Multiorgan immunohistochemical endothelial expression of E-selectin in a forensic case of sepsis.

Sepsis is one of the major threats for the survival and prognosis of patients in intensive care units. In cases where detailed clinical data and monitoring is available, the diagnosis of sepsis is reliable. But when clinical data are incomplete or missing and sepsis is only suspected based on the autopsy results, the picture is often equivocal. This report describes the gross pathological findings obtained from the autopsy of a 48-year-old woman with Crohn's disease after surgical intervention. Macroscopically, we found intestinal perforation and signs of peritonitis. Histologically, the pulmonary/bronchial arteries were lined with E-selectin (CD 62E)-positive endothelial cells, which are an established postmortem histological marker of sepsis. We extended our investigations to the cerebral cortex and subcortical medullary layer. The endothelium of the cortical vessels and those in the cerebral medullary layer were likewise immunopositive for E-selectin. Furthermore, numerous TMEM119-positive, highly ramified microglial cell profiles were found in the grey and white matter. Microglial cells were lining the vascular profiles. In addition, TMEM119-positive microglial profiles were abundant in the cerebrospinal fluid (CSF). Multiorgan E-selectin positivity of the vascular endothelia provides further evidence for the postmortem diagnosis of sepsis.

The polychromatism of postmortem cerebrospinal fluid.

Based on the assumption that postmortem cerebrospinal fluid (CSF) is contaminated depending on the chosen sampling technique in the forensic setting resulting in bloody or at least hemolytic CSF samples, we systematically documented a total of 183 postmortem CSF samples. These samples were all assessed for their quality and color, regardless of the cause of death or the postmortem interval. The investigations were carried out through subjective assessment of color and turbidity, as well as objective measurements of the optical density (OD) of the CSF supernatants after centrifugation of each sample, with standardized photographic documentation. The observations revealed that in 28 cases the CSF was absolutely (crystal-) clear and transparent. Most of our samples showed color changes ranging from xanthrochromic to rose. Intensive staining of the supernatants was only found in a small proportion of the examined collective. We found that postmortem CSF has no uniform appearance but rather a diverse range of color spectra, and the color, as well as the OD of the CSF, correlates significantly with the postmortem interval (p < 0.001) when sampled using the proposed standard procedure.

Density of TMEM119-positive microglial cells in postmortem cerebrospinal fluid as a surrogate marker for assessing complex neuropathological processes in the CNS.

Routine coronal paraffin-sections through the dorsal frontal and parieto-occipital cortex of a total of sixty cases with divergent causes of death were immunohistochemically (IHC) stained with an antibody against TMEM119. Samples of cerebrospinal fluid (CSF) of the same cases were collected by suboccipital needle-puncture, subjected to centrifugation and processed as cytospin preparations stained with TMEM119. Both, cytospin preparations and sections were subjected to computer-assisted density measurements. The density of microglial TMEM119-positive cortical profiles correlated with that of cytospin results and with the density of TMEM119-positive microglial profiles in the medullary layer. There was no statistically significant correlation between the density of medullary TMEM119-positive profiles and the cytospin data. Cortical microglial cells were primarily encountered in supragranular layers I, II, and IIIa and in infragranular layers V and VI, the region of U-fibers and in circumscribed foci or spread in a diffuse manner and high density over the white matter. We have evidence that cortical microglia directly migrate into CSF without using the glympathic pathway. Microglia in the medullary layer shows a strong affinity to the adventitia of deep vessels in the myelin layer. Selected rapidly fatal cases including myocardial infarcts and drowning let us conclude that microglia in cortex and myelin layer can react rapidly and its reaction and migration is subject to pre-existing external and internal factors. Cytospin preparations proved to be a simple tool to analyze and assess complex changes in the CNS after rapid fatal damage. There is no statistically significant correlation between cytospin and postmortem interval. Therefore, the quantitative analyses of postmortem cytospins obviously reflect the neuropathology of the complete central nervous system. Cytospins provide forensic pathologists a rather simple and easy to perform method for the global assessment of CNS affliction.

