrast occurred with significant lower ratios. Hardy-Weinberg equilibrium (HWE) was observed for all markers except for MID1361 and MID329. This was attributed to an insufficient number of samples. For two known linkage groups within the 26-indel set (MID357-MID356 and MID3690-MID3719-MID2089), haplotype data were determined. A pairwise comparison of German and Baltic allele frequencies did not show significant deviation. This result indicates a possible genetic association between all four population groups. The calculated biostatistical parameters show high forensic efficiency for this set of indel markers. In a segregation analysis investigating 194 meiosis, no mutations have been detected regarding expected transmission patterns.

Unusual planned complex suicide committed with a muzzle-loading pistol in combination with subsequent hanging.

In Germany, suicides by firearms are not very common in contrast to deaths by hanging and intoxications. The use of historical muzzle-loading firearms in the context of suicides is a rarity. Contact shots from muzzle loaders cause an unusual wound morphology with extensive soot soiling. We report the case of a 59-year-old man, who committed a planned complex suicide by shooting into his mouth with a replica percussion gun in combination with hanging. The gunshot injury showed strong explosive effects in the oral cavity with fractures of the facial bones and the skull associated with cerebral evisceration (so-called Krönlein shot). Due to the special constellation of the case with hanging immediately after the shot, external bleeding from the head injuries was only moderate. Therefore, the head injuries could be assessed and partially reconstructed already at the scene.

[On the forensic relevance of orphan diseases]. / Zur forensischen Relevanz seltener Erkrankungen.

A 40-year-old woman died shortly after complaining of non-specific symptoms after a pharmacist had accidentally given her the wrong medication. The woman's partner was not familiar with her medical history and the medical file had to be obtained from the family doctor. Autopsy findings and histological examination confirmed the clinically diagnosed autoimmune polyglandular syndrome without a tangible cause of death. Poisoning could not be demonstrated and no relation between the dosage error and death could be established. Laboratory tests revealed diabetic coma with ketoacidosis as the cause of death, which was probably caused by a prolonged lack of insulin administration. In addition to the clarification of legal issues, the complete post-mortem examination of orphan diseases is also relevant for achieving a better understanding of differential diagnostic aspects and complex pathophysiological contexts. Moreover, the genetic background often underlying such diseases should be a reason to inform the family of the deceased about the autopsy results. Only then can secondary preventive measures be taken in time.

Does the Quantification of Δ9-Tetrahydrocannabinolic Acid A in Serum/Plasma Provide Any Additional Information About Consumption Pattern from Drivers Under the Influence of Cannabis?

Introduction: Δ9-tetrahydrocannabinolic acid A (THCA-A) is one of the main ingredients of cannabis plants and is converted to the psychoactive substance Δ9-tetrahydrocannabinol (THC) by decarboxylation during heating above ∼90°C. During the consumption of cannabis, a varying proportion of THCA-A is absorbed into the body. Therefore, the quantification of THCA-A in serum/plasma might provide additional information on consumption behavior in driving under the influence of cannabis cases. Materials and Methods: In this study, an already established gas-chromatography mass-spectrometry (GC-MS) method for the quantification of THC, 11-OH-THC, and THC-COOH in serum and plasma samples was extended to include THCA-A. This validated method was then applied to 1228 routinely achieved serum/plasma samples from drivers suspected of cannabis consumption in Western Saxony. Two different grouping systems for chronic/occasional consumption, one system for acute/subacute consumption, Huestis formulas, and the cannabis influence factor (CIF) were used for evaluation. Results: Method validation showed appropriate results for forensic toxicological routine analysis. Limit of detection and lower limit of quantification (LLOQ) for THCA-A were 0.3 and 1.0 ng/mL, respectively. Reproducibility was <11% and accuracy ranged between 104% and 107%. THCA-A was stable in native samples at least for 2 weeks at room temperature or 4°C as well as 1 month at -20°C. Freeze-thaw stability for three cycles and processed sample stability over 3 days was proven. A total of 865 cases with a THC concentration above the German analytical cutoff of 1 ng/mL as well as the analytical LLOQs of 0.9 and 2.5 ng/mL for 11-OH-THC and THC-COOH, respectively, were included in further statistical analysis. In 407 (47.1%) of these samples, THCA-A was quantifiable. Different statistical analyses indicated a correlation between THCA-A and THC concentrations in cases of chronic and acute consumption. In addition, an increase of chronic and acute cases with increasing THCA-A concentrations was observed. However, no correlation between THCA-A and CIF was found. Discussion: These data show that THCA-A might be an additional indicative marker to provide information about consumption frequency and acuteness. Additional studies with known consumption frequencies and times are required to verify these findings.

Systematic Workup of Transfusion Reactions Reveals Passive Co-Reporting of Handling Errors.