Axonal injury is detected by ßAPP immunohistochemistry in rapid death from head injury following road traffic collision.

The accumulation of ßAPP caused by axonal injury is an active energy-dependent process thought to require blood circulation; therefore, it is closely related to the post-injury survival time. Currently, the earliest reported time at which axonal injury can be detected in post-mortem traumatic brain injury (TBI) tissue by ßAPP (Beta Amyloid Precursor Protein) immunohistochemistry is 35 min. The aim of this study is to investigate whether ßAPP staining for axonal injury can be detected in patients who died rapidly after TBI in road traffic collision (RTC), in a period of less than 30 min.We retrospectively studied thirty-seven patients (group 1) died very rapidly at the scene; evidenced by forensic assessment of injuries short survival, four patients died after a survival period of between 31 min and 12 h (group 2) and eight patients between 2 and 31 days (group 3). The brains were comprehensively examined and sampled at the time of the autopsy, and ßAPP immunohistochemistry carried out on sections from a number of brain areas.ßAPP immunoreactivity was demonstrated in 35/37 brains in group 1, albeit with a low frequency and in a variable pattern, and with more intensity and frequency in all brains of group 2 and 7/8 brains from group 3, compared with no similar ßAPP immunoreactivity in the control group. The results suggest axonal injury can be detected in those who died rapidly after RTC in a period of less than 30 min, which can help in the diagnosis of severe TBI with short survival time.

Quantitative evidence of suppressed TMEM119 microglial immunohistochemistry in fatal morphine intoxications.

The aim of this pilot study was to investigate the diagnostic potential of TMEM119 as a useful microglia-specific marker in combination with immunostainings for phagocytic function and infiltrating capacity of monocytes in cases of lethal monosubstance intoxications by morphine (MOR), methamphetamine (METH), and of ethanol-associated death (ETH) respectively. Human brain tissue samples were obtained from forensic autopsies of cases with single substance abuse (MOR, n = 8; ETH, n = 10; METH, n = 9) and then compared to a cohort of cardiovascular fatalities as controls (n = 9). Brain tissue samples of cortex, white matter, and hippocampus were collected and stained immunohistochemically with antibodies against TMEM119, CD68KiM1P, and CCR2. We could document the lowest density of TMEM119-positive cells in MOR deaths with highly significant differences to the control densities in all three regions investigated. In ETH and METH deaths, the expression of TMEM119 was comparable to cell densities in controls. The results indicate that the immunoreaction in brain tissue is different in these groups depending on the drug type used for abuse.

Metabolomics in postmortem cerebrospinal fluid diagnostics: a state-of-the-art method to interpret central nervous system-related pathological processes.

In the last few years, quantitative analysis of metabolites in body fluids using LC/MS has become an established method in laboratory medicine and toxicology. By preparing metabolite profiles in biological specimens, we are able to understand pathophysiological mechanisms at the biochemical and thus the functional level. An innovative investigative method, which has not yet been used widely in the forensic context, is to use the clinical application of metabolomics. In a metabolomic analysis of 41 samples of postmortem cerebrospinal fluid (CSF) samples divided into cohorts of four different causes of death, namely, cardiovascular fatalities, isoIated torso trauma, traumatic brain injury, and multi-organ failure, we were able to identify relevant differences in the metabolite profile between these individual groups. According to this preliminary assessment, we assume that information on biochemical processes is not gained by differences in the concentration of individual metabolites in CSF, but by a combination of differently distributed metabolites forming the perspective of a new generation of biomarkers for diagnosing (fatal) TBI and associated neuropathological changes in the CNS using CSF samples.

Myelin basic protein and neurofilament H in postmortem cerebrospinal fluid as surrogate markers of fatal traumatic brain injury.

The aim of this study was to investigate if the biomarkers myelin basic protein (MBP) and neurofilament-H (NF-H) yielded informative value in forensic diagnostics when examining cadaveric cerebrospinal fluid (CSF) biochemically via an enzyme-linked immunosorbent assay (ELISA) and comparing the corresponding brain tissue in fatal traumatic brain injury (TBI) autopsy cases by immunocytochemistry versus immunohistochemistry. In 21 trauma and 19 control cases, CSF was collected semi-sterile after suboccipital puncture and brain specimens after preparation. The CSF MBP (p = 0.006) and NF-H (p = 0.0002) levels after TBI were significantly higher than those in cardiovascular controls. Immunohistochemical staining against MBP and against NF-H was performed on cortical and subcortical samples from also biochemically investigated cases (5 TBI cases/5 controls). Compared to the controls, the TBI cases showed a visually reduced staining reaction against MBP or repeatedly ruptured neurofilaments against NF-H. Immunocytochemical tests showed MBP-positive phagocytizing macrophages in CSF with a survival time of > 24 h. In addition, numerous TMEM119-positive microglia could be detected with different degrees of staining intensity in the CSF of trauma cases. As a result, we were able to document that elevated levels of MBP and NF-H in the CSF should be considered as useful neuroinjury biomarkers of traumatic brain injury.

TMEM119 as a specific marker of microglia reaction in traumatic brain injury in postmortem examination.

The aim of the present study was a refined analysis of neuroinflammation including TMEM119 as a useful microglia-specific marker in forensic assessments of traumatic causes of death, e.g., traumatic brain injury (TBI). Human brain tissue samples were obtained from autopsies and divided into cases with lethal TBI (n = 25) and subdivided into three groups according to their trauma survival time and compared with an age-, gender-, and postmortem interval-matched cohort of sudden cardiovascular fatalities as controls (n = 23). Brain tissue samples next to cortex contusions and surrounding white matter as well as samples of the ipsilateral uninjured brain stem and cerebellum were collected and stained immunohistochemically with antibodies against TMEM119, CD206, and CCR2. We could document the highest number of TMEM119-positive cells in acute TBI death with highly significant differences to the control numbers. CCR2-positive monocytes showed a significantly higher cell count in the cortex samples of TBI cases than in the controls with an increasing number of immunopositive cells over time. The number of CD206-positive M2 microglial cells increased survival time-dependent. After 3 days of survival, the cell number increased significantly in all four regions investigated compared with controls. In sum, we validate a specific and robustly expressed as well as fast reacting microglia marker, TMEM119, which distinguishes microglia from resident and infiltrating macrophages and thus offers a great potential for the estimation of the minimum survival time after TBI.

Cerebellar heterotopia of infancy in sudden infant death syndrome: an observational neuropathological study of four cases.

Sudden infant death syndrome (SIDS) is the sudden unexpected death of an infant < 1 year of age that remains unexplained after comprehensive workup including complete autopsy and investigation of the circumstances of death. The triple risk hypothesis posits that SIDS results as a combination of both intrinsic and extrinsic factors on the background of a predisposing vulnerability. Neuropathological examination in the past has focussed mainly on the brainstem as the major player in respiratory control, where subtle findings have been linked to the chain of events leading to death in SIDS. The cerebellum has received less attention, probably due to an assumed negligible role in central cardiorespiratory control. We report four cases of SIDS in which neuropathological investigation revealed cerebellar heterotopia of infancy, a distinct malformation of the cerebellum, and discuss the potential impact of this condition on the aetiology and pathogenesis of SIDS.

Post-mortem cerebrospinal fluid diagnostics: cytology and immunocytochemistry method suitable for routine use to interpret pathological processes in the central nervous system.