Introduction: Reporting of transfusion reactions is good practice and required by many guidelines. Errors in the transfusion chain can also lead to severe patient reactions and depend on active error reporting. We aimed to characterize transfusion incidents and asked whether workup of transfusion reactions may also contribute to revealing logistical errors. Methods: Transfusion medical records from 2011 to 2019 at our tertiary medical centre, as well as forensic autopsy reports, digitized sections, and court records from 1990 to 2019 were analysed. A total of 230,845 components were transfused between 2011 and 2019 at our own institution. Results: Overall, 322 transfusion incidents were reported. Of these, 279 were from our own institution, corresponding to a frequency of 0.12% of all transfusions. The distribution of reaction types is consistent with the literature, with allergic reactions (55.9%), febrile-non-hemolytic reactions (FNHTR, 24.2%), hemolytic reactions (3.4%) and other types at smaller frequencies (<3%). Twenty-nine (10.4%) of the 279 reports revealed logistical errors, including hemoglobin above guideline threshold (4.3%), incorrect or non-performed bedside tests (3.2%), inadequate patient identification (2.5%), laboratory and issuing errors, missed product checks or failure to follow recommendations (1.1% each). Eight of 29 (27.5%) of the logistical errors were detected by serendipity during workup of incident reports. In addition, 8/932 autopsy cases under code A14 (medical treatment errors) were found to be transfusion-associated (0.9%). Conclusion: Systematic workup of transfusion incidents can identify previously undetected errors in the transfusion chain. Passive reporting of errors through the recording of side effects may serve as a tool to assess more closely assess the frequency and quality of handling errors in real life, and thus serve to improve patient safety.

Quantification of (9R)- and (9S)-hexahydrocannabinol (HHC) via GC-MS in serum/plasma samples from drivers suspected of cannabis consumption and immunological detection of HHC and related substances in serum, urine, and saliva.

The semisynthetic cannabinoid hexahydrocannabinol (HHC) is currently getting a lot of media attention because the legal status in many countries is not clearly specified. In this study, a GC-MS method for the quantification of Δ9-tetrahydrocannabinol (THC), 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC), and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH) was extended to (9R)- and (9S)-HHC. The applicability was proven by serum/plasma samples from drivers suspected of cannabis consumption. Limit of detection (LOD) and lower limit of quantification (LLOQ) were 0.15 and 0.25 ng/mL, respectively. Within-run imprecision was <6.5% and between-run imprecision was <10.0%. Inter-injection stability, processed sample stability (3 days), freeze-thaw stability (three cycles), and storage stability (1 week room temperature; 1 month 4°C, -20°C) could be proven. Both HHC diastereomers could be detected in 17 (5.3%) out of 321 analyzed samples from traffic controls in Western Saxony. The mean ratio between (9R)- and (9S)-HHC was 1.99 (CV = 14.6%). Quantification resulted in concentrations between

Sensitive and simultaneous quantification of 16 neurotransmitters and metabolites in murine microdialysate by fast liquid chromatography-tandem mass spectrometry.

The sensitive and simultaneous measurement of multiple neurotransmitters in microdialysate (MD) of freely moving mice is a prerequisite to study neurochemical imbalances in specific brain regions. The quantitative analysis of 16 neurotransmitters and metabolites, including serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), melatonin (ME), dopamine (DA), levodopa (l-DOPA), 3-methoxytyramine (3-MT), norepinephrine (NE), epinephrine (EP), homovanillinic acid (HVA), acetylcholine (ACh), deoxy carnitine (iso-ACh), choline (Ch), and É£-aminobutyric acid (GABA), adenosine (ADE), glutamine (Gln), and glutamic acid (Glu) was achieved within a chromatographic separation time of 6.5 min by the application of a biphenyl column coupled to an API-QTrap 5500 (AB SCIEX) mass spectrometer. Optimized chromatographic separation as well as high sensitivity allow the simultaneous analysis and precise quantification of 16 neurotransmitters and metabolites in artificial cerebrospinal fluid (CSF). Sample preparation procedure consisted of simply adding isotopically labeled internal standard solution to the microdialysis sample. The limits of detection in aCSF ranged from 0.025 pg (Ch) to 9.75 pg (Gln) and 85.5 pg (HVA) on column. Recoveries were between 83 and 111% for neurotransmitter concentrations from 0.6 to 45 ng/ml or 200 ng/ml with a mean intra-day and inter-day coefficient of variation of 7.6% and 11.2%, respectively. Basal extracellular concentrations of the following analytes: 5-HT, 5-HIAA, ME, DA, 3-MT, HVA, ACh, iso-ACh, Ch, GABA, ADE, Gln, and Glu were determined in the striatum of mice with a MD flow rate of 0.5 µl/min. This LC-MS/MS method leads to an accurate quantification of ACh and its isobaric structure iso-ACh, which were detected in the MD samples at ratios of 1:8.6. The main advantage of the high sensitivity is the miniaturization of the MD protocol with short sample collection times and volumes down to 5 µl, which makes this method suitable for pharmacological intervention and optogenetic studies to detect neurochemical changes in vivo.