Due to its protected anatomical location, cerebrospinal fluid (CSF) is a very stable fluid which undergoes comparatively little change in the early post-mortem phase. While many immunohistochemical markers already established for clinical diagnostic issues in tissue samples obtained by biopsy could meanwhile be translated also to post-mortem tissue, no systematic immunocytochemical investigations have generally been conducted on post-mortem body fluids and for CSF specifically, have not been established at all. CSF as the fluid directly surrounding the brain should also be examined to allow a more detailed characterization of processes in the central nervous system. Comparing traumatized tissue and CSF can complete forensic assessment and complement neuropathological evaluation.

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.

Forensic Pathology of Traumatic Brain Injury.

Traumatic brain injury constitutes a significant proportion of cases requiring forensic examination, and it encompasses (1) blunt, nonmissile head injury, especially involving motor vehicle accidents, and (2) penetrating, missile injury produced by a range of high- and lower-velocity projectiles. This review examines the complex pathophysiology and biomechanics of both types of neurotrauma and assesses the macroscopic and histologic features of component lesions, which may be used to determine the cause and manner of death resulting from an intentional assault or accident. Estimation of the survival time postinjury by pathologic examination is also important where malicious head injury is suspected, in an attempt to ascertain a time at which the traumatic event might have been committed, thereby evaluating the authenticity of statements made by the alleged perpetrator.

Traumatic basal subarachnoid haemorrhage or ruptured brain aneurysm in 16-year-old boy? - case report

Traumatic basal subarachnoid haemorrhage (TBSAH) represents only 1.8% of all subarachnoid haemorrhage cases diagnosed during autopsy. This report presents such a case from the current practice of the authors. Sixteen-year-old boy was beaten by the aggressors. Suddenly he lost his consciousness and fall after he received a single blow in the neck. He was resuscitated immediately, but died at the scene. During the external examination we did not find any significant external injuries. Autopsy revealed large contusion of right sternocleidomastoid muscle. In the cranial cavity we found extensive subarachnoid haemorrhage, located mainly on brain basis, in the posterior cranial fossa and covering the subtentorial structures. During the preparation of blood vessels we noticed a slight change of morphology suggesting damaged vessel or aneurysm, or vascular malformation located in the basilar artery bifurcation, which was taken to detailed microscopic evaluation using the special stainings. Histological examination showed vital interruption of the basilar artery wall with massive haemorrhage, without the presence of general microscopic pathology. From the medico-legal viewpoint, to determine traumatic background of haemorrhage it is necessary to find the coexistence of the following circumstances: a sustained trauma, post-mortem findings consistent with a time of injury, the presence of temporal relationship between injury and death, and morphological vital injury of the brain vessel, as well as the absence of prior vascular malformations. For this purpose Verhoeff-van Gieson's, Masson's, Turnbull's and Gomori' histological stainings may be successfully used.

Macroscopic and Microscopic Cerebral Findings in Drug and Alcohol Abusers: The Point of View of the Forensic Pathologist.

Drug abuse still represents a significant challenge for forensic pathologists; it must always be considered during the autopsy examination when the brain morphological alterations observed are not characteristic of any known disease of the central nervous system (CNS). Nonetheless, no specific brain lesions had been found to characterize the precise drug that caused the poisoning. In fact, a broad spectrum of changes affecting the CNS are seen in drug abusers. Thus, forensic pathology plays a key role in identifying the encephalic morphological alterations underlying the death. The aim of this review is to present an updated overview of the literature regarding the correlation between the main substances of abuse and the morphological alterations of the CNS to help the forensic pathologist to discriminate drug-induced alterations of the brain. The authors used the PRISMA criteriology to perform the bibliographic search for the present review. Among the articles identified according to the selected search criteria, 116 articles were chosen which allow us to define a picture of the main macroscopic and microscopic alterations of the brain in drug abuse.

Neuroforensomics: metabolites as valuable biomarkers in cerebrospinal fluid of lethal traumatic brain injuries.

Traumatic brain injury (TBI) is a ubiquitous, common sequela of accidents with an annual prevalence of several million cases worldwide. In forensic pathology, structural proteins of the cellular compartments of the CNS in serum and cerebrospinal fluid (CSF) have been predominantly used so far as markers of an acute trauma reaction for the biochemical assessment of neuropathological changes after TBI. The analysis of endogenous metabolites offers an innovative approach that has not yet been considered widely in the assessment of causes and circumstances of death, for example after TBI. The present study, therefore, addresses the question whether the detection of metabolites by liquid-chromatography-mass spectrometry (LC/MS) analysis in post mortem CSF is suitable to identify TBI and to distinguish it from acute cardiovascular control fatalities (CVF). Metabolite analysis of 60 CSF samples collected during autopsies was performed using high resolution (HR)-LC/MS. Subsequent statistical and graphical evaluation as well as the calculation of a TBI/CVF quotient yielded promising results: numerous metabolites were identified that showed significant concentration differences in the post mortem CSF for lethal acute TBI (survival times up to 90 min) compared to CVF. For the first time, this forensic study provides an evaluation of a new generation of biomarkers for diagnosing TBI in the differentiation to other causes of death, here CVF, as surrogate markers for the post mortem assessment of complex neuropathological processes in the CNS ("neuroforensomics").

Traumatic Aneurysm Involving the Posterior Communicating Artery.

Traumatic intracranial aneurysms (TICAs) are rare, accounting for less than 1% of all intracranial aneurysms. However, they are associated with a mortality rate of over 50%. The case presented herein focuses on a posterior communicating artery TICA caused by violent aggression. A 41-year-old man with massive subarachnoid hemorrhage (SAH), on admission to hospital, had a CT angiography that showed a ruptured left posterior communicating artery aneurysm with continuous blood loss and underwent neurosurgical cooling. The CT scan also showed fractures of the mandible, mastoid and left styloid process, as well as brain contusions caused by blows and kicks. Despite medical treatment and surgery, after four days, he died. The assault dynamics were recorded by a camera in the bar. The damage was caused by kicks to the neck and head. The forensic neuropathological examination showed the primary injury (SAH, subdural hemorrhage, cerebral contusions, head-neck fractures), as well as secondary damage following the attack (cerebral infarcts, edema, supratentorial hernia, midbrain hemorrhage). The coil was intact and well positioned. In this case, circumstantial information, medical records, and the type of injury could shed light on the mechanism of the production of a TICA. In addition, the CT angiography and histological investigations helped to distinguish a recent and traumatic aneurysm from a pre-existing one. Following precise steps, the study of aneurysms can be helpful in clarifying their traumatic origin even when the victim was taking drugs. The aim of this study is also to share the diagnostic process that we used in the forensic field for the assessment of suspected traumatic aneurysms.

Temporal lobe uncal herniation with contralateral superior cerebellar artery infarct.

Herniation of the temporal lobe uncus typically leads to the compression of the ipsilateral oculomotor nerve, resulting in ipsilateral mydriasis, as well as compression of the ipsilateral posterior cerebral artery, leading to infarction in the posterior inferior temporal lobe and medial occipital cortex. In this report, we present the case of a 45-year-old man with a large left subdural hematoma. At autopsy, we observed left cingulate and uncal herniations, along with the characteristic lesions of Kernohan notch phenomenon due to compression of the contralateral cerebral peduncle. Additionally, a hemorrhagic infarct was identified in the right cerebellar hemisphere in the distribution of the superior cerebellar artery (SCA). This case provides the first autopsy report of uncal herniation with contralateral SCA infarct, an extremely rare condition. Importantly, this vascular complication may often go unnoticed in patients with Kernohan notch phenomenon although it may carry a grave clinical prognosis.

Prevalence of secondary brain injuries and association with trauma circumstances in neuropathologically examined medico-legal autopsy cases with primary head trauma.