Entomological identification of the post-mortem colonization of wolf cadavers in different decomposition stages.

Human-driven biodiversity loss is progressively becoming a problem with dramatic consequences for the conservation of vital ecosystems. The increasing number of illegal killings of the grey wolf (Canis lupus, Linnaeus, 1758), a threatened species, displays the need for investigation and prosecution of such offences. Forensic entomology makes use of the knowledge about necrophagous insects to estimate a minimum time-since-death interval of the deceased person or animal, which can give important information on a possible perpetrator. The cadaver fauna along five decomposition stages of wolves in Germany was investigated in the period 2014-2021. The insects from 70 wolf cadavers, originating from all over Germany, were provided by the Leibniz Institute for Zoo and Wildlife Research Berlin. The accumulated degree day (ADD) model was applied for the post-mortem interval estimation on wolf cadavers for the first time. A total of 20 coleopteran species and 14 different dipteran species were discovered and identified. Almost 99 % of all insect specimens were from the order of Diptera, and beetles (Coleoptera) accounted for only 1 % of the cadaver fauna. The blowflies (Calliphoridae) are of particular importance for forensic issues, accounting for about 66 % of all families. Carrion beetles (Silphidae) were found as the second most abundant family (about 21 %). In addition, combining all cases, a steadily increasing insect species richness S was detected from early decay to advanced decay (fresh S = 8; bloated S = 12; active decay S = 21; advanced decay S = 34). In the following remains stage, the species number decreased again (S = 24). However, no significant difference in the number of species was found between the stages of decay when the cases were considered individually. The temporal pattern of insect appearance was found to be congruent with those of previous studies. Furthermore, a time of death was determined for each case and compared to the pathologist's estimates. This study provides insights into the arthropod fauna of wolf remains for the first time, applies the ADD-Model for post-mortem interval estimation, and discusses the suitability of forensic entomology for wildlife death investigations.

Survived ileocecal blowout from compressed air.

Industrial accidents with compressed air entering the gastro-intestinal tract often run fatally. The pressures usually over-exceed those used by medical applications such as colonoscopy and lead to vast injuries of the intestines with high mortality. The case described in this report is of a 26-year-old man who was harmed by compressed air that entered through the anus. He survived because of fast emergency operation. This case underlines necessity of explicit instruction considering hazards handling compressed air devices to maintain safety at work. Further, our observations support the hypothesis that the mucosa is the most elastic layer of the intestine wall.

Neonatal freshwater drowning after birth in the bathroom.

The macroscopic and microscopic findings in neonatal freshwater drowning are demonstrated on the basis of 3 exemplary cases of birth in the bathroom after concealed pregnancy. If the newborn is expelled under water and remains there until death, the lungs cannot be ventilated with air. In case of incomplete or only temporary submersion, aeration of the lungs may develop in the immediate postpartum period. The morphologic criteria of separate existence are critically discussed with respect to the presented findings.

An avoidable death in police custody?

An obviously inebriated 36-year-old man was found in a helpless condition on a pavement. When he was brought to a police station, a doctor certified the individual to be fit to be held in custody. He was unresponsive when the officers tried to wake him the next morning but was allowed to sleep on until he was found dead in his cell at mid day. An autopsy revealed as cause of death an epidural hematoma and cerebral contusion with fracture of the calvarium. The expert report stressed the point that the monitoring of the state of consciousness had been objectively insufficient throughout the entire time period, both in terms of frequency as well as in terms of the monitoring methods. However, neither the police officers involved nor the doctor, was subjected to prosecution mainly because of the high burden of proof required by German criminal law. The article will discuss conclusions for the avoidance of such deaths (such as training of police officers, legal regulations on monitoring procedures, etc).

Survival-time dependent increase in neuronal IL-6 and astroglial GFAP expression in fatally injured human brain tissue.

Knowledge on trauma survival time prior to death following a lethal traumatic brain injury (TBI) may be essential for legal purposes. Immunohistochemistry studies might allow to narrow down this survival interval. The biomarkers interleukin-6 (IL-6) and glial fibrillary acidic protein (GFAP) are well known in the clinical setting for their usability in TBI prediction. Here, both proteins were chosen in forensics to determine whether neuronal or glial expression in various brain regions may be associated with the cause of death and the survival time prior to death following TBI. IL-6 positive neurons, glial cells and GFAP positive astrocytes all concordantly increase with longer trauma survival time, with statistically significant changes being evident from three days post-TBI (p < 0.05) in the pericontusional zone, irrespective of its definite cortical localization. IL-6 staining in neurons increases significantly in the cerebellum after trauma, whereas increasing GFAP positivity is also detected in the cortex contralateral to the focal lesion. These systematic chronological changes in biomarkers of pericontusional neurons and glial cells allow for an estimation of trauma survival time. Higher numbers of IL-6 and GFAP-stained cells above threshold values in the pericontusional zone substantiate the existence of fatal traumatic changes in the brain with reasonable certainty.