Primary head injury is often followed by secondary brain damage. However, the association between injury circumstances and the prevalence of secondary injuries remains unclear. We report the prevalence and association of secondary brain injuries with the circumstances in which a head injury was sustained. The sample comprised 76 neuropathologically examined medico-legal autopsy cases with an acute primary head injury. Neuropathology reports were analysed to determine the prevalence of various secondary injuries, i.e., hypoxic-ischaemic neuronal injury, brain oedema, and vascular axonal injury (VAI). The prevalences were compared between cases from three distinct injury circumstances, i.e., fall, assault, and strangulation. The sample had a median age of 49 years (interquartile range 27-73) and 71.1% were identified as male. As for distinct injury circumstances, the sample comprised 14 fall cases, 21 assault victims, and 6 strangulation victims. The prevalence of hypoxic-ischaemic neuronal injury was highest in strangulations (100.0%), followed by assaults (81.0%) and falls (64.3%); of specific brain regions, statistically significant differences between the three case groups were found in frontal and parietal cortex (p ≤ 0.018) and the hippocampus (p = 0.005). Brain oedema was present in approximately half of assault (47.6%) and strangulation cases (50.0%), contrastingly to the lower prevalence in falls (7.1%; p = 0.024). The prevalence of VAI appeared higher among assault (23.8%) and strangulation cases (16.7%) compared to falls (7.1%), but the differences were not statistically significant. We conclude that hypoxic-ischaemic neuronal injury and brain oedema were more prevalent among assault and strangulation cases compared to falls.

Tissue sample analysis for post mortem determination of brain edema.

The post mortem evaluation of a brain edema is routinely performed by pathologists based on the macroscopic signs during autopsy. This method represents the current gold standard, but is subjective and observer dependent. Therefore, three post mortem evaluation methods of brain samples were analyzed in this work: histology, wet-dry weight and normalized cerebral weight, which was described in 2020 by Bauer et al. Tissue samples from six different regions of 34 brains were collected and examined both by rating of histological slides and by measuring the water content by using a drying oven. The rating of the histological slides, stained with hematoxylin and eosin, was performed by two pathologists independently. For the water content, the wet weight and the dry weight of each sample were set in relation. The normalized cerebral weight was calculated by dividing the brain weight by the brain volume, which were both determined during autopsy and in computed tomography images, respectively. A fair to moderate interrater agreement was obtained for the histologic evaluation and a significant correlation was present between one rater and the wet-dry weight and the normalized cerebral weight method. When classifying according to the gold standard, a significant difference was detected between the edematous and nonedematous cases by using the wet-dry weight method in the cerebral cortex and by using the normalized cerebral weight method. However, the significant correlations and group differences were limited to the aforementioned results. In conclusion, both the histological and the wet-dry weight method show limited benefits for the classification of brain edema and the histology analysis is highly observer dependent. The normalized cerebral weight method, however, reveals a significant effect between the edematous and nonedematous cases when classifying according to the gold standard. Therefore, we suggest to apply this method for the assessment of brain edema since it is objective and rater independent. Nevertheless, the exact evaluation of brain edema remains a challenging task, especially due to the continuous transition between no edema and edema.

Abordaje del trauma cráneo encefálico en patología forense / Addressing head trauma in forensic pathology

En el presente artículo se describen los mecanismos de trauma y las principales lesiones que se encuentran en una muerte violenta ocasionada por traumatismo cráneo encefálico con el fin de contribuir a un abordaje práctico de estos casos en Patología Forense, tanto para los médicos de planta como para los que están en formación, con el propósito de homogenizar y unificar criterios y siempre con el objetivo final de contribuir con una adecuada administración de justicia y así cumplir con el objetivo de la especialidad en Medicina Legal.

In this paper we describe the main mechanisms of trauma and major injuries that are founded in a violent death caused by head trauma in order to contribute to a practical approach to these cases in Forensic Pathology for both staff physicians and residents, in order to standardize and unify criteria; and always with the ultimate goal of contributing to the proper administration of justice that is the final purpose in Forensic Medicine